The first is an on-farm loss caused by cluster thinning to reduce berry variability during ripening, a practice presumed to improve wine quality. Cluster thinning at veraison is a common practice performed in California wine grape cultivation, and the timing is dependent on variety, vineyard location, and cultivation practices. The second by-product is grape marc, which consists of grape skins, stems, seeds, and pulp accumulated either after extracting the juice to ferment white wine or after fermentation and pressing for red wine . Several studies on red and white wine grape varieties have shown that concentrations of total phenolic content, -catechin, -epicatechin, procyanidin dimers and trimers, and proanthocyanidins of wine grapes would generally decrease and then stabilize between veraison and harvests . Thus, thinned at veraison are considered rich sources of flavanols. Nonetheless, thinned clusters are traditionally made into verjus, sour grape sauce, and unripe grape syrup in European and Middle East countries or simply left behind in many vineyards as soil fertilizers in spite of the increased production costs and lost yields . The health-promoting properties of flavanol monomers, an important subgroup of the dietary polyphenols, particularly -catechin and -epicatechin, and their oligomeric procyanidins, have been extensively studied in cocoa and chocolate . Epidemiology studies have suggested that the consumption of flavanol-rich diets and plant extracts is associated with reducing risk of cardiovascular disease , lowering blood pressure, especially in pre-hypertensive and hypertensive individuals, and enhancing their physical activity . Cocoa oligomeric procyanidins were found to be effective at preventing weight gain, development of glucose intolerance, and insulin resistance during a long-term high-fat feeding study by Dorenkott et al. . Lotito and Fraga demonstrated in an in vitro study, dutch bucket for tomatoes that the addition of -catechin and -epicatechin would delay lipid oxidation, and α-tocopherol and β-carotene depletion in human oxidized plasma induced by a radical generator.

The biological activities of -epicatechin and -epicatechin-containing foods are of particular interests in recent years because -epicatechin causes multiple actions that may provide beneficial synergy for cardiovascular and neuropsychological health . It is also worth mentioning that a predominance of -catechin, rather than the more bio available -catechin , was found in commercial chocolate samples. -Catechin is the naturally occurring form in foods such as berries and cacao beans, however, the Dutching process converts cocoa flavanols to the -catechin enantiomer . Grapes are known for their rich polyphenols including abundant -catechin, -epicatechin, and procyanidins. In an evaluation of anti-platelet activity of grape marc extracts, the ethanolic extract with catechin, epicatechin, and quercetin being the most abundant phenolic compounds was capable of inhibiting platelets aggregation in a wide range of agonist concentrations . Another recent study on the digested fractions of seedless red and white wine grape marc revealed that all digested fractions prevented the hyperglycemic actions in the cell viability and nitric oxide /reactive oxygen species balance, suggesting the protective effects of grape marc products on the vascular endothelial barrier function . Holt et al. also conducted a comprehensive literature review on using chardonnay marc, a plant-based natural product, as a new model for upcycled co-products in food applications and comparing with considerable clinical data generated from cocoa products on human cardiometabolic health in the context of healthy dietary patterns. Thus, it is logical to explore upcycling solutions for flavanol-rich thinned clusters in food applications, especially in cocoa-based products that have been perceived as flavanol-rich by consumers. The upcycling of wine-making by-products in food applications has been generating more interest in recent years with the main focus on using dried grape marc as a bulking agent to change textural and rheological properties in foodstuffs such as cookies , soft candies , and chocolate spread . In a chocolate ice cream feasibility study conducted by Soukoulis and Tzia , grape molasses was used as a potential sucrose substitution that exhibited the best chocolate color and mouth-coating ability in the final product.

Bolenz and Glöde evaluated the processing aspects and the impact on chocolate properties like particle size, total phenolic content, and sensory perception of milk chocolate enriched with grape marc and grape seed flour. However, concentration data on bio active flavanols such as -epicatechin are beyond the study scope. When cocoa-based products are analyzed for their flavanol content, individual ingredients, such as thinned clusters in various forms , are generally not separated from the matrix to gain insights on the individual contribution from each ingredient. Our study utilized improved ultra-performance liquid chromatography-fluorescence detector methods that provided new reference information on health-promoting flavanol monomers -catechin and -epicatechin, and procyanidins of thinned clusters alongside cocoa flavanols. The idea of incorporating grape thinned grape clusters into cocoa-based food products as functional ingredients opens new doors to upcycle underutilized grapevine by-products and to improve cocoa-based products with potentially more health-promoting and positive sensory properties with fewer calories.Fresh Chardonnay and Pinot noir thinned clusters were collected at veraison from the Los Carneros American Viticultural Areas in Sonoma County in mid-August 2019. The clusters were then categorized into light and dark for Chardonnay, and red and green for Pinot noir, respectively, based on berry colors. To gain more in-depth understanding of the flavanol composition of the thinned clusters, the seed and seedless fractions were manually separated, freeze-dried, and milled as described in Sinrod et al. .Flavanols and procyanidins of grape thinned grape clusters and cocoa powders were analyzed and reported following a single laboratory validated UPLC-FLD method by Bussy, Hewitt, et al. and Bussy, Ottaviani, and Kwik-Uribe with modifications. Cocoa powders were defatted three times by sonicating 5 g powder in ~45 mL hexane at 50°C for 5 min each time and completely dried in a fumehood prior to extraction. Defatting was skipped for grape thinned clusters due to low-fat content of seedless samples and concerns of phenolic degradation during extensive hot hexane extraction for seed fractions.

For subsequent extraction of flavanols and procyanidins, 0.05 g defatted Acticoa™ cocoa powder, 0.2 g defatted Mullica™ cocoa powder, and 0.2 to 0.25 g thinned clusters were dissolved in 5–10 mL acetone:water:acetic acid , sonicated at 50°C for 5 min, and centrifuged at 5000 rcf for 10 min, respectively. Supernatants were further cleaned on Oasis PRiME MCX cartridges , diluted, and filtered through PTFE filters for UHPLC-FLD analysis. A Waters™ Torus diol column kept at 50°C was used for separation on an Agilent 1290 Infinity UPLC coupled with a 1260 Infinity FLD. Mobile phase A was acetonitrile: acetic acid and mobile phase B was methanol:water:acetic acid . The gradient was following 0% B for 0.37 min to 45% B in 10.03 min to 95% B in 0.25 min and held for 2.35 min. The flow rate was 1 mL min−1 with a sample injection volume of 2 μL. The FLD condition was optimized at a photo multiplier tube gain at 11 with an excitation wavelength of 230 nm and an emission wavelength of 321 nm. The quantification was determined using NIST RM 8403 as external standards.Flavanol enantiomers – and -epicatechin as well as – and -catechin were characterized using a modified method from Machonis et al. For cocoa powders, 0.05 and 0.1 g of defatted Acticoa™ and Mullica™ samples were dissolved in 5 mL methanol:water:acetic acid , respectively,blueberry grow pot while for grape thinned clusters, 0.1 to 0.15 g freeze-dried samples were dissolved in 5 mL MWAA. All mixtures underwent sonication at 50°C for 5 min and centrifuged at 5000 rcf for 10 min to obtain supernatants. Supernatants were further diluted and filtered for UPLC-FLD analysis. An Astec® Cyclobond® I2000 RSP kept at 35°C was used for separation on an Agilent 1290 Infinity UPLC-FLD. The isocratic mobile phase was 20 mM ammonium acetate buffer :methanol . The flow rate was 1 mL min−1 for a total run time of 30 min with a sample injection volume of 10 μL. The FLD was set at an excitation wavelength of 276 nm and an emission wavelength of 316 nm. Five-point calibration curves of each enantiomer were plotted for quantification.The moisture content of freeze-dried thinned grape clusters was determined according to Thakur et al. for reporting TPC, and flavanols and procyanidins of thinned clusters in DW.The total phenolic content was measured in triplicate for thinned cluster fractions. All data are expressed as mean ± standard deviation . One-way analysis of variance followed by Tukey & Dunnett multiple comparisons were applied using XLSTAT to evaluate significant differences among thinned clusters and cocoa powders flavanol content. For all comparisons, differences were considered statistically significant at a value of p< .05.The two wine grape varieties, Chardonnay and Pinot noir, were categorized into four different thinned cluster fractions based on berry color. Each variety that was analyzed in the current study included: Chardonnay seed and seedless dark , Chardonnay seed and seedless light , Pinot noir seed and seedless red , and Pinot noir seed and seedless green . To help illustrate the potential of using Chardonnay and Pinot noir grape thinned clusters as functional ingredients in cocoa-based products, two commercial cocoa powder samples were also analyzed and used as reference samples for their monomeric and oligomeric flavanols and procyanidins content. The total phenolic content determination was the starting point for the thinned grape cluster characterization because it provided important information, although unspecific, about the relative composition of the thinned grape cluster sample .

The composition and concentration of phenolics in grapes vary greatly due to variety and degree of ripeness , and environmental and vineyard management factors such as soil type and irrigation strategy . Thus, it is not surprising to see a 2.1-fold and 1.7-fold variations of TPC in Chardonnay and Pinot noir fractions, respectively. As shown in Figure 1, Chard_S_Light, which was considered the least mature among all fractions, had the highest TPC at 70.1 ± 1.1 mg g−1 in dry weight . The highest TPC for Pinot noir was detected in its green seedless fraction at 62.4 ± 1.1 mg g−1 DW. Within the Chardonnay fractions, the less mature fractions had higher TPC compared to the more mature fractions in both seed and seedless forms although no significant difference was found in two seedless fractions . The same trend was also observed in Pinot noir fractions as the less mature green fractions for both seed and seedless had higher TPC compared to the more mature red fractions. Pantelić et al. also found that TPC values were significantly higher in seeds than in skins and pulps in seven red and six white grapevine varieties grown in Serbia; our Chardonnay Dark and Pinot noir Green fractions were on the opposite where the seedless fractions yielded higher TPC for both varieties. Typically, the amount of TPC in the white grape varieties is lower than that of red grapes due to lack of anthocyanins synthesis , yet, previous studies have found that the TPC of grapes mainly depends on the varietal differences, not on grape skin color . Furthermore, in this study, thinned clusters for Pinot noir were collected at the beginning of veraison before anthocyanins accumulation reached its peak. Variations among grape fractional sample preparation , TPC extraction , and data reporting format could result in significant TPC differences from study to study for the same varieties . Flavanol monomers and procyanidins are abundant in cocoa and grapes. In cocoa, procyanidins are the major flavanols which consist of oligomers as well as polymers of catechins and epicatechins commonly bonded through a C4-C6 or C4-C8 linkage . Grapes are also rich sources for oligomeric and polymeric procyanidins with studies showing oligomeric procyanidins such as dimers and trimers being easier to be absorbed in vivo . Although cocoa-based products can be perceived as “healthy” among consumers because cocoa flavanols exhibited numerous health-promoting properties , the amount of bioactive and bioavailable cocoa flavanols, mainly -epicatechin, -catechin, and procyanidin dimers and trimers, vary greatly among cocoa-based products thus are unlikely to achieve the same level of healthpromoting effects in vivo. Therefore, it is crucial to quantify the flavanol monomers and oligomeric procyanidins content in the grape thinned clusters to determine their bioactive potential when compared with cocoa ingredients such as cocoa powder. A new cocoa extract reference NIST RM8403 was used as an external standard in the recently approved 2020.05 AOAC official method which allows more accurate and reliable determination of cocoa flavanols and procyanidins using UPLC-FLD . The current study successfully applied this method to the Chardonnay and Pinot noir thinned cluster fractions as well as two commercial cocoa powders in the context of catechin, epicatechin, and their oligomeric procyanidins . An example of chromatogram is shown in Figure 2.

These overt statements of support for Lewis’s misogynistic motif may appear starkly different from scholarly assessments, but I will argue that a similar acceptance of the novel’s attitude toward women emerges in scholarly treatments of The Monk if we look closely enough. For instance, in some of the pioneering gothic studies, though scholars do not express approval of the way the novel’s female characters are brutalized, they evoke a similarly brutal attitude toward femininity when they write about the novel’s genre. Even today, scholars traditionally discuss Lewis and Ann Radcliffe together because of the common narrative that The Monk responded to The Mysteries of Udolpho and The Italian responded to The Monk, and revisiting some of the earliest instances of this comparison reveals that implicit in it can be a misogynistic attitude. The gendered genre binaries that scholars utilize when contrasting Radcliffe and Lewis sometimes cast the two authors as a sheltered, timid woman and a wild, bold man. For instance, in 1978 Howells writes that “Lewis is shocking and subversive in a way that Mrs Radcliffe never was in his exploration of the dark irrational hinterland of the human mind,” as if he is an intrepid explorer.More troublingly, some early scholars create scenarios in which the Lewisian man menaces the Radcliffean woman. Montague Summers writes that “Mrs. Radcliffe shrank from the dark diablerie of Lewis,” and Michael Sadleir narrates, “Into the firelit refuge of the Radcliffian novelist the follower of Lewis would fain intrude, haggard and with water streaming from his lank hair, shrieking . . . then,nft growing system when he had struck the company with silent fear, he would wish to vanish once again into the howling darkness.”45 The seemingly inoffensive idea that Lewis’s novel unsettled the status quo looks not quite so inoffensive when coupled with imagery that recalls the way his male characters violate his female ones.

Even scholars who object to the novel’s treatment of female characters can end up aligning themselves with the perspective they censure through their own interpretive methods. Feminist critic Judith Fetterley, writing in 1978 about the masculine tradition of American authorship, argues that “a female reader is co-opted into participation from which she is explicitly excluded; she is asked to identify with a selfhood that defines itself in opposition to her; she is required to identify against herself.”Building on this description of coercive reading, she writes, “Powerlessness is the subject and powerlessness the experience,” as female readers are forcibly alienated from the classic literary plots of men dominating women.Using a similar framework to Fetterley’s, feminist critics have repeatedly called attention to the novel’s tendency to objectify and show contempt for women,but some suggest that readers have no choice but to inhabit a masculinized, heterosexual, lascivious, dehumanizing point of view while reading, as if the novel forces readers to take a position they find abhorrent. Under the control of Lewis’s narrative, they imply that there is no possibility of reading his abused female characters sympathetically. In describing the rape scene, Howells observes that it reads like pornography, and though she clarifies that its carnal account is not sexually exciting, she maintains that “there is for us the voyeuristic fascination of watching how far conventional limits can be transgressed,” as if the depiction of a taboo compels all readers to become mesmerized spectators and leaves no option of withdrawing in disgust.Elizabeth Napier concurs, arguing that Lewis’s writing inspires “a kind of prurient curiosity that depends upon withdrawal and distance.”Though the novel appears to advocate for compassion, she claims, the way it aestheticizes sex and violence makes pity impossible for readers.

She writes that Lewis “puts the reader in the novel’s most crucial moments in the position of a voyeur” and makes his victimized characters “undergo punishments so extreme as to excite horror rather than pity,” and she hints that Antonia desires the man who rapes and kills her and thus prevents readers from having “[a] correct response to this purest of characters.”Napier disallows the possibility of readers having a simultaneously aesthetic and emotional response, or being capable of feeling both revolted at the violence and sympathetic for the victim, or reading Antonia’s warmth toward Ambrosio as that of a sibling, or being capable of a “correct” response to a victim even if that victim is not irreproachable, or even reading against Lewis’s authorial prompts in order to feel into fictional suffering. These scholars’insistence that Lewis’s distancing techniques allow for no emotional connection with his female characters ends up sounding like an acceptance of the novel’s voyeurism and misogyny rather than the objection that they likely intend. Close reading, which could provide an antidote to the distance and disconnection that many feminist critics identify, can end up performing an uncomfortable sympathy with the text as well. Fredric Bogel recently championed close reading as a practice of what he calls “true infatuation”—intense attention to the text that is comparable to erotic obsession.This erotic textual obsession, when applied to The Monk, can become unseemly. When scholars take time to analyze the passages in which they find Lewis’s writing most effective, their careful literary analysis begins to sound like they are reveling in the sensational violence and sex. Robert Kiely observes Lewis’s tendency toward abstract, indirect writing and descriptions of Ambrosio’s sensible bewilderment, in contrast to which “the encounters with female corpses are, in addition to their intrinsic unpleasantness, stylistically striking.”

KIiely meditates on the power of Lewis’s specificity and simplicity of style in these scenes where women are dead or dying, writing that passages like the one where Ambrosio chokes and suffocates Elvira or the one where a mob tramples the prioress interrupt the conventional and “possess an energy and realism for which the reader is not fully prepared.”Though Kiely means to make the point that the directness of Lewis’s violent writing makes the violence more awful and the victims more human, his appreciation of forceful style sounds like an appreciation of violent action in this context. George Haggerty similarly venerates Lewis’s skill with violent scenes, analyzing how his combination of abstractions and vivid carnage in his description of the prioress’s mutilation makes the scene conceptually and viscerally effective. He goes so far as to claim that this philosophical brutality is the only effective technique Lewis could have used to make readers feel this scene without having another character’s reactions to prompt them: “Without the filter of a responding consciousness, there is no other way to assure affective success. If we are disgusted, Lewis has succeeded.”The tendency of literary scholars to write as if their own response is the only possible one can be jarring when they linger appreciatively over scenes of violence or sex that obviously provoke very different responses in other readers. Writing in this mode, Ahmet Süner delves into the sexuality of a moment that I will revisit shortly, in which Matilda bares her breast, contending that Lewis’s language enhances the erotic effect of the scene even as it appears to be modestly euphemistic. He writes, “The sensation . . . overtakes the metaphor, taking over the object; consequently, the object is surrendered to the bare mechanics and bare metaphoricity of sensation . . . all these clichéd metaphors are already chewed and devoured so that they may unobtrusively give way to the free flow of desire.”His own language as he analyzes Lewis’s sounds like a sexual encounter, or a sexual assault: “overtakes,” “taking over,” “surrendered,” “bare,” “devoured,” “free flow of desire.” Süner likely intended this sexualized analytical language to reinforce his point about the passage’s prurience, but it ends up seeming as if Süner has been absorbed into the voyeurism and sensual ecstasy he was trying to parse. When scholars analyze Lewis’s sensational writing,nft hydroponic system whether they chafe at it or embrace it, they often implicate themselves. Because of the ways that normative scholarly methodologies shape discussions of sensation, critiques or close readings of The Monk’s depictions of violent rage and transgressive desire can implicate scholars, but they are also limited in the ways they can address Lewis’s portrayals of other feelings. The suspicious critical mood informs the way scholars approach Lewis’s less embodied descriptions of emotion, which they tend to find false. For instance, scholars have written that the hero Raymond’s horror upon encountering a ghost has “the behaviouristic detail of a theatrical performance,” that Ambrosio’s frequent confusion of mixed emotions makes him “flutter and reel” like a sensible heroine, that Agnes’s grief over her dead child is part of an “over-the-top tableau” typical of Lewis’s “theatrical excess,” and that the sadness of Antonia’s reunion with her beloved and her mother’s ghost as she dies “is undercut by Lewis’s parodic manipulation of Sentimental conventions.”Whether these scholars assume that Lewis is forced to write in the style of drama or sentimental novels because of a lack of available fictional options or that he is performing a critique of these modes, they generally do not find these emotional moments worthy of sustained critical attention.

What options exist for scholars who want to write about the novel’s emotions but don’t have an acceptable scholarly method for doing so without either dismissing its portrayals of fear, confusion, and sadness as ineffective or analyzing its portrayals of lust and rage in ways that become disturbingly aligned with those feelings? This is the bind I found myself in after rereading The Monk with the voices of these scholars in my head. A lay critic’s comparatively broad range of affective choices do not fit comfortably within a scholarly role. What ended up working for me was to approach emotional scenes not through feeling but through seeing, by first considering how artists have illustrated them. My next section will show how this kind of consideration can reveal a variety of affective and interpretive possibilities in a single textual moment, and my final section will explore what these possibilities can open up in the text itself.In this section, I will return to visual analysis, focusing on illustrations rather than book covers. Rachel Schmidt writes that illustrations of narratives require that the artist engage in a process of interpretation that will enhance certain effects of the text for readers.In the case of Don Quixote, Schmidt argues that early illustrations were involved in shaping the novel’s critical reception. Many of the illustrated editions of The Monk that I could find actually appear to have little in common with the novel’s critical reception, unlike its twentieth- and twenty-first-century book covers. Whereas the book covers represent extremes of sensationalism or abstraction and thus illuminate the mixture of extremes that I have argued are operating uncomfortably in certain scholarly treatments of the novel, the illustrations present a wider range of feelings, and many are indeterminate in their affect. In contrast to book covers, which govern readers’ impressions of the book as a whole early in the reading experience, illustrations represent carefully chosen moments and provide visual cues for understanding them. The moments that are chosen for illustrations and the ways particular artists interpret them provide multiple possibilities for contextualizing an emotional situation at a particular point in the text. After viewing every illustration I could locate, from soon after the novel’s publication to the 1980s, I discovered that the moment in which Matilda exposes her breast and threatens to end her life is by far the most common visual representation from the main plot of the novel.This is perhaps in part because it allows artists to titillate with nudity, though what is arguably the most sensual scene, when Ambrosio spies on Antonia bathing, was illustrated in only one of the editions I found. The moment of threatened suicide, occurring in the scene where Rosario reveals herself to be Matilda, is also likely a subject of fascination because the scene is when the stable ground of gender identity gives way in the novel, which some artists emphasize by portraying both figures in their entirety, so viewers can see that they are dressed identically in monks’ robes. Interdependent with Lewis’s portrayal of sexuality and gender in the scene, though, is his portrayal of power. As I will examine in my close readings of the next section, the drawn-out scene in the novel features complex, shifting power dynamics . The illustrations I will compare all freeze the scene in the urgent standoff before Ambrosio surrenders his control over Matilda’s fate, and the choice to represent this moment of possibility implies that this is one of the key decisions that determines the course of the novel.

The modern edition states that it reproduces the 1793 version exactly, except for what the editors deem typographical errors, including “seemingly random and nonsensical commas.”This omitted comma is far from random, though, as it makes Matilda’s temporal and experiential situation either restrictive or nonrestrictive . Thus, the recent version implies that the Matilda who has suffered is a changed Matilda—her situation has been absorbed into her identity. The original version implies that Matilda is not that closely identified with her circumstances, just as it refuses to state something like “she was sad.” In the syntax of the Parsons’s emotional curriculum, situations can supplant a person, but they cannot become subordinate or equal to her. In this happy moment, Matilda’s travails remain, temporarily minimized but independent of her. It is the fact of these travails that matter most in the novel’s world—the relatable information, not the verbal expression of a sufferer’s response to them, which is too personal to be expressed or sympathized with. Indeed, words may be not only insufficient but also inappropriate, as suggested by a poem that Matilda finds scratched on the window of the room where the Countess of Wolfenbach was imprisoned by her husband after the murder of her lover and theft of her baby. The verse is necessarily brief, reading, “I am dumb, as solemn sorrow ought to be; / Could my griefs speak, my tale I’d tell to thee.”This passage of Wolfenbach is the one most often analyzed by scholars: Gillian Beer uses these lines as an example of the rhetorical force of incapacity,Karen Morton asserts the effectiveness of the window poetry as a mediated articulation of grief,grow bag for blueberry plants and Angela Wright quotes another of the verses to illustrate the novel’s powerful critique of marriage.

Deidre Shauna Lynch writes that when Matilda encounters this verse and others along with a handprint on the blood-stained floor of the room without knowing the Countess’s history, the writing “accentuates the other disappearances that this site of inscription has witnessed . . . The unknownness of that hand and the inaccessibility of the backstory behind the words are arresting.”In addition to way the discovered poetry makes the scene comparable to Radcliffe’s more reputable work, this is one situation in the novel where Parsons’s emotional writing can meet with approval from a modern audience, because the fact that Matilda encounters these words in writing, apart from the mysterious poet, removes the pressure of the fictional writer or the actual author to make some greater attempt at articulating emotion.In the context of these disembodied, departicularized, ghostly words, we do not expect a modern articulation of physical sensation or precise emotion from the composer or the narrator, so in this instance the lack of information can be as effective for us as it always is for the inhabitants of Parsons’s school of affliction. Many people today believe that the powerful and personal feelings that arise in response to extreme situations can never truly be explained to those outside that experience. In real life, we rarely expect someone who has lost a child to wrap up every feeling involved in that excruciating event and its aftermath in language and hand it to an uninvolved person. We are more likely to accept that the horrific nature of the circumstance makes the sufferer worthy of sympathy or aid even without knowing the details, and to believe that the feelings a person in that situation would experience are unimaginable, only knowable to those who have experienced the same event. In this way, Parsons’s style more accurately reflects our current beliefs than our own fiction, which routinely enters experiential territory that its readers and even its authors have not traversed firsthand. In this rare moment in the Parsons’s novel, our ethical convictions and aesthetic sensibilities are in accord with Wolfenbach’s school of affliction. Despite the inadequacy of language in Parsons’s emotional curriculum, stories can supplement the firsthand experiences of suffering by educating the audience about the types and proportions of human misery.

Soon after meeting Matilda, Mother Magdalene tells her, “Another time you shall know my sad story, and will then confess, of the two, I have been most wretched.”When she does elaborate on her various distresses, Mother Magdalene asks Matilda the seemingly rhetorical question, “[H]ave your troubles ever equalled mine?”Mother Magdalene’s storytelling helps teach Matilda how to measure and rank suffering, to subordinate her own troubles to the superior affliction of another. Though Santos argues that the stories in the novel perform a similar didactic function for readers, “as a mediating device that anticipates and regulates the reader’s response to intense feeling,” the form does not necessarily result in the intended effect. Napier, for example, criticizes the fact that the most dramatic moments in the novel reach the reader at a distance, through these stories of pain and woe, which she reads as the mistaken prioritizing of a moral message over vivid action.Both these readings disregard the fact that apart from any intended or actual effect on a reader, within the world of the novel the stories themselves play their own essential role in plot and character development. Deidre Lynch writes that Wolfenbachelevates books as “essential props for its protagonists’ projects of memory and mourning,” as “the pages the characters behold are often conceptualized as their legacies, either familial or cultural.”and I would argue that in the case of oral stories in the novel, this phenomenon is doubly true. Over the course of the novel, Matilda gains wisdom and perspective in part by listening to several tales of woe. The climactic scene even focuses on the telling of a sad story, as Matilda’s uncle slowly spins the tale of how he wronged her parents and deprived her of her birthright. The resolution of the novel consists not only of the typical restoration of Matilda’s status, mother, and lover, but also, and essentially, her learning more of the story of suffering that shaped her life. It is a story that gives her identity, and it also completes the process of educating her about the extreme forms of affliction others experience, to the point where she can see her own troubles in the proper proportions taught by the school of affliction and become a more altruistic person. Through all these messages and the techniques Parsons uses to convey them, the novel itself acts as its own school of affliction, with Parsons teaching her readers how to properly respond to her stories of suffering. The novel’s frequent assertions that emotions are indescribable mark off the narrative territory that the novel will not cover and attempts to recalibrate readers’ expectations, if they are not already primed to read incapacity for speech as a sign of deep feeling.

Instead of attempting to enter into the particulars of her characters’ feelings, Parsons encourages her readers to learn a culturally appropriate response to these types of situations—one that she models in the novel through her listening characters. Santos’s observation about the modeling of sympathetic response in the novel is correct as far as the intention built into the convention, if not correct about its actual effect on all readers. However, an essential part of this modeling is the way Parsons’s listening characters do not make a sympathetic response conditional to details; the telling and hearing of stories is not only conventional in itself but also conventional in its reliance on types alone for effect. In responding to Matilda’s story of evading her lustful uncle, the Countess of Wolfenbach is a perceptive and experienced reader who recognizes the character type of the damsel in distress and responds with the expected attention and sympathy. As these scenes in which a sufferer relates her sad story and a listener responds with compassion recur, Parsons uses repetition to teach her readers how to recognize generic categories and respond appropriately—to feel with the formula. Whereas The Castle of Wolfenbach represents a school of affliction that teaches a method of feeling with the formula of stories of suffering, its reception teaches a method of feeling against that formula. This is a different kind of literary emotional training, but one that still teaches that we do not need to know particulars in order to respond correctly. As I discussed in the first section of this chapter,blueberry grow bag eighteenth-century discourse about novels, and especially Minerva novels, created an atmosphere in which a work like Wolfenbach could be perceived as a part of a broader threat—one of a mass of new gothic fiction that appeared to cater to a public appetite for extreme emotion and to dull readers’ powers of aesthetic discrimination, and even, some feared, to encourage immoral behavior by portraying vice. Eighteenth-century critics considered Parsons’s writing to be somewhat better than most of the novels published during the growth of disreputable circulating libraries, and publishers reprinted it until the mid-nineteenth century in editions alongside reputable novels, but after this time it was forgotten. By the twentieth century, scholars knew Wolfenbach only as one of Northanger Abbey’s Horrid Novels, a label that implies it is both exhilarating and bad. It is this latter quality that scholars and amateur critics tend to focus on when they approach Wolfenbach through Northanger, and they often treat Jane Austen as a model for how to condescend to the emotional and aesthetic excesses of Wolfenbach and other novels of its kind. Scholars of the twentieth and twenty-first centuries rarely mention Wolfenbach apart from its context as a Northanger Horrid Novel. In this context, scholars sometimes characterize Austen as a book critic who uses Northanger as a venue for teaching her readers to discriminate between Ann Radcliffe’s worthwhile gothic novels and her imitators, including Eliza Parsons. For example, Bette Roberts argues that the list of seven novels that Isabella recommends to Catherine “enumerates the negative extremes to which the reader may contrast what Austen considers to be higher expressions of the Gothic in the notably superior novels of Ann Radcliffe.”These novels fall far short of Radcliffe, Roberts explains, because although they “pretend” to advocate for morality, rationality, and self-improvement, the “disastrous stories” they include are not truly meant for edification of the “already perfect” characters but merely to “appeal to the readers’ emotions.”This critique overlooks the way that in Wolfenbach, Matilda demonstrates her growth not only by gaining her identity through the disastrous story that relates to her origins but also through learning to subordinate her own disasters to those of others and access universal wisdom.

Other scholars have argued that Austen’s novel teaches not only proper literary taste but also life lessons through aesthetic means. Robert Kiely demonstrates how Henry Tilney challenges Catherine’s indiscriminate usage of words as a means to enable her to transcend her own naïve perspective and exercise better judgment of people and situations, similar to how I argue Parsons uses stories of suffering in Wolfenbach. For Kiely, however, Austen’s un-gothic restraint and distinction are the key pedagogical tools, as he finds that “as a display of the disciplined mind and the well-chosen word [Northanger Abbey] does more than all the hysterical criticism of the periodicals to deflate some of the poses and excesses of Romanticism.”Jacqueline Howard argues that Kiely’s reading misses the ways that Austen not only ridicules gothic novels but also skewers the criticism of novels, making Henry an object of critique by styling his instruction as the masculine condescension “of those authoritative, male-authored pronouncements addressed to young women in conduct books and reviews.”Kiely’s analysis is the sort of scholarship on Jane Austen that Eve Kosofsky Sedgwick describes as being sadistically concerned with “the spectacle of a Girl Being Taught a Lesson.”This critical fascination with the way Austen’s protagonists become chastened by experience has likely placed even more emphasis than Austen does on the problematic reading habits of the young Northanger women, underscoring the idea that Wolfenbach and the other Horrid Novels are pleasures that should not be indulged. Wolfenbach’s own pedagogical narrative appears to register as insufficiently harsh to these scholars, perhaps because Matilda does not experience the kind of shame that Catherine does when she gains perspective on her youthful feelings, or perhaps because Parsons herself indulges in too many aesthetic and emotional excesses in Wolfenbach.Emotional involvement of the sort that Wolfenbach portrays and encourages appears to some scholars as the fault that Austen intends to educate readers out of with Northanger.

The scholarly distrust that Kramnick mentions and that Lynch complicates has become an object of scholarly concern in the last two decades. In her 2003 book Touching Feeling, Eve Kosofsky Sedgwick reintroduces Paul Ricoeur’s “hermeneutics of suspicion,” observing that this style of criticism resembles paranoia and that the use of it is now “widely understood as a mandatory injunction rather than a possibility among other possibilities.”Perhaps because many of us consider it mandatory, scholars have had difficulty defining this mode of scholarly critique or describing what other methods readers employ. Michael Warner, prompted by Sedgwick’s essay, attempts to delineate what critical reading is and what it is not, concluding that our immersion in critical reading has meant that “whatever worlds are organized around frameworks of reading other than critical protocols remain, for the most part, terra incognita.It is this largely unmapped territory that I explore in my dissertation. I will compare responses to the same novels—and often the same features of those novels—from scholars working in the last few decades, earlier scholars, periodical critics, and amateur critics, which will make their different techniques and affective protocols apparent. The current affective protocols of the hermeneutics of suspicion have been most thoroughly demonstrated by Rita Felski, whose 2015 book The Limits of Critique arrived after I began this project but has been essential in helping me explain some of the problems I see with today’s critical practices. Felski uses the term “critical mood” to describe the suspicious orientation of most scholars toward texts that has pervaded otherwise disparate methodologies and become so dominant that it obscures or invalidates other attitudes and methods.

Felski writes that in the opinion of many humanistic scholars, “To refuse critique . . . is to sink into the mire of complacency, credulity,grow bag gardening and conservatism.”She acknowledges that this critical mood allows for pleasure of the disciplinary sort that Lynch describes, but she objects to the way it disallows receptivity to “the multifarious and many-shaded moods of texts,” as a suspicious critic is “fearful of being tricked or taken in. Locked into a cycle of punitive scrutiny and self-scrutiny, she cuts herself off from a swathe of intellectual and experiential possibility.”It is worth emphasizing that for groups that are routinely attacked by ideologies and policies, suspicion is self-preservation, especially considering the ways that literature informs our awareness of the world. But Felski’s point that this affective orientation limits scholars is important. In my dissertation, by devoting attention to the emotional qualities of numerous responses, both professional and nonprofessional, older and newer, I hope to suggest alternatives to the dominant scholarly attitude. For reasons I will address later, I am not attempting to offer a historicist account of emotion and criticism in this dissertation. However, I will briefly discuss some of the ideas of eighteenth-century philosophers and critics that influenced the reception of gothic novels. British eighteenth-century writers were actively engaged in defining and describing both emotions and taste, which they often treated as subjective phenomena that could nonetheless be assessed according to standards of appropriateness. For some, these standards were universal, deriving from God and humans’ common sensory makeup For others, they were social, established by what was beneficial to society or what had been consistently judged by others over hundreds of years .Many eighteenth century British writers shared an interest in moderation and were invested in disciplining excess, which helped create a cultural atmosphere in which many gothic novels would be judged as inappropriate for their extremes of character and situation, and even more so for their extravagant portrayals of feeling. To illustrate how these eighteenth-century judgments of appropriateness in art and emotion have been applied to literature and the slipperiness of this application, we can consider Adela Pinch’s analysis of a passage from Henry Home, Lord Kames.

Kames, the author of a thorough eighteenth-century treatment of aesthetics, discusses the need for writers to consider propriety when representing a characters’ feelings. For Pinch, this is an issue specific to eighteenth-century conceptions of emotion, but a similar problem in judging literary emotion continues beyond that historical period. For example, in 1968 critic Northrop Frye writes of Radcliffe’s heroines, “We may wonder why any literary convention should have produced these absurd creatures, drizzling like a Scotch mist and fainting at every crisis in the plot.”And in 2009, a Goodreads reviewer writes to Radcliffe’s Emily, “STOP CRYING YOU STUPID WHINY BITCH.”When critics judge the failure of emotional portrayals, these judgments often sound like indictments of the emotional people portrayed. This sort of judgment of literary feeling was vigorously exercised in the eighteenth century, as periodical critics, moralists, authors, and the general public argued about the effects of new fictional forms. William Warner writes that in the early debate about novels, “the novel reader is characterized as a susceptible female whose moral life is at risk” due to the dangerous pleasures she supposedly received from early amatory fictionBy the mid eighteenth century, Warner argues, the works of Henry Fielding and Samuel Richardson participated in a project of elevating the status the novel, and as the perceived cultural threat of novels in general diminished, critics and authors shifted focus to the emotional and moral value of particular kinds of novels.Those who wrote about gothic fiction in its early years often drew on this discourse to make claims for the emotional and moral benefits of these works or to condemn the novels for their dangers. In 1773, poet Anna Letitia Barbauld and her husband, John Aiken, describe the emotional conflict of reading narratives of terror, in which the pain induced by the miseries described is rivaled by the pain of suspense. However, they find that the exceptions are “well-wrought scenes of artificial terror which are formed by a sublime and vigorous imagination.”These sorts of fictional terror, of which The Castle of Otranto is “a very spirited modern attempt,” “elevate the soul” and provide pleasure through the combination of the terrible and the marvelous.Similarly, these writers recommend works that evoke pity with a delicate hand, in which authors lighten scenes of suffering with “strokes of pleasantry and mirth,” as excessive exposure to distress can “render us insensible to every thing” and even make people incapable of feeling pity for real people.

The novels of Ann Radcliffe, published later, would accord to these rules, but Matthew Lewis’s Monk would violate them, which helps explain their very different contemporary reception. Barbauld and Aiken’s concern about desensitization would take a more urgent form in William Wordsworth’s 1802 preface to Lyrical Ballads. Wordsworth decries the emotional danger of modern life, which “blunt[s] the discriminating powers of the mind” and “produces a craving for extraordinary incident” that is satisfied by “frantic novels” that many scholars assume to be gothic.The horror that early critics express at the proliferation of poorly written, formulaic gothic novels in the 1790s, which I will discuss inchapter 1, is thus in part horror over the power of literature to shape and be shaped by the baser feelings of readers.E. J. Clery identifies 1797 as “the year in which reviewers and critics began to put a name to the category of fiction we now call Gothic or the fantastic, although the name varied: ‘modern Romance’; ‘the terrible school’; ‘the Terrorist System of Novel Writing’; ‘Terrorist Novel Writing’; ‘the hobgoblin-romance’.James Watt argues against the critical tendency to unify the genre because “‘Gothic’ fiction was far less a tradition with a generic identity and significance than a domain which was open to contest from the first, constituted or structured by the often antagonistic relations between different writers and works.”Though I contest Watt’s own contestation in my second chapter, I am less invested in judging the legitimacy of the generic categorization of gothic novels than in examining what this categorization implies for critics, especially in terms of quality and feeling. Thus, what matters most for the selection of books in this study is that critics have labeled them as gothic. Scholars of gothic fiction have argued that, above all, gothic is a genre defined by its emotional characteristics. Coral Ann Howells names “feeling as the distinctive attribute of Gothic—feeling as it is explored and enacted in the fictions themselves, and feeling as the primary response elicited from the reader.”George Haggerty even claims that “Gothic form . . . is affective form. It almost goes without saying that these works are primarily structured so as to elicit particular responses in the reader. Perhaps, however, plastic grow bag it is not so obvious that Gothic fiction therefore cannot have specific meaning . . . it is central to the nature of Gothic fiction that differing interpretations will seem equally valid.”Haggerty’s argument here inspires my approach in this dissertation, as I try to determine how readers’ situated interactions with the texts allow for different affective, evaluative, and interpretative possibilities. For example, a twenty-first-century reader who picks up The Monk because Stephen King recommended it will have a different experience than an eighteenth-century reader who saw an advertisement suggesting the novel would be like The Mysteries of Udolpho, and the audience for which either of those readers would write a review would shape their responses differently as well. Haggerty himself, however, turns his argument to different ends, writing that “Gothic works only become fully intelligible when we understand the extent of their affective rationale.”Haggerty and most other scholars of gothic and similarly emotionally dependent fiction turn either to the text itself or to its contemporary context in order to determine a novel’s “affective rationale,” investigating the formal, structural, verbal, and conceptual ways that the text prompts certain responses. Barbara Benedict elucidates some of this affective rationale in her examination of how sentimental fiction “frames” feeling in a way that simultaneously encourages and discourages it. She argues, “The conventional language, pictorial diction, tonal instability, structural fragmentation, and multiple narrative voices work to externalize these interior experiences, to deprive them of authority, and to subordinate them within a social frame.”For example, she writes that Ann Radcliffe’s “disjunctive styles and structures . . . aestheticize emotional reactions with the discourse of spectatorship while they also exhibit control of sentimental excess.”Stephen Ahern thoroughly explores the contradictions that Benedict identifies, explaining that amatory, sentimental, and gothic fiction are all rooted in the unstable concept of sensibility and thus share “an ambivalence toward excess” which “arises from two fundamental yet conflicted imperatives: sensibility is an idealist discourse that is ineradicably rooted in physiological response, and sensibility is coded according to unstable categories of class and gender difference.”The effects of the portrayals of extreme emotion in sensibility narratives are unpredictable, he writes, “for overwrought descriptions of pathos can quickly descend into bathos if the audience . . . is unconvinced of the authenticity of the emotion expressed. Theatricality is a necessary condition of literary forms that find their truth in scenes of heightened emotion, but it is also a destabilizing force.”These scholars and many others have done valuable work in clarifying the emotional functions of gothic novels, but even though they acknowledge readers’ volatile responses to the mechanisms they describe, they still suggest that these novels evoke a fairly small range of emotions. For example, Napier’s analysis of gothic form leads her to conclude, “A successful response to the Gothic is based on instability: one must be pleased by what one dreads, take pleasure from distress, luxuriate in terror.”When scholars attempt to explain what kinds of responses the emotional conventions of fiction dictate, even when they allow for instability, they can end up suggesting that there are only a certain number of correct responses. Most scholarly accounts of feeling in literature do not recognize actual readers’ idiosyncrasies because they focus on what the construction of concepts and conventions enable. Even when the construction is built with internal contradictions, actual responses always exist in a more complicated world of influences beyond the text, and the cultural norms that may have caused contemporary readers to feel a certain way do not necessarily obtain for readers distant from the circumstances in which the text was written. Various scholars have explained the origins of the English gothic genre, its popularity, and its early critical reception as being attributable to a range of political, economic, philosophical, and social forces: anxieties about the French Revolution, foreign influences, or consumerism; dissatisfaction with Enlightenment rationality or women’s position in society; and worries about the preservation of middle-class sensibilities or the growing power of the lower classes, among others.This historical research allows us to situate gothic fiction in its contemporary context in several different ways, which is vital for my dissertation and scholarship on the gothic in general.

The plants for our experiments were artificially inoculated with race 1 or 2 isolates of F. oxysporum f. sp. fragariae using previously described protocols . The AMP132 isolate originated in California, whereas the MAFF727510 isolate originated in Japan . To produce spores, the pathogen was grown on potato dextrose agar or Kerr’s broth under continuous fuorescent lighting at room temperature, as previously described . Crude suspensions were passed through two layers of sterilized cheesecloth to remove hyphae. Spore densities were estimated using a haemocytometer and diluted with either sterile DI water or 0.1% water agar to a final density of 5 × 106 spores/ml. Seedling and bare-root plants were inoculated by submerging their root systems up to the crown in the spore suspension for 7–8 min prior to planting. The individuals in these studies were visually phenotyped for resistance to Fusarium wilt over multiple post-inoculation time points using an ordinal disease rating scale from 1 to 5 . For our field studies, individual plants were phenotyped once per week for four to eight consecutive weeks beginning in early June. Symptoms were observed on plants 26- to 36-weeks post-inoculation. For greenhouse and growth chamber studies, entries were phenotyped weekly for 6 to 12 weeks post-inoculation. For field, greenhouse,square black flower bucket and growth chamber experiments, the onset and progression of disease symptoms among resistant and susceptible checks were used as guides for initiating and terminating phenotyping.Our race 1 resistance screening experiments were conducted over a three year period at the UC Davis Plant Pathology Farm. The plants for these experiments were artiificially inoculated with the AMP132 isolate of the pathogen. Strawberries had not been previously grown in the fields selected for our studies.

The fields were tilled and disked prior to fumigation and were broadcast fumigated in October of each year with a 60:40 mixture of chloropicrin:1,3-dichloropropene at 560.4 kg/ha. The entire field was sealed with an impermeable plastic film for one-week post-fumigation before shaping 15.3 cm tall × 76.2 cm center-to-center raised beds. Sub-surface irrigation drip tape was installed longitudinally along the beds followed by black plastic mulch with a single row of planting holes spaced 30.5 cm apart. Artifcially inoculated plants were transplanted in mid-November both years. The fields were fertilized with approximately 198 kg/ha of nitrogen over the growing season and irrigated as needed to prevent water stress. For the 2016–17 field experiment, 344 germplasm accessions were screened for resistance to AMP132 and were part of a study that included 565 germplasm accessions developed at UC Davis, which is hereafter identified as the ‘California’ population. The resistance phenotypes for the latter were previously reported by Pincot et al. . Collectively, 981 germplasm accessions were screened in the 2016-17 field study. These were arranged in a square lattice experiment design with four single-plant replicates per entry . The experiment design and randomizations of entries within incomplete blocks were generated with the R package agricolae . For the 2017-18 and 2018- 19 field experiments, 144 ‘host diferential panel’ individuals were screened for resistance to AMP132 . These individuals were arranged in a 12 × 12 square lattice experiment design with four single-plant replicates per entry as described above. Guardian, Wiltguard, and Earliglow S1 and 61S016P006 S2 populations were screened for resistance to AMP132 in the 2016-17 field study. Ninety-nine Guardian S1 and 98 Wiltguard S1 individuals were phenotyped and genotyped and 85 Earliglow S1 and 77 61S016P006 S2 individuals were phenotyped. Nine-month-old S1 or S2 plants started as seedlings and asexually multiplied bare-root plants of the parents were artificially inoculated with AMP132, transplanted to the field in March 2018, and visually phenotyped weekly for six to 11 weeks post-inoculation.

The 12C089P002 × PI602575 , PI552277 × 12C089P002 , and PI612569 × 12C089P002 full-sib families, 17C327P010 S1 family, and parents of these families were screened for resistance to AMP132 in greenhouse experiments at UC Davis. Two- to four-month-old seedlings of the progeny and bare-root plants of the parents were artificially inoculated with AMP132 and planted in February 2019 , June 2019 , or November 2019 into 10.2 × 10.2 × 15.2 cm plastic pots filled with 3 parts coir : 1 part perlite and phenotyped weekly for six to 12 weeks post-inoculation. Four uninoculated and four inoculated single-plant replicates of the parents were arranged in completely randomized experiment designs. The plants were irrigated with a dilute nutrient solution as needed to maintain adequate soil moisture. The 12C089P002 × PI602575 and PI552277 × 12C089P002 populations were genotyped with a 50K Axiom SNP array .We screened a host diferential panel for resistance to the MAFF727510 isolate of Fof race 2 in a growth chamber at the UC Davis Controlled Environment Facility in 2018-19. Two single-plant replicates/individual were arranged in a randomized complete block experiment design. The entire experiment was repeated twice, resulting in four clonal replications/individual. The bare-root plants for these experiments were produced in high-elevation nurseries, preserved in cold storage, artificially inoculated with the MAFF727510 isolate, transplanted into 10.2 × 10.2 × 15.2 cm plastic pots filled with a 4 parts sphagnum peat moss : 1 part perlite , and phenotyped weekly for six to 12 weeks post-inoculation. The plants were grown under a 12-hour photoperiod with a 20 °C night temperature and 28 °C day temperature and irrigated with a dilute nutrient solution as needed to maintain adequate soil moisture. Because these experiments utilized a non-California isolate of the pathogen, the experiments were quarantined and conducted in compliance with federally-mandated bio-safety regulations .

DNA was isolated from newly emerged leaves harvested from field grown plants using a previously described protocol . Leaf samples were placed into 1.5 ml tubes or coin envelopes and freeze-dried in a Benchtop Pro . Approximately 0.2 g of dried leaf tissue/sample was placed into wells of 2.0 ml 96-well deep-well plates. Tissue samples were ground using stainless steel beads in a Mini 1600 . Genomic DNA was extracted from powdered leaf samples using the E-Z 96®Plant DNA Kit according to the manufacturer’s instructions. To enhance the DNA quality and yield and reduce polysaccharide carry-through, the protocol was modifed by adding Proteinase K to the lysis bufer to a final concentration of 0.2 mg/ml and extending lysis incubation to 45 min at 65 °C. Once the lysate separated from the cellular debris, RNA was removed by adding RNase A. The mixture was incubated at room temperature for 5 min before a final spin down. To ensure high DNA yields, the sample was incubated at 65 °C for 5 min following the addition of elution bufer. DNA quantification was performed using Quantifor dye on a Synergy HTX . The individuals phenotyped in these studies were genotyped with either 50K or 850K Axiom® SNP arrays . SNP markers on the 50K Axiom array are a subset of those on the 850K Axiom array. The probe DNA sequences for SNP markers on both arrays were previously physically anchored to the ‘Camarosa’ and ‘Royal Royce’ reference genomes . The ‘Camarosa’ genome assembly has been deposited in the Genome Database for the Rosaceae and Phytozome . The ‘Royal Royce’ genome assembly has been deposited in the Genome Database for the Rosaceae and Phytozome . The assemblies for each ‘Royal Royce’ haplotype have been deposited in a Dryad repository . The physical addresses for the SNP markers are provided in our online resources . We utilized both reference genomes as needed to cross-check and compare statistical findings and search genome annotations. The results presented in this paper utilized the haplotype-resolved ‘Royal Royce’ reference genome FaRR1 unless otherwise noted. SNP genotypes were called using the Affymetrix Axiom Suite . Samples with call rates exceeding 89-93% were included in genetic analyses. The haplotypes for 71 50K Axiom array-genotyped SNPs within a 1.60 Mb haploblock on chromosome 2B were imputed and phased using BEAGLE software version 5.3 for 651 individuals phenotyped for resistanceto Fusarium wilt race 1. The individuals were classified as resistant or susceptible ,square black flower bucket wholesale where ̄y is the estimated marginal mean for resistance score calculated from replicates. We ran BEAGLE with 10 burnin iterations for imputation and 25 subsequent iterations for phasing on sliding windows of 5 Mb with a window overlap of 2 Mb.The R package lme4 was used for linear mixed model analyses of the germplasm screening experiments . LMMs for square lattice experiment designs were analyzed with entries as fixed effects and incomplete blocks, complete blocks, years, entries × years, and residuals as random effects . LMMs for randomized complete block experiment designs were analyzed in parallel to estimate the relative efficiency of the square lattice to the randomized complete block experiment designs .

We did not observe an increase in efficiency by using incomplete blocks; hence, the statistics reported throughout this paper were estimated using LMMs for randomized complete block experiment designs . Estimated marginal means for entries were estimated using the R package emmeans . Genome-wide assocation study analyses were carried out to search for the segregation of loci affecting resistance Fusarium wilt races 1 and 2 among individuals genotyped with either the 50K or 850K Axiom SNP array . GWAS analyses were applied to estimated marginal means for resistance phenotypes using physical positions of SNP markers in the ‘Camarosa’ and ‘Royal Royce’ reference genomes . SNP marker genotypes were coded 1 for AA homozygotes, 0 for heterozygotes, and -1 for aa homozygotes, where A and a are the two SNP alleles. GWAS analyses were performed using the GWAS function in the R package rrBLUP. The genomic relationship matrix was estimated from SNP marker genotypes for each population using the rrBLUP A.mat function . The genetic structure of the GWAS population was investigated using hierarchical clustering and principal components analysis of the GRM as described by Crossa et al. . To correct for population structure and genetic relatedness, a Q + K linear mixed model was used where Q is the population stratification structure matrix and K is the GRM . The first three principal components from eigenvalue decomposition of the GRM were incorporated into the Q + K model. Bonferroni-corrected significance thresholds were calculated for testing the hypothesis of the presence or absence of a significant effect. GWAS was repeated in the California population by ftting a SNP marker in LD with FW1 as a fixed effect using the rrBLUP::GWAS function .SNP markers with ≤ 5% missing data, high quality codominant genotypic clusters, progeny genotypes concordant with parent gentoypes, and non-distorted segregation ratios were utilized for genetic and quantitative trait locus mapping analyses. The linkage phases of the SNP markers were not known a priori. The arbitrarily coded SNPs in the original data were in mixed coupling and repulsion linkage phases. The linkage phases of the SNP markers were ascertained using pair-wise recombination frequency estimates, and recoded so that the 100% of the SNP markers were in coupling linkage phase. This was only necessary in the S1 populations. The recoded SNP markers were genetically mapped in S1 populations using phase-known F2 mapping functions. SNP markers were genetically mapped in full-sib populations using phase-known back cross mapping functions from the subset of SNPs that were heterozygous in the resistant parent and homozygous in the susceptible parent. Genetic maps were constructed using the R packages onemap and BatchMap and custom PERL scripts for binning co-segregating SNP markers, calculating pairwise recombination frequencies, and grouping markers using LOD threshold of 10 and maximum recombination frequency threshold of 0.05. The custom PERL scripts are available in the Dryad repository for this paper . Linkage groups were aligned and assigned to chromosomes using inter-group linkage disequilibrium statistics and percent-identity against the reference genome . Marker orders and genetic distances were estimated in parallel using the RECORD algorithm in Batchmap with a 25-marker window, window overlap of 15 markers, and ripple window of six markers . For smaller linkage groups, the window size was reduced incrementally by five to ensure at least two overlapping windows. We used the checkAlleles, calc.errorlod, and top.errorlod functions of the R package qtl and custom R scripts to identify and eliminate spurious SNP markers and successively reconstruct linkage groups as described by Phansak et al. . Genetic distances were estimated from recombination frequencies using the Kosambi mapping function .

A sub-sample of shoots and fine roots was collected for organ-dried biomass estimation and sugar and starch analysis. Harvest index was calculated after oven drying the samples.Subsamples of leaves, shoots, and roots were oven-dried at 70◦C to a constant weight. Dried tissues were ground with a tissue lyser . Thirty milligrams of the resultant powder was extracted in ethanol:water solution. Briefly, 1.5 ml was added to each sample and extracted for 10 min at 90◦C in a water bath. Then, they were centrifuged at 10,000 rpm for a minute, and the supernatant was collected for sugar determination. Total soluble sugars and individual sugars were determined in the shoot, leaf, and root ethanolic extracts and in the diluted berry must samples . Samples were filtered with PTFE membrane filters and transferred into high-performance liquid chromatography vials and subjected to reversed-phase HPLC analysis. The equipment consisted of an Agilent 1100 system coupled to a diode array detector and an Infinity Refractive Index Detector . The reversed-phase column was Luna Omega Sugar with a guard column of 5 mm. The temperature of the column compartment was maintained at 40◦C and the RID flow cell was kept at 35◦C. The mobile phase consisted of isocratic elution with acetonitrile:water at a flow rate of 1.0 ml/min with a run time of 22 min. Standard solutions of 10 mg/L of D-glucose, D-fructose, Dsucrose, and D-raffinose were injected to obtain the retention time for each compound,flower buckets wholesale and detection was conducted by RID. Sugar standards were purchased from VWR International . Sugar concentration of each sample was determined by comparison of the peak area and retention time with standard sample curves.

Starch content of the roots, shoots, and leaves was conducted using the Starch Assay Kit SA-20 in accordance with the manufacturer’s instructions. Briefly, pellets of different tissues were dissolved in 1 ml DMSO and incubated for 5 min in a water bath at 100◦C. Starch digestion commenced with the addition of 10 µl α-amylase and then incubated in boiling water for another 5 min. Then, the ddH2O was added to a total volume of 5 ml. Next, 500 µl of the above sample and 500 µl of starch assay reagent were mixed and incubated for 15 min at 60◦C. Negative controls with the starch assay reagent blank, sample blank, and glucose assay reagent blank and positive controls with starch from wheat and corn were performed. Reaction started with the incubation of 500 µl of each sample and 1 ml of glucose assay reagent at 37◦C and was stopped with the addition of 1 ml of 6 M sulfuric acid after 30 min. The reaction was followed with a Cary 100 Series UVVis Spectrophotometer and starch content was expressed as percent of starch per tissue dried weight. Weather data for the 2019 and 2020 growing seasons are shown in Table 1. Compared with the 2019 growing season, 2020 had 17 days more with temperature over 30◦C, a maximum daily temperature of 1.1◦C higher, and almost 800 mm less of precipitation, leading to an ETo of 23 mm higher. On the other hand, the lower available water for grapevine growth resulted in smaller canopy development decreasing the ETc, which explained the lower irrigation amount of 2020 compared with 2019 . Petiole mineral nutrients were not affected by irrigation amounts in the 2018–2019 growing season . Conversely, total N increased in 100% ETc, while the K content in 25% ETc vines decreased in the 2019–2020 growing season. The micronutrients were not affected by the applied water amounts in either year of the study. The plant water status decreased throughout the season . In 2019, the 100% ETc treatment had the highest SWP, while 25% ETc had the lowest SWP as expected. Conversely, there were no significant differences during the 2020 season between treatments. Likewise, we measured significant differences between the different irrigation amounts in gs and AN in both growing seasons .

We measured higher gs and AN in grapevines subjected to 100% ETc treatment from the second half of July, coinciding with the veraison, to harvest,compared with 25% ETc. The gs and AN of 50% ETc were transiently lower than those of 100% ETc, but consistently greater than those of 25% ETc. The WUE differed between irrigation amounts at harvest in 2019 and at mid-ripening in 2020 with 100% ETc grapevines showing the highest WUE . The enhancement of the photosynthetic performance in 100% ETc grapevines was accompanied by increased total chlorophyll and carotenoid content in the leaves . Calculation of the seasonal integral of SWP and gas exchange variables allowed to establish the seasonal-long trend for grapevine physiological response. Thus, SWP seasonal integrals for both seasons were affected by the interaction between irrigation amount and year. During the 2019 season, there was a significant increase of SWP with 100 and 50% ETc siSWP compared with 25% ETc siSWP . However, in the 2020 growing season, no difference in seasonal pattern was measured. On the other hand, seasonal integrals of gs , AN, and WUE were significantly different between years. The AN and WUE were significantly lower in 2020 compared with 2019 . Grapevine growth was monitored for different organs as shown in Table 3 and Supplementary Table S2. Leaf, shoot, and root fresh weights increased with increased irrigation amounts . The biomass of the leaves, roots, and shoots increased in the grapevines subjected to 100% ETc irrigation compared with 50 and 25% ETc . The applied irrigation treatments affected the harvest index . The greatest harvest index was measured in 100% ETc, while the lowest was measured in 25% ETc, respectively. Cluster number was not affected by the replacement of different fractions of ETc . An increase in the yield per grapevine was observed in both seasons with a highly significant increase in yield per grapevine in 100% ETc treatment.

Likewise, the linear increase in yield was evident from 25% ETc to 50% ETc as well, in both years. We also measured linear increases in leaf area to fruit ratio and berry size as the amount of irrigation increased from 25% ETc to 100% ETc. There was a significant increase of SS and starch content in the leaves as affected by the applied water amount . This increase in leaf SS was attributed to the increases in glucose, fructose, and raffinose content of the leaves . The total sugar and starch content of the shoots were not affected by the applied water amount . However, sucrose and raffinose in the shoots increased in 50 and 100% ETc treatments compared with 25% ETc . Root carbohydrate content and composition were not affected by irrigation treatments, with sucrose being the main soluble sugar found in root tissues . Our analysis of the different carbohydrates found in grapevine tissues indicated that starch was the main NSC in the shoots and roots, which accounted for >50% regardless of the applied water affecting their proportions . In the leaves, starch content was the less abundant NSC, but a significant effect of irrigation treatments was observed with the 100% ETc treatment reaching the highest amount. Finally, the proportions of sucrose and raffinose in the shoots decreased when water application was restricted to 25% ETc . Regarding the sugar composition of the must, fructose and glucose were the main sugars found , and their ratio ranged between 0.62 and 0.78 with no difference between treatments . In spite of the warming trends recorded for the study area within the two growing seasons covered by this study, the plant water status recorded in both growing seasons was optimal for grapevine growth as indicated by the midday SWP and the gs . Thus, seasonal integrals of SWP ranged between -0.8 and -1.1 MPa, while gs ranged between 150 and 250 mmol m−2 s −1 , in accordance to the midday SWP and gs values considered as well-watered conditions . Moreover,flower harvest buckets water status of the grapevines subjected to less applied water amount never reached values lower than -1.5 MPa for SWP and/or 50 mmol m−2 s −1 for gs , which have been reported to impair grapevine performance and berry ripening . As Keller et al. reported before, in warmer years, 100% ETc treatment may suffer from mild water deficit. Thus, under our experimental conditions, at the end of the season, especially in 2020, grapevines reached SWP values to ca. -1.2 MPa; however, they are not sufficient to impair grapevine physiology and metabolism in warm climates . Previous studies highlighted that plant water status is closely related to leaf gas exchange parameters . Thus, low values of SWP were related to decreased gs likely because plants subjected to mild to moderate water deficit close their stomata as an early response to water scarcity to diminish water loss and carbon assimilation . Accordingly, in both growing seasons, a higher SWP promoted increased stomatal conductance and, consequently, net carbon assimilation rates in grapevines subjected to 100% ETc. AN and gs peaked around veraison and then declined in all the treatments similar to several studies conducted in a warm climate before . Thus, previous studies have pointed out that limited photosynthetic performance, hence lower gs and AN values, may be triggered by passive or active signals .

Nevertheless, AN in 50% ETc treatment was not severely decreased presumably by increases in WUE, which have been related to improvements in stomatal sensitivity to water loss and vapor pressure despite the hormonal signaling from roots to shoots . Likewise, Tortosa et al. suggested that differences in WUE between Tempranillo grapevine clones were more explanatory of the variations in carbon assimilation rather than a different stomatal control. Finally, it is worth mentioning that WUE was significantly lower in the driest and hotter growing season regardless of the irrigation treatment as previously reported . Regarding intrinsic WUE , no effect due to growing conditions was observed in contrast to previous studies on vines subjected to mild water stress . The water deficits applied in this study were from moderate to severe based on SWP values; thus, it is expected that the vegetative and reproductive growth of vines will be impacted accordingly. Thus, in previous studies, higher water deficits resulted in reductions of yield and berry size . The reduction in berry mass has been associated with the inhibition of cell expansion and the diminution of inner mesocarp cell sap . The detrimental effects of 25% ETc were reported previously, suggesting that this applied water amount Vegetative growth was also impaired by water deficits applied in this study, as indicated in the decrease of leaf and root dry biomasses measured in 25 and 50% ETc treatments. Diminution of root growth under water stress has been related to the loss of cell turgor and increased penetration resistance of dried soils . In addition, a recent study suggested that the loss of leaves could decrease the supply of carbohydrates and/or growth hormones to meristematic regions, thereby inhibiting growth . In accordance with previous studies, severe water deficits led to lower shoot to root ratio because root growth is generally less affected than shoot growth in drought-stressed grapevines . Given that grapevine vegetative growth occurs soon after bud break in springtime, our results corroborated the crucial role of water availability during that period on vine development, physiological performance, and yield components reported in previous studies . Thus, irrigation of grapevines during summer could not be sufficient to fulfill water requirements when rainfall has been scarce in spring , and precipitation amounts prior to bud break result in cascading effects for the rest of the growing season that cannot be overcome with supplemental irrigation . The allocation of NSC varied between organs for which roots accounted 30%, shoots 25%, and leaves 40% of the whole plant NSCs at harvest, slightly differing from those reported for several fruit trees but similar to the works in grapevine . The NSC composition was highly dependent on the grapevine organs, with starch being the main NSC in the roots and shoots. Previous studies reported that roots accumulated the largest amounts of starch in plastids, namely amyloplasts, which is fundamental to allow rapid vegetative development during the next spring .

The task was to eliminate or minimize the flicker in the visual field three times by turning a dial that changed the intensity of a 460 nm light. Each participant performed the test while looking directly at the flickering light at 0.25, 0.5, 1, and 1.75 RE degrees, representing the MPOD level from the center to the periphery of the macula. Skin carotenoid content was measured by reflection spectroscopy . After cleaning, the tip of the right index finger was inserted into the spectrophotometer and three measurements were collected. A skin carotenoid score was calculated by the system software. Carotenoids that exist in human plasma, including β-carotene, lycopene, L, Z, and their isomers have been successfully detected in toto and quantified by this device, which has been validated to reflect fruit and vegetable consumption.Sample size was based on a study that assessed the impact of a Z supplement on MPOD in 24 healthy people. Statistical analyses were performed with JMP version 16 . Two-tailed t-tests evaluated potential between group differences at baseline. The MPOD and skin carotenoid data were analyzed with mixed-effects models using time and treatment as the main factors, with age and sex as the covariates, and participant ID as the random effect. For main effects, student t-tests determined significance within group pairs. p-Values of 0.05 or less were considered statistically significant. The mean values of the dietary intake data were compared by two-tailed t-tests, which were log-transformed when necessary,black plastic plant pots wholesale and presented as the mean ± S.E.M. or the back-transformed mean with 95% confidence intervals . Reported protocol compliance was greater than 96% for both groups, and no adverse symptoms were noted other than minor intestinal gas from one participant in the goji berry group. Table 1 presents the reported average intake of select nutrients in the habitual diet that may have affected eye health over the study period. No significant differences between groups were noted.

The composition of the goji berries is presented in Table 2. A daily goji berry serving provided 28.8 mg of Z, which was substantially higher than the 4 mg of Z present in the supplement. Although sufficient extraction of L from our goji berry samples could not be obtained, previous work by others estimated a L content of 0.15 mg in 28 g of goji berries from six different goji berry samples collected in the Ningxia province of China, the same region from which the goji berries used in this study were obtained. Baseline MPOD measures were similar between the goji berry and supplement groups . No significant interaction effects for treatment and time were observed in any REs. A significant main effect of time was found for MPOD at 0.25 RE . In a sub-analysis, intake of goji berries, but not LZ, significantly increased MPOD at 0.25 RE at day 90 compared to baseline . There was also a significant main effect of time for MPOD at 1.75 RE , with a significant increase at day 45 compared to the baseline , and again between day 90 . No significant MPOD changes were noted at any REs in the LZ group.Ninety days of 28 g of goji berry intake significantly increased the optical biomarker MPOD in healthy adults at 0.25 and 1.75 REs. These results suggest that even in a healthy population with no evidence of small drusen or early AMD, goji berry intake can improve eye health. Our results are consistent with data of improved MPOD after a similar amount and intake period of goji berry in a Chinese population at risk for intermediate AMD. Moreover, our trial is consistent with reports of protection against macular hypopigmentation and drusen development in a population of generally healthy and older individuals who were provided Z at approximately a third of the amount of Z provided in the current trial. Our findings suggest that a higher intake of Z relative to L may be useful in reducing the risk of AMD. This is consistent with increased MPOD levels after 4 months of supplementation with 20 mg Z or 26 mg Z with 8 mg L plus 190 mg of mixed omega-3 fatty acids by young healthy adults. Interestingly, we observed a significant increase in MPOD at 1.75 RE, but not at 0.5 or 1 RE, in the goji berry group.

A possible explanation for this trend is the relatively low macular pigment at 1.75 RE compared to the other REs, which may increase the potential for improved MPOD in this peripheral area of the macula. Our results are also consistent with data from 11 randomized controlled trials where supplementation with at least 10 mg of the macular carotenoids was effective at increasing MPOD. Significant correlations were observed between the overall skin carotenoid score and MPOD, which is consistent with clinical results of carotenoid supplementation. Further analysis demonstrated that L and Z, but not goji berry intake, was significantly influencing this trend. Previous work has shown an association between serum L and Z in skin and blood with macular pigment carotenoid accumulation. Data from the current trial are consistent with this observation as goji berry intake was significantly associated with the skin carotenoid score. However, in contrast to data with L and Z supplements, MPOD score was not correlated with changes in skin carotenoids with goji berry intake. The skin photometer detects overall carotenoid content, and as goji berries are also rich in β-carotene, neoxanthin, and cryptoxanthin, these carotenoids likely influenced the skin measurements, and would not reflect the selective carotenoid accumulation of L and Z in the macula. Other goji berry components such as taurine, vitamin C, zinc, and LBP may influence the results by lowering oxidant stress and improving eye health. For example, studies in animals and cell lines suggest that LBP can protect against AMD by reducing oxidative stress and cell apoptosis in retinal pigment epithelium. Taken together, under the conditions tested, it is reasonable that MPOD may not fully correlate with skin carotenoids in the goji berry group. To our knowledge, the impact of goji berry intake on MPOD in healthy middle-aged people has not been previously reported. While others have noted improved MPOD after LZ supplementation among people with low MPOD baseline levels, our findings suggest that even in populations with normal MPOD values, a significant increase can be detected after goji berry consumption at the most central part of the macula .

A meta-analysis regarding the effects of L, Z, and meso-Z supplementation noted that the MPOD at baseline was inversely associated with macular responses,black plastic plant pots bulk suggesting individuals with a relatively lower macular pigment status may receive more benefit with higher amounts of L or Z. The Age-Related Eye Disease Study 2 trial assessed the impact of dietary supplements containing 10 mg of L, 2 mg of Z, 500 mg of vitamin C, 400 IU of vitamin E, 80 or 25 mg of zinc, 2 mg of copper, and/or 350 mg of docosahexaenoic acid plus 650 mg of eicosapentaenoic acid. The results showed a significantly reduced rate of progression from intermediate- to late-stage AMD after 5 years. Secondary analyses of the study indicated protective roles of L and Z. We did not use the AREDS2 supplement for the comparison group because this formula has only been shown to be effective for those with intermediate AMD, and no clinical evidence exists for its efficacy in our study population of healthy people. In addition, we note that 80 mg of zinc in the AREDS2 supplement is twice the upper limit of recommended daily intakes for zinc. In epidemiological studies, L and Z intakes have been inversely associated with the development of AMD. In the current study, the reported dietary intake of L plus Z, not including the berries or supplement, was 3.1 and 1.9 mg/d in the goji berry and supplement groups, respectively, which is higher than the typical estimated intakes in the US of 1.6–1.86 mg/d. Three to five mg/d of L and Z have been recommended to help support normal macular function, although no recommended dietary allowance values yet exist. A few studies have explored the effects of L and Z from a whole food on MPOD. Daily consumption of one Hass avocado containing 0.5 mg of L over 6 months was associated with a significant increase in MPOD in healthy adults. In contrast, no increase in MPOD was observed after consuming one Hass avocado daily for 3 months. Daily consumption of egg yolks providing 1.38 mg L and 0.21 mg Z resulted in a significant increase in MPOD and other measures of visual acuity in older adults with signs of early stage AMD after 12 months. Another study giving older adults two egg yolks/day for 5 weeks, followed by four egg yolks/day for 5 weeks, reported increases in MPOD, but only among those with low baseline MPOD values. The addition of either spinach or corn , or the combination, for 14 months significantly increased the MPOD among the majority of healthy individuals. Our study has some limitations. Choice of a control is always a challenge in whole food studies, since masking is an issue.

A commercially available LZ supplement was used, rather than an inert capsule, since our research design was intended to compare options available to consumers and explore the role of goji berries over and above the intake of purified L and Z. The actual amount of L and Z in the supplement was not confirmed. A previous report noted that the carotenoid content of some powder-based supplements tested in 2017 did not meet label claims, while oil-based supplements did. Since L and Z are preferentially deposited at different eccentricities in the retina, the different amounts of Z in the goji berries and supplement may not be ideal. Volunteers were not screened for low MPOD as an inclusion criterion. Although the relatively modest number of participants in each group may raise some concerns, these numbers are similar to those reported by Obana et al. and are consistent with an initial probe study. Finally, although MPOD was the primary outcome measure, other ocular measurements such as contrast sensitivity and best corrected visual acuity were not assessed. Future studies on goji berry intake and eye health ideally should combine functional and anatomic measurements.Grapevine is an economically important fruit species worldwide, and has a historical connection with the development of human culture. Grapevine comprises cultivated and wild forms . More than 6000 accessions are recorded as individual varieties. Some are rare and have only a few unique vines that are important to national heritage, and are valuable as resources for cultivation and breeding. In the last few decades, the cultivated grapevine has experienced a drastic reduction in diversity due to the increased focus of the global wine industry on a few major cultivars. Moreover, the loss of natural habitat is adversely affecting the genetic diversity of the wild V. vinifera species, with some populations on the verge of extinction. Hence, immediate action to conserve indigenous grapevine germplasms is required. Vitis germplasm conservation can be achieved either in situ or Ex situ . In situ conservation refers to preserving a species in its native environment, and preserving and recovering viable populations in their natural habitat. However, anthropogenic activities and abiotic or biotic stress may lead to the extinction of the germplasm. On the other hand, ex situ conservation refers to preserving germplasm outside their native habitat. The methods include using slow growth tissue culture, cryopreservation, and seed banks; or preservation of the whole plant in a botanical garden or in a field gene bank; and via greenhouse cultivation of plant material. Using a slow growth tissue culture under in vitro conditions on minimal nutrient medium ensures minimum maintenance costs by retarding growth rates. However, this approach has several limitations, including the need for technical expertise, plant loss due to contamination of cultures, high labor costs, and the possibility of obtaining somaclonal variations. Cryopreservation in liquid nitrogen also provides an opportunity for the long-term preservation of Vitis germplasm, which can then be used as a backup for field collections for important indigenous cultivars. However, exposing cells to extremely low temperatures can result in freezing injury; hence, cells must be carefully handled and prepared before being frozen in liquid nitrogen.

These 10 alleles were examined for evidence of IHR by comparing them to the previously described non-IHR X. fastidiosa subsp. multiplex alleles and to the known X. fastidiosa subsp. fastidiosa and sandyi alleles . Of these 10, 4 alleles were found to be derived in their entirety from X. fastidiosa subsp. fastidiosa, and 3 were found to be chimeric for X. fastidiosa subsp. multiplex and fastidiosa sequences, with significant evidence of one or more recombination breakpoints. These 7 alleles encompassed 4 loci: leuA, cysG, holC, and pilU. The locus most strongly implicated in IHR wascysG, since all of the 9 recombinant-group STs were characterized at this locus by 1 of 3 cysG alleles unique to the group. The involvement of IHR in the genesis of all 3 of these alleles is illustrated by their close genetic relationship to X. fastidiosa subsp.fastidiosa and sandyi alleles . Allele 12, apart from being found in the recombinant group, is an X. fastidiosa subsp. fastidiosa allele . The other two alleles were found to be chimeric: allele 18 contains a single recombinant region at the 3= end of 342 bp, while allele 6 has two short recombinant regions, one at the 5= end of at least 23 bp and another toward the 5= end of at least 35 bp . The DNA sequence variation defining these patterns is shown in Table 2. The patterns seen in the DNA sequences of the 3 cysGalleles are consistent with the hypothesis of a single IHR that introgressed donor allele 12 into X. fastidiosa subsp. multiplex, followed by subsequent intrasubspecific recombination reintroducing X. fastidiosa subsp. multiplex sequence to create alleles 6 and 18 . There are no inconsistent sites, container size for blueberries provided the 5= recombination breakpoint in allele 18 starts after position 71. Introgression of X. fastidiosa subsp. fastidiosa sequence into X. fastidiosa subsp. multiplexwas found in alleles at 3 other loci .

In the case of pilU, 7 of the 9 recombinant STs carried either an allele identical to a known X. fastidiosa subsp. fastidiosa allele or 1 bp different from it . Allele 1 is an allele that characterizes most U.S. isolates as well as several STs found in Costa Rica, while allele 9 is unique to the recombinant group. The leuA locus has a single statistically significant recombinant allele, allele 4 . It differed by 2 bp from the X. fastidiosa subsp. fastidiosa allele 9 but by 8 bp from the most similar nonIHR X. fastidiosa subsp. multiplex allele. X. fastidiosa subsp. fastidiosa allele 9 could be the donor for its entirety , although if the recombination region started after site 10 but before position 520 , then only one site would be unexplained. That remaining site carries a base unique to this allele and is probably a novel mutation. If the recombination breakpoint was 3= of position 295 then X. fastidiosa subsp. fastidiosa allele 1 provides as good a match as allele 9 . Similarly, holC allele 7 was also 8 bp different from the most similar non-IHR X. fastidiosa subsp. multiplex allele, providing clear evidence that the 5= end was derived from X. fastidiosa subsp. fastidiosa . The pattern can be explained if X. fastidiosasubsp. fastidiosa allele 19 is the donor of the 5= region ending somewhere between positions 183 and 286, since it leaves no inconsistent bases . The loci leuA and holC each had an additional allele that were unique to the recombinant group, as was an allele at another locus, nuoL4. Although these last 3 alleles did not show statistically significant evidence of introgression , they all showed a grouping of 2 or 3 nucleotide changes that were not found in non-IHR X. fastidiosa subsp. multiplex but were present in X. fastidiosa subsp. fastidiosa. Of these 3, the strongest case for IHR is holC9, where a region of possible IHR can be seen at the 3= end of the sequence .Analysis of the recombinant group ofX. fastidiosa subsp. multiplex showed three important results. First, intersubspecific recombination was shown to have occurred in 50% of 8 loci scattered throughout the genome that were chosen independently of the data . Second, it was shown that the donor of the introgressed sequence was X. fastidiosa subsp. fastidiosa, a subspecies introduced from Central America into the United States as a single strain .

However, the introgressed sequence at two of the loci did not come from any of the X. fastidiosa subsp. fastidiosa genotypes that have been found in the United States. This result suggests that another introduction of X. fastidiosa subsp. fastidiosa must have occurred, an introduction that resulted in successful IHR, after which the donor genotype seems to have disappeared. This involvement of an unexpected X. fastidiosa subsp. fastidiosa strain supports the hypothesis that the members of the recombinant group share a single ancestral IHR event. Third, the hypothesis that IHR has facilitated a shift to new hosts is strongly supported by the example of blueberry, where 10 isolates have been typed and potentially supported by the example of blackberry .Of course, the direct causation of this link can never be proved without knowledge of the genetic changes driving this shift. It can always be argued that the link is fortuitous and that one or more point mutations in the nonrecombined X. fastidiosa subsp. multiplex genome are causal in the host shift. Arguing against this possibility are 2 additional pieces of information. First, both blueberry and blackberry are native to the United States, so if only a simple genetic change was required to infect these species, why did the native non-recombinant X. fastidiosa subsp. multiplex apparently never acquire these changes? Second, a similar but even more extensive mixing of the genomes of X. fastidiosa subspp. fastidiosa and multiplex is found in the only form of X. fastidiosa that infects another U.S. native plant, mulberry . Furthermore, in other bacterial species, it has been demonstrated that recombination can drive rapid evolution, both in the laboratory and, in the case of Helicobacter pylori, in mice . Similarly, McCarthy et al. concluded that lineages of Campylobacter jejuni in chickens versus cattle and sheep were able to shift host type, because rapid adaptation was facilitated by recombination with the resident host population.

In the study by Nunney et al. , it was shown that the recombinant genotypes formed a well-defined group , demonstrating that intersubspecific homologous recombination was not randomly distributed across the X. fastidiosa subsp. multiplex isolates. This work was based on a survey of 143X. fastidiosa subsp. multiplex isolates using just 8 loci. There were 33 isolates that showed some evidence of IHR in at least 1 locus: all but 2 showed statistically significant evidence in at least 2 loci, while the remaining 110 showed no such evidence . The generality of this discrete group of recombinant forms was supported by our analysis presented here of the sequence data from 9 more loci sequenced by Parker et al. . These loci divided isolates into 2 groups that appeared to correspond to the recombinant and non-IHR groups, respectively ,raspberry grow in pots even though Parker et al. found no evidence of IHR. Upon reanalysis, we found statistically significant IHR in 6 of the 9 loci in the clade A data but no evidence of IHR in the clade B data. Clade A included 6 isolates that we had typed in the present study, and each of these showed evidence of IHR in 4 or 5 of the additional 9 loci. Thus, in two independent samplings that together examined 17 loci, there was clear evidence of substantial genomewide IHR in the recombinant group isolates, amounting to 50% of the genes showing IHR across the MLST locis plus the pilU locus . The average was higher when based on the loci sequenced by Parker et al. ; however, this was probably biased upwards by the manner in which the loci were chosen . None of the IHR events in 6 of the 9 loci identified using the targeted introgression test, or in the case of complete introgression, a chi-square test, were detected by Parker et al. using PHI and the 9 tests implemented in RDP . This failure of the standard tests of recombination to detect IHR was previously noted by Nunney et al. , motivating the development of their introgression test. We examined the hypothesis that the recombinant group STs were derived from a single IHR event involving a X. fastidiosa subsp. multiplex recipient and an X. fastidiosa subsp. fastidiosa donor. The distribution of allelic differences among the recombinant STs was consistent with them all being derived from a single initial event, but a small number of other intersubspecific and intrasubspecific recombination events would also be needed . More importantly, the genotypes seen in the recombinant group can be accounted for entirely, or very nearly so, based on a single X. fastidiosa subsp. fastidiosa donor genotype. For example, the substantial variation in cysGcan all be accounted for by an ancestral introgression of X. fastidiosa subsp. fastidiosa allele 12 followed by subsequent intrasubspecific recombination of X. fastidiosa subsp. multiplex sequence to form the other two alleles . In contrast, variation at pilU could be accounted for by a second donor contributing the X. fastidiosa subsp. fastidiosa pilU9 allele, but it could also have arisen by a single mutation in pilU1 unique to the recombinant group. A possible single X. fastidiosa subsp. multiplex recipient genotype was also identified . This genotype is consistent with a known ST: setting cysG to allele 3 makes the recipient identical to ST45, which was sampled from the states of California, Kentucky, and Texas .

Elsewhere, we consider a slightly different hypothesis regarding the origin of the recombinant group in which the donor and recipient subspecies are reversed—i.e., that it was derived from a single IHR event, but involving an X. fastidiosa subsp. multiplex donor and an X. fastidiosa subsp. fastidiosa recipient; however, apart from the role reversal, the conclusions are unaltered . The ancestral reconstruction allows us to consider the second question posed earlier: is the donor consistent with the X. fastidiosa subsp. fastidiosa genotypes found in the United States? The answer is very clearly “no.” The original donor carried cysG12 and holC19 . These alleles are both found in isolates from Central America, but no X. fastidiosa subsp. fastidiosa isolate found in the United States comes close to matching this criterion: the most similar U.S. ST has a 12-bp mismatch. There has been extensive sampling of X. fastidiosa subsp. fastidiosa within the United States, based on 85 isolates sampled across the United States from 15 different host plants . There is very little variation within X. fastidiosa subsp. fastidiosa isolates from the United States, consistent with the hypothesis that all X. fastidiosa subsp. fastidiosa isolates currently found in the United States are derived from a single strain introduced from Central America . Based on these data, we conclude that the X. fastidiosa subsp. fastidiosa donor was introduced into the United States from Central America and recombined with a native X. fastidiosa subsp. multiplex genotype similar to ST45; however, this donor lineage of X. fastidiosa subsp. fastidiosa was ultimately unsuccessful and died out. We can never conclusively prove the absence of this genotype from North America. However, X. fastidiosa has been extensively sampled from many plant species throughout the United States, and no isolates of X. fastidiosa subsp. fastidiosa have been found that carry alleles similar to the inferred donor alleles cysG12 and holC19 ; indeed all X. fastidiosa subsp. fastidiosa isolates so far found in the United States are consistent with the introduction into the United States of just a single genotype . The transient presence of the donor genotype is consistent with a single large-scale introgression event founding the recombinant group. This raises the possibility that conjugation might have been involved; however, if this was the case, the genomic DNA was broken into pieces prior to homologous recombination, since the data show short regions of recombination. The data from the MLST loci plus pilU show 7 significant recombination events , and 3 of them included at least one recombination break point.

The values for glutamate reported for pomegranate herein are greater than values reported for grape juice , indicating increased importance for consumers regarding marketing fraud in the pomegranate juice industry because often grape juice is used as the primary adulterating agent in pomegranate juices in the USA, along with apple and pear juice. Glutamine is the amino acid of the highest concentration in human blood , which may play into the folklore regarding pomegranate juice as a “blood tonic” . Ethanol is of importance in the food and beverage industries because it is an indicator of anaerobic respiration and metabolism in post harvest fruit products. Along with other alcohols, ethanol can contribute to off flavors or even enhance the flavor of fruit if concentrations are low . The freshly expressed juice had ethanol values that were far less than the levels prohibited by countries for religious and food safety reasons, which is typically required to be less than 0.5% ethanol in the USA for the beverage to be considered non-alcoholic; religious restrictions are typically stricter.This work demonstrates the high level of phenotypic diversity of pomegranate juices that exists in approximately 5% of the USDA available pomegranate germplasm collection. This study is the first of its kind in utilizing 1H NMR coupled with conventional post harvest juice quality methodologies to assess differences among pomegranate cultivars for metabolic, physicochemical and nutritional traits. The results indicated a great complexity of juice quality and nutritional differences among the cultivars analyzed, with many fitting the profile of Wonderful, but others differing greatly from this standard. As a replacement,blueberry production alternate or substitute candidate for ‘Wonderful’ in juice markets, ‘Al Sirin Nar,’ ‘Blaze,’ ‘Desertnyi,’ ‘Parfianka,’ ‘Phoenicia,’ ‘Purple Heart,’ and ‘Sakerdze,’ all meet a host of juice quality parameters and mostly fit the nutrition composition of ‘Wonderful.’

These varieties should be considered for further investigation via cultivar trials to determine phenotypical traits important to growers and taste panels to determine preferences of consumers. There were striking differences between the commercial juice and fresh-squeezed juices as well as significant differences between juice extraction methods for many parameters, including amino acid content, phenolic content and antioxidant activity. Potassium concentration varied greatly among cultivars, which can affect the flavor and nutritional composition of these fruits and their juices. This work also presents further evidence that pomegranate is not only a potentially healthy fruit in terms of phenolics and antioxidant activity, but also for nutrition as it relates to amino acids and mineral nutrition . It is important to note that these juice quality traits and nutritional factors can be significantly different among cultivars. Whether the cultivars with unique quality profiles appeal to consumers will need to be investigated in future research. This study is the first of its kind comparing fresh-pressed juice quality of the industry standard, Wonderful, with 13 other NCGR pomegranate cultivars using 1H NMR coupled with other physicochemical analytical techniques. Pomegranate is a deciduous tree crop that has been domesticated for thousands of years for its fruit, flowers, bark, and leaves , all of which have been believed to possess medicinal properties . Despite its long history of cultivation, limited horticultural information is available for growers, breeders, and the food and beverage industries about when fruit of a given cultivar is ready for harvest, processing and consumption . In the United States, Wonderful, the industry standard, is a tart, acidic, moderately hard-seeded fruit that has been reported to have astringent and bitter juice compared to other cultivars previously analyzed from the collection at the United States Department of Agriculture – Agricultural Research Service National Clonal Germplasm Repository , Davis, CA .

Despite these negative fruit quality traits, pomegranate cultivation in the United States remains predominantly a monoculture of ‘Wonderful.’ It is believed that cultivars in the national germplasm with desirable traits, such as soft seededness and low acidity may be candidates for commercial production. Studies have demonstrated a large variation in mature fruit size within commercial orchards of ‘Wonderful,’ which poses a problem for fresh market growers and packers. Wetzstein et al. reported a greater than five-fold range in mature fruit volume and weight in commercial ‘Wonderful’ pomegranate groves. Factors that influence fruit size and yield include aril number , cultivar , cultural practices , and plant maturity . Finding cultivars with better uniformity than Wonderful would be beneficial to the industry In addition to variable fruit size, dates of fruit maturity can play a major role in fruit quality. Late season harvests run the risk of fall rains, which have been associated with greater numbers of split fruit . Typical commercial harvest windows for ‘Wonderful’ range from late September to early November, but fruit in the Central Valley of California, USA, where the most pomegranate cultivation occurs, are typically ready to harvest starting in late October. Usually by November, effects of weather, especially rains, and pests will begin to damage the fruit. Therefore, harvest date can determine whether a cultivar is a good candidate for commercial production. In addition to the fresh fruit market, pomegranates are also utilized for juice. The beverage and wine industries utilize different fruit juices that have sufficient quantities of organic acids, carbohydrates , and phenolic compounds. Concentrations of total soluble solids , often expressed in ºBrix, for commercial pomegranates range from 12% to 16% at maturity. It is recommended that ‘Wonderful’ have at least 15% TSS at harvest , but above 17% is preferable . Hasnaoui et al. reported that citric acid is the determinant of sour flavor in pomegranate juice, despite sugar concentration. Sweet pomegranates typically have been reported to have citric acid concentrations less than 0.50% . Standards for fruit maturity of sweet cultivars are being investigated because growers often pick early-season cultivars too early in order to increase profits .

There are no known imposed regulations on growers in any country, meaning they can harvest early before fruit maturity without short-term consequence. The effect of picking early on consumer perception and acceptance of pomegranate fresh fruit has been shown to be associated with astringency and a low flavor preference score . Standards have been proposed for titratable acidity and total soluble solids for ‘Wonderful’ pomegranate . Generally, citric acid is the most abundant organic acid in pomegranate juice, so TA is generally expressed in citric acid equivalents. ‘Wonderful’ pomegranate fruit is considered mature when juice is lower than 1.85% TA , so fruit are picked when fruit measure below that threshold. Maturity index is a standardized measure of maturity in many fruit crops. For pomegranate, a commonly used MI is the ratio of °Brix to TA, also known as the sugar to acid ratio. This ratio is often used to determine fruit maturity, but it has been found to not be usable for sweet cultivars. Instead, fresh aril weight is used at the indicator of fruit maturity and consumer acceptance in the sweet cultivars . The optimum MI for ‘Wonderful’ has been calculated to be greater than 8.1, at which point the fruit is ready for harvest. Other cultivars may have significantly different quantities of organic sugars and acids, so MI is logically different for different types of pomegranates . Fruit quality is not only related to sugar content, titratable acidity and spoilage, but also to phenolic compounds that contribute to the fruit’s flavors, antioxidant activity, and color . Cultivar is more influential in determining fruit juice composition than site of cultivation, year of harvest, or length of storage ,blueberry in container so it is important to study differences in traits among cultivars to identify superior cultivars in germplasm resources and make them available to growers and consumers. Determining levels of phenolics and the antioxidant activity of a cultivar’s juice is important to the beverage industry and consumers, because advertisements promote high antioxidant activity as the main selling point of juice products. If any cultivar were to demonstrate similar antioxidant activity to Wonderful, it would possibly be competitive in the pomegranate market were it to meet other consumer preferences. Having lower antioxidant activity than ‘Wonderful’ could make for an undesirable candidate for commercial production, although the public is unlikely to be able to detect differences in antioxidant activities. The objectives of this research were: 1) to evaluate fruit and juice quality traits of NCGR germplasm by comparing commercial quality parameters to the industry standard, Wonderful; and 2) to determine potential harvest windows of ten preselected pomegranate cultivars, based on seasonal trends of potential maturity indices with harvest dates. Fruit were harvested from the USDA-ARS National Clonal Germplasm Repository for Tree Fruit and Nut Crops and Grapes in Davis, CA, USA for two seasons. The trees were all over nine-years-old and in full maturity. The pomegranate cultivars analyzed included: Ambrosia, Desertnyi, Eversweet, Golden Globe, Green Globe, Haku Botan, Loffani, Parfianka, Phoenicia, and Wonderful . The cultivars in this study that have been described as soft-seeded included Desertnyi, Eversweet and Parfianka. Low acid cultivars included Ambrosia, Desertnyi, Eversweet, Golden Globe, Green Globe, and Loffani. Wonderful fresh fruit was included as a control and as the standard to compare the other cultivars in this study. All cultivars are of American origin except for Haku Botan and Desertnyi and Parfianka . Up to twelve fruit of each cultivar were hand-harvested in mid-September, midOctober, and mid-November in 2014. This was repeated in 2015.

Fruit were ground shipped, and then stored at 6 °C and 98% relative humidity for 3-4 weeks until processing. Fresh market quality fruit, as defined by being well-filled, mature, and unblemished, were chosen for juice analysis from each of the 10 cultivars. Three cultivars, Golden Globe, Green Globe and Parfianka, had fruit damage in November, so less fruit were available for these cultivars during November harvest dates. ‘Golden Globe’ and ‘Parfianka’ had no fresh market quality fruit available in November 2015 and ‘Green Globe’ had no fresh market fruit available for 2014 and 2015. At this last season harvest, the trees of these cultivars had fruit that were either cracked or infested with insect pests, especially leaf footed bug.Up to 12 fruit per cultivar were weighed using a tared digital scale. The equatorial diameter and stem- to blossom-end length of each fruit and calyx length and diameter were measured with a digital caliper. After weighing and measuring, fruit were opened by scoring the peels of the fruit longitudinally with a scalpel. Arils were then manually extracted from a subsample of five fruit. Weight of 100 arils and total aril weight was determined with a tared digital scale. Only intact, non-damaged arils were weighed for the weight of 100 arils to reduce error. Edible fruit fraction was determined by dividing weight of all arils by the total weight of fruit and converting the result to percent. For juice processing, fruits were halved and 100 undamaged arils were manually removed and placed in a polyethylene bag. These arils were pressed manually with approximately 480 Newtons of force to express the juice directly into a 15 mL test tube. Raw juice samples were transferred to a centrifuge tube via pipette and were centrifuged at 1000 g for 5 min using a Becton Dickinson DYNAC Centrifuge . Aliquots of the supernatants were used for all chemical analyses. 5.2.3 °Brix Degrees Brix, also known as total soluble solids, were quantified with a Vee Gee Scientific PDX-1 Digital Refractometer , utilizing 0.5 mL sample of expressed, centrifuged juice. Individual juice samples, each one representing a single whole fruit, were transferred to the refractometer sample reservoir with a hand pipettor. The sample reservoir was cleaned by spraying with deionized water with a wash bottle and wiped dry with Kimwipes before each subsequent sample. Results were reported in °Brix and represented the relative percent sugar content of the centrifuged pomegranate juices. One measurement was taken for each juice sample. Juice TA was measured with a Hanna HI 84532 fruit juice TA mini-titrator . Samples were prepared by mixing individual centrifuged juice aliquots with 45 mL of deionized water. Each sample represented the juice of one whole fruit and 3 to 5 samples were analyzed for TA per cultivar. Because the predominant organic acid in most pomegranates is citric acid, results are expressed in citric acid equivalents, which represent percentage citric acid in juice solution.

Some of these early cultivars include Foothill Early, Granada, and Early Wonderful. These cultivars reach maturity long before fall rains, which can be devastating to pomegranate crop yields due to fruit splitting . However, the early cultivars listed have been described as inferior compared to the fruit and juice quality of Wonderful, specifically having lower internal color quality . However, it is important to note that different cultures prefer differing flavor profiles for pomegranates. Pomegranate is utilized for many products, ranging from fresh fruit to various types of value-added products. Value added products can be from the seeds, which contain fleshy, juice-containing arils, which are the outer integument of the seed, or from the fruit peel. Maestre et al. reported that uses for pomegranate fruit include juice, jams, preserves, jellies, refrigerated arils, frozen arils, liquors, syrups and soft drinks. These various uses for pomegranate can be dependent on the cultivar that is being utilized. For example, jellies produced with ‘Borde’ experience fewer losses than ‘Mollar’ pomegranate when stored at higher temperatures. Yoshimura et al. reported that pomegranate is a successful skin whitening agent in mammals, which could be utilized as a plant-source material in the cosmetic or pharmaceutical industries. Per Stover and Mercure , as a fresh fruit, consumer demand for pomegranate may increase with the introduction of cultivars with soft-seeded arils and uniquely colored and sweeter arils. Day and Wilkins also believe that both commercial growers and consumers may express interest in cultivars other than Wonderful if they were to have “unusual” or “unique” traits. As a whole fresh market fruit, pomegranates should be medium to large at harvest and their exterior color should be pink or red , but it should be noted that market preferences have changed in the past, as with Malus and Prunus crop species. In the United States,big plastic pots the most important product produced from pomegranates is juice . The beverage industry has a market value of $187.4 billion, with total fruit juice sales equal to $50 billion .

Previous market research found that pomegranate juice was ranked first in the super premium juice category in grocery store sales and that PomWonderfulTM juice was the leader in pomegranate juice sales, with $36.5 million in sales during the time of the study . Market research also suggested that for juice blends, pomegranate-blueberry blends had the best sales. In addition to consumer acceptance of the fruit, to be a successful commercial cultivar, a cultivar must be easily propagated in the nursery and readily established in a commercial orchard; otherwise, it would be difficult to implement commercial production for that genotype. Compared to all other methods of propagation, utilizing dormant hardwood cuttings is the most inexpensive and convenient method to propagate pomegranate. As a result, it has become the standard for the industry for the production of clonal bare root or container-propagated trees . Perhaps the most important reason pomegranate trees are propagated via stem cuttings is that the seeds of pomegranate are the product of sexual reproduction and if propagated by seed, the progeny are not true to type . As a result, seeds are not utilized in the nursery industry for production of commercial pomegranate trees. Historically, pomegranate was propagated commercially by dormant hardwood cuttings in California long before the first scientific reports on pomegranate were published by the University of California . Although many studies have been conducted on propagation of pomegranate via hardwood cuttings , none of these studies is known to have included any of the cultivars from the USDA-ARS NCGR. In addition, there are no data in the literature regarding dormant cutting propagation of the most important commercial cultivar in the United States, Wonderful. Another omission from the literature is the lack of information on vegetative growth parameters of the cultivars in the collection. These parameters may elucidate important horticultural traits, such as plant vigor, branching habit, and the proportion of buds that produce vegetative shoots, including the proportion of buds below the soil-line that produce both vegetative shoots and roots. Success rate and vegetative growth parameters are important characteristics for professionals in the nursery industry and commercial growers.

Research on pomegranate propagation typically utilizes hardwood cuttings with stem lengths of 20 cm or more . Whereas it may be optimal to use 20 cm of stem length for hardwood cuttings, the most limiting factor to crop expansion of pomegranate in California is the lack of nursery stock . Alikhani et al. reported that cuttings with three buds or more givethe best results for clonal hardwood propagation; Heidari et al. found evidence that single node cuttings worked better than those having two or four buds. This finding was ascribed to the endogenous hormonal balance in the propagule. Reports on the use of the auxin indole-butyric acid to increase rooting are contradictory. Polat and Caliskan reported that 1 g×L -1 of IBA was sufficient to root pomegranate cuttings, but stated that this concentration was not optimal. Saroj et al. reported that 2.5 g×L -1 IBA was an optimum rate of auxin for high rooting success in pomegranate. However, much higher doses of IBA have been demonstrated to increase pomegranate propagation success, with a treatment of 12 g×L -1 resulting in a lower rooting success rate . To be considered for commercial pomegranate production, a pomegranate cultivar would need to produce high yields and be as precocious as Wonderful. Wonderful typically starts producing an economic crop in year three after planting , so any variety that produces fruit in year three or earlier could possibly be a competitive cultivar relative to the industry standard. The cultivar would also need and acceptable establishment rate, i.e. to quickly reach a tree size that partially fills the 4 m x 5.8 m spacing typical of pomegranate groves within the same time or earlier than Wonderful, excluding cultivars with dwarf and semi-dwarf traits that could be farmed in high density plantings. Another trait of benefit to a commercial cultivar is having an earlier harvest date than Wonderful, which is typically harvested for the fresh market in late September, October, or early November, depending on climate and growing conditions. For juice production, ‘Wonderful’ trees are stripped at the end of the commercial fresh fruit production season, which can be as late as November. The cultivar must also maintain good fruit quality over this period to be competitive with Wonderful. Having an earlier harvest date is ideal because it will increase the chances of avoiding fall rains, which are known to cause devastating losses due to fruit splitting.

Orchard establishment rates for pomegranate cultivars under drip irrigation are unknown. Day et al. found that ‘Wonderful’ trees will produce a commercial crop by the third year in the field under furrow irrigation in the San Joaquin Valley in Central California, but plant establishment data were not collected. No data are available for tree height, canopy width within a row or across the row or trunk diameter, the traits which were measured by Webster et al. to determine establishment rates in apple cultivars. These traits are important in all tree crops. Comparisons of tree establishment rates across different climates are also not found in the literature. For a commercial fruit tree grove to be sustainable and profitable, the trees need to be precocious, i.e., to produce as many flowers and fruit of marketable quality, as early as possible . Pomegranate is monecious and produces two flower types, functionally “male” and functionally “female.” Male flowers only produce pollen and have an underdeveloped gynoecium, whereas the female flowers are perfect and are able to produce fruit . To ensure that precocity is not a false measure of the ability of a tree to set fruit, the number of male and female flowers during peak bloom must be counted on each tree . Pomegranate is a crop that can survive in relatively harsh conditions and it has been classified as a drought- and heat-tolerant crop . Glozer and Ferguson reported that pomegranate trees tolerate temperatures as low as -11° C. The geographical suitability of cultivars for commercial production at a given site can also be assessed by quantifying physiological parameters influencing tree health, growth and productivity. These parameters include photo inhibition, stomatal conductance, transpiration and photosynthetic rate. To determine satisfactory geographic suitability and success,growing berries in containers a cultivar would have to perform physiologically at an equal or greater level than Wonderful. Hepaksoy et al. found evidence that leaf transpiration and water-use efficiency were correlated with fruit splitting in pomegranate. Additionally, being able to forecast establishment rates and precocity of the cultivars is crucial to assess whether a cultivar is a candidate as a commercial cultivar and it allows growers to anticipate establishment of their trees. In addition, information on tree size and growth habit is important to evaluate a cultivar’s suitability for trellising or high density planting.

The pomegranate fruit is often classified as a berry , or it is considered berry-like , having a “leathery” exocarp that ranges in color from light yellow to black , although in the market, pomegranate fruit are typically a bright red color. Peel texture can range from soft to tough. Mature fruit are spherical with a calyx at the blossom end , which closely resembles a crown. After fertilization, sepal color typically changes from orangered to green, but can vary among cultivars. As the fruitlet develops, the fruit’s color typically changes from green to the color of that genotype at maturity . Pomegranate seeds are arranged in locules that are separated by white to yellowish septa, inedible mesocarp tissue , which are not evenly distributed in the fruit . The septa are inedible because they are comprised of insoluble fiber . Locules at the stem end are generally more numerous than those at the calyx end . The epidermis of each seed is fleshy and is botanically referred to as a sarcotesta or aril. The flavor and color of the sarcotesta is cultivar-dependent, ranging from sour to sweet and white to deep purple, respectively. The hardness of the seeds, amount of juice contained in the arils, and aril size are also cultivar dependent . A large pomegranate fruit can contain up to 1200 to 1300 seeds . Pomegranate is a non-climacteric fruit, meaning that it matures and does not ripen . Differences in seed number may affect a cultivar’s ability to be successful in the prepackaged aril market, which has developed considerably over the last five years. Studies have demonstrated a large variation in mature fruit size within commercial orchards of ‘Wonderful,’ which poses a problem for fresh market growers and packers. Wetzstein et al. reported a greater than five-fold range in mature fruit volume and mass in commercial ‘Wonderful’ pomegranate groves. At maturity, pomegranate fruit are typically 3.5 to 6.5 cm in diameter with a mass of 30 g to several hundred grams , although commercially-valuable pomegranates typically weigh more than 400 g and the largest-sized ‘Wonderful’ fruit from mature trees can have a diameter of over 100 mm . Factors that influence fruit size and yield include aril number , cultivar , cultural practices , and tree age . Cultivars that show greater uniformity in fruit size would be beneficial to the industry due to the fresh market sizing problems reported for Wonderful by Wetzstein et al. . Typical commercial harvest windows for ‘Wonderful’ range from late September to early November, but fruit in the San Joaquin Valley of California, USA, where the most Punica cultivation occurs, are typically ready to harvest starting in late October. Usually by November, the effects of weather and pests will begin to take a toll on mature fruit. Harvest date can determine whether a cultivar is a candidate as a commercial cultivar. Cultivars with an earlier fruit maturity date have a better chance of competing with Wonderful in the market. Later cultivars run the risk of fall rains, which have been associated with greater numbers of split fruit and a loss in fresh market sales . The beverage and wine industries utilize fruit juices that have sufficient quantities of organic acids, carbohydrates , and phenolic compounds. For commercial pomegranate fruit, concentrations of total soluble solids range from 12% to 16% at maturity, but ‘Wonderful’ should have at least 15% TSS at harvest . Hasnaoui et al. reported that citric acid is the determinant of tart flavor in pomegranate juice, and this is said to be independent of the sugar concentration.