Different execution strategies also make it difficult to compare validation results between papers. Additionally, there remain some problems with the RWR method, particularly its reliance on the known gene distribution, which is unlikely to reflect the true distribution of genes associated with the trait. It is also apparent that even with the bidirectional extension of QTL there remains a tendency to over represent the centers of chromosomes. Given the central importance of gene distribution to RWR, addressing this particular failing will produce a method that is much more effective at identifying QTL of interest, and will therefore improve the rate at which breeding and fine mapping can be accomplished. The probability associated with the selection of a particular marker, P, as the origin for a particular QTL is calculated by assessing the number of locations in the genome on which the QTL can be placed. P is determined by the number of markers that can be used as the origin O of the QTL of length L, a number which excludes all markers that would result in the QTL extending beyond the end of the chromosome. This origin-based model of QTL mapping is an approximation for the true process of QTL placement, wherein QTL are roughly centered on a marker and are terminated at a marker on either end which represent the 95% confidence intervals for that QTL. In the case where the original QTL were not terminated at markers, it is preferable to use a model in which the QTL is centered on the chosen marker. Models requiring that the QTL must both start and end on a marker are not feasible, vertical farming aeroponics because the distance between markers is not uniform; with this constraint, a QTL of a given length might have only one possible genomic location. Markers are used in a direction-independent manner to avoid under representation of the ends of the chromosomes.

Under the null hypothesis, the probability of using any marker is {0,1,2}/M, which can take on any value between 0 and 1, inclusive. Here, the numerator depends on whether the marker can serve as O with unidirectional QTL extension, bidirectional QTL extension, or with neither; M refers to the total number of markers from which the QTL can extend to the left plus the total number of markers from which the QTL can extend to the right . Although the denominator could be adjusted to take into account the fact that a single QTL can only be mapped to one chromosome, this is unnecessary, because a QTL is not equally likely to be mapped to every chromosome. To understand why, consider the most obvious strategy to account for differences in chromosome length and the number of usable markers: a weighting scheme. We would divide the number of usable markers on each chromosome by the total number of usable markers in the genome . If this weighting is then used to adjust the probability P of using any marker on that chromosome, which is already proportional to 1/MC, the chromosome-specific marker counts cancel out and leave only the whole-genome marker count. To best represent the topology of the genome, the SPQV simulates genetic loci by selecting genes at random from the whole genome gene distribution to represent the genetic basis of the trait of interest. The use of the whole genome gene distribution as a source accounts for the topology of the genome, including the decrease of gene density at the centromere and telomeres . This strategy assumes that the true distribution of genes associated with a particular trait is approximately the same as the distribution of genes on a whole, rather than the assumption used in the simplest instance of RWR: that the distribution of known, previously associated genes reflects the true distribution of genes associated with that trait. We argue that this novel assumption is more likely to be accurate because trait-related genes can easily be discovered in a spatially biased manner : tandem arrays promote clustered discovery, some transposons involved in transposon-mediated mutagenesis preferentially target certain sequences , and genes in regions close to the centromere tend to be difficult to identify through methods that rely on recombination .

Additionally, the genome is interconnected; many traits rely on the interaction between multiple, seemingly disparate biological processes. Use of a random distribution for simulating genes with the SPQV is also possible, but fails to capture the genomic topography. Because genes within functional groups are not randomly arranged, duplication events and gene clusters in the original set of known genes are taken into account by considering genes without a marker between them as one genetic unit. The SPQV values are clearly most similar to those produced by the RWR experiment that was closest to biological reality: marker-only QTL origins, bidirectional mapping, and no bounce back . This makes sense, as the SPQV method is designed as a smoothed version of an experiment with these characteristics. Restriction of QTL origin to the markers that were used in mapping leads to an increase in EGN for RWR . This effect occurs for all QTL lengths. It is likely that the increase of identified genes in the context of restricted QTL placement is attributable to the physical structure of chromosomes: the markers selected for QTL mapping have a similar distribution to the genome wide distribution of genes , and are therefore relatively sparse in gene-poor regions such as the centromere. Similarly, the use of bounce back leads to an increase in RWR identified genes for all lengths of QTL, though this increase is particularly noticeable for some of the larger QTL . It is likely that the relatively large number of genes situated close to the ends of chromosomes is the main contributor to the impact of bounce back on identified gene number. It is possible that this reduction is due to a smoothing of the distribution, as the occurrence of bounce back is effectively split in half over the two separate tails of the chromosome. The presence of long and short arms on chromosomes, and the corresponding lopsidedness of the gene distribution, might also contribute to this phenomenon .

The use of bidirectional mapping appears to result in fewer genes identified by RWR, though this effect is relatively minor when compared to the effects of origin restriction and bounce back. In spite of the prominence of dark colors on the left side of the heat map, the confidence intervals identified for small and medium QTL by the SPQV and by RWR are fairly similar regardless of RWR method . The CIs in this range were consistently far below 1 regardless of method. In all, the SPQV 95% confidence limit for small QTL tends to be slightly smaller than the one produced by the RWR method that takes the same biological realities into account . However, this makes little difference in practice, because they are both less than 1: because observed gene counts are integers, EGNs from either method will be rounded up . In other words, if an SPQV confidence limit is defined as 1.2, the QTL of interest must have an observed gene content of 2 or more genes to be considered significant during general use. For larger QTL, SPQV values tend to outsize those produced by RWR. These large QTL approach the size of a full chromosome, and can indeed be larger than several chromosomes within the S. italica genome. It is the authors’ opinion that this is not an overestimation for the true distribution of genes associated with the trait of interest, as the true distribution likely has more than the known number of genes. Because of this, significance is unlikely for very large QTL,vertical indoor hydroponic system except for in the case of a true distribution of genes that is extremely uneven at the chromosomal level. A reduction in tiller number is a classical domestication trait in maize . Modern maize lines have been bred to grow as single stalked plants to facilitate high-density planting, while the maize progenitor, teosinte, is highly tillered. The genetic network associated with tiller suppression is controlled by the teosinte branched1 gene that also controls several other aspects of maize morphology , including inflorescence and floral architecture. Several mapping populations made from crosses between the W22 maize inbred line and teosinte were recently described and used to map several domestication traits . As expected, several domestication traits associate tightly with tb1 pathway. Here, we use the QTL reported by Chen et al. 2019 to illustrate the utility of the SPQV. Only the QTL with the same effect direction in both maize/teosinte mapping populations were assessed. Seven genes closely associated with the tb1 pathway in maize were located in the Zm-W22 NRGene 2.0 assembly and analyzed using SPQV. Notably, these genes were selected based on their strong, known associations with the branching pathway in maize. Since only high-confidence genes can be used accurately with our method, if any gene were not truly associated with the trait of interest, its presence will render the SPQV more stringent than necessary.

The QTL identified for the traits BARE , EB , GLUM , KRN , STAM and TILN have previously been connected with the tb1 pathway in maize. These QTL were therefore assessed in relation to the seven genes in Table 1. The markers found in the W22 x TIL01 RIL sub-population were used to determine the base pairs associated with the CIs of these QTL. Where the end points of the QTL did not have an exact match to a marker, the next closest marker was used so as to mimic ‘extension’ style mapping. The results of this analysis are reported in Table 2-2. Four of the assayed QTL identified a gene from the tb1 pathway, corresponding to four out of six of the represented traits. If the various adjustments described in this paper are applied to the RWR-based assessment of QTL mapping experiments, a more apt confidence limit for the expected number of genes will be identified. These adjustments do not account, however, for all of the issues associated with the application of RWR to this particular variety of question. RWR not only continues to rely on the distribution of known genes, but also results in gene-count distributions that nearly always fail to meet the requirement for smoothness . These distributions, in other words, have a tendency to change abruptly, and are frequently binary in the case of small and very large QTL. The ‘unsmoothness’ of any given distribution will be exaggerated by small QTL size and short lists of known genes; a known gene list with fewer than one gene per chromosome, for example, would produce a binary distribution even for large QTL. Additionally, RWR continues to exhibit a reduced likelihood of the QTL falling in the regions [1,1+L] and [C–L, C] even with the adjustment for bidirectional QTL extension. Finally, a practical weakness of the application of considered RWR is that this procedure requires a great deal of thought, effort, and expertise, and there are many points in the procedure at which simple errors can produce dramatic changes in the confidence limits that are ultimately produced. In light of the flaws of naive RWR, and the complexity of making the suggested adjustments, we recommend using the SPQV to assess the quality of QTL mapping experiments. The function provided, SPQValidate, requires only a few lists of data; the function itself accomplishes the analytic work that might be a stumbling block in RWR. Many of the other problems inherent to RWR are overcome by the SPQV’s probabilistic nature. This tool is potentially overly conservative, however, in the case of short QTL. It is extremely unlikely that the SPQV will produce a value of 0 for the confidence limit, as any locus is likely to be within range of at least one marker for even the shortest identified QTL. Because of this, the minimum confidence limit is, in practice, 1, which might be misleading for small QTL. Additionally, the total number of genes in a QTL is not necessarily an authoritative measure of a QTL’s validity; one can imagine that a QTL located on a single gene of high impact might be considered non-significant if the SPQV is the only method of validation used.

As of July 2012, the GENERA database listed 583 scientific studies on the safety of GMO crops and their food ingredients. In addition, the experiential evidence of billions of meals consumed by persons around the world since commercial release of genetically-engineered crops in 1996 supports the safety of genetically-modified foods. Since 1996, there has not been one verified health complaint to humans, animals or plants from genetically-engineered crops, raw foods, or processed foods. Despite some published attempts to deny this overwhelming scientific evidence in support of genetically engineered foods, the scientific consensus is clear —genetically-engineered crops, foods, and processed ingredients do not present health and safety concerns for humans, animals, or plants. SPS Agreement Article 3 sets forth provisions that could save Proposition 37. Paragraph 3.2 affirms a SPS measure that conforms to international standards relating to health and safety. However, Paragraph 3.2 does not protect Proposition 37 because there are no international standards that categorize genetically-engineered raw or processed foods as unsafe or unhealthy. Comparing Proposition 37 to the legal standards in the SPS Agreement shows that Proposition 37 almost assuredly is not compliant with the SPS Agreement. Indeed, the WTO SPS claim against Proposition 37 is so strong that its proponents are probably not going to defend it as meeting the legal standards of the SPS Agreement. Despite its textual language and the electoral advertising emphasizing food safety and health concerns,vertical farming aeroponics proponents will argue that Proposition 37 cannot properly be characterized as a labeling requirement “directly related to food safety.” Proponents of Proposition 37 will seek to have it classified as a technical barrier to trade in order to avoid the SPS Agreement and its scientific evidence standards.

The TBT Agreement applies to technical regulations, including “marking or labelling requirements as they apply to a product, process or production method.” As Proposition 37 imposes mandatory labels, Proposition 37 is a technical regulation under the TBT definitions. TBT Article 2 sets forth several provisions against which to measure technical regulations for compliance with the TBT Agreement. It states, “Members shall ensure that technical regulations are not prepared, adopted or applied with a view to or with the effect of creating unnecessary obstacles to international trade. For this purpose, technical regulations shall not be more trade-restrictive than necessary to fulfill a legitimate objective, taking account of the risks non-fulfillment would create. Such legitimate objectives are, inter alia, … the prevention of deceptive practices; protection of human health or safety, animal or plant life or health, or the environment. …” Article 2.2 expressly lists three legitimate objectives: national security requirements; protection of human health or safety, animal or plant life or health, or the environment; and prevention of deceptive practices. As for health and safety, Proposition 37 does not provide a label giving consumers information about how to use a product safely or a safe consumption level or any other health and safety data—unless the warning-style label against genetically-modified food itself is considered a valid warning. But, as discussed with regard to the SPS Agreement, there is no scientific evidence available to indicate that genetically modified foods have negative health or safety implications for humans, animals, or the environment. Proposition 37 does not assert a legitimate health and safety objective under TBT Article 2.2.Proposition 37 can be defended as upholding the third legitimate objective—prevention of deceptive practices. Indeed, the Proposition is titled the “California Right to Know Genetically Engineered Food Act,” indicating that labels will assist California consumers in knowing what they are purchasing and avoiding purchases that they desire to avoid. Those who would challenge Proposition 37 for noncompliance with the TBT Article 2.2 will argue that Proposition 37 is not a protection against deceptive practices. Opponents can point to the structure of the proposed Act and its exemptions to provide evidence that Proposition 37 will actually confuse consumers more than inform them accurately.

Proposition 37 exempts foods that lawfully have the USDA Organic label. Under the USDA National Organic Program , organic foods can contain traces of unintentional genetically-modified crops or ingredients without losing the organic label. Simultaneously, those California consumers still will be eating unlabeled food products containing genetically modified crops or ingredients at trace levels, except those products will carry the label “USDA Organic.” In other words, opponents of Proposition 37 will argue that Proposition 37 is itself the deceptive labeling practice and, thus, fails to promote a legitimate objective under TBT Article 2.2. Proponents of Proposition 37 will respond by citing to the recent WTO Dispute Resolution Appellate Body relating to the challenge of Canada and Mexico against the United States country-of-origin label for meat. The WTO Panel ruled against COOL on the grounds of a violation of TBT Article 2.2 because the COOL law would confuse consumers. But the WTO Appellate Body reversed this Panel ruling and determined that COOL did provide information as a legitimate objective under Article 2.2.Aside from “legitimate objectives,” TBT Article 2.2 also requires that technical regulations not be “unnecessary obstacles to international trade” and “not more trade-restrictive than necessary.” Opponents of Proposition 37 will argue that it violates these TBT obligations primarily because consumers already have labels that provide the same level of consumer protection from deception. Opponents will point to the existence of the Non-GMO label and the USDA-Organic label that allow consumers to choose foods which will have minimal levels of genetically-engineered content. These Non-GMO and USDA-Organic labels are voluntary labels that do not impose legal and commercial burdens upon other food products in international trade. TBT Article 2.1 also provides a standard against which to measure Proposition 37 by stating, “Members shall ensure in respect of technical requirements, products imported from the territory of any Member shall be accorded treatment no less favorable than that accorded like products of national origin and to like products originating in any other country.”TBT Article 2.1 requires Members to treat “like products” alike and to refrain from favoring either domestic or other international “like products” as against the products of the Member bringing the Article 2.1 complaint.

Obviously, proponents of Proposition 37 consider genetically-engineered agricultural products as fundamentally different than organic and conventional agricultural products. Proponents will argue that Proposition 37 deals with genetically-engineered agricultural products that constitute a class of products of their own.Opponents of Proposition 37 will respond with two arguments. Opponents can argue that regulatory agencies around the world have considered genetically-engineered raw agricultural products to be substantially equivalent in every regard to conventional and organic agricultural products. Opponents will argue that the substantive qualities of genetically-engineered agricultural products are “like products” and that the process producing the “like products” does not create a separate product classification. Opponents will argue “product” over “process” as the appropriate TBT Article 2.1 interpretation. Opponents of Proposition 37 will also present a second argument. More precisely, opponents of Proposition 37 will highlight the fact that Proposition 37 imposes labels, testing, and papertrail tracing on vegetable oils even though the oil has no DNA remnants of the crop from which the oil came. Soybean oil is soybean oil regardless of what variety of soybean the food processor crushed to produce the oil. With regard to the TBT Article 2.1 arguments,vertical indoor hydroponic system opponents of Proposition 37 may gain support from the Canada and Mexico WTO complaints against the U.S. COOL law. Both the WTO Panel and the WTO Appellate Body determined that Canadian and Mexican meat was a “like product” to United States meat. As a “like product,” the WTO reports ruled that the U.S. COOL law violated TBT Article 2.1 by imposing discriminatory costs and burdens on meat imported into the United States.TBT Articles 2.4 and 2.5 provide a safe harbor for technical regulations if those technical regulations adopt international standards. However, the Codex Alimentarius Commission, the international standards body for food labels, has not created an international standard which proponents of Proposition 37 can claim as its origin and safe harbor.SPS Agreement Article 11 and TBT Agreement Article 14 are both titled “Consultation and Dispute Settlement.” Thereby the SPS Agreement and the TBT Agreement make explicit that Member States to these agreements can complain using the WTO Dispute Settlement Understanding Agreement. For example, Argentina or Brazil or Canada—all likely to be affected by Proposition 37 for the export of soybeans and canola, especially for cooking oils—have the treaty right to file a complaint within the WTO dispute resolution system. Bringing a WTO complaint is fraught with difficulties. Members must think politically and diplomatically about whether it is worthwhile to bring a complaint—even a clearly valid complaint. Members must be willing to expend significant resources in preparing, filing, and arguing WTO complaints. Finally, even if a Member prevails in the Panel or Appellate Body reports, Members recognizes that its WTO remedies are indirect and possibly not fully satisfactory. Although the United States is a Member of the WTO Agreements, the United States, in contrast to Argentina, Brazil and Canada, is not an exporting Member to California.

Consequently, the United States cannot file a WTO complaint invoking the DUS Agreement against California. But by being a Member of the WTO Agreements, the United States has ratified these treaties as part of the law of the United States, transforming these treaties into the supreme law of the land under the U.S. constitution. Moreover, under the WTO Agreements, the United States has the duty to ensure that local governments comply with the WTO Agreements. Therefore, the United States has the legal authority to challenge Proposition 37 in order to protect its supreme law of the land and to avoid violating its WTO obligations.Opponents of Proposition 37 are likely to challenge Proposition 37 immediately if California voters adopt it in November 2012. As indicated in the introduction, these opponents are likely to bring challenges on three different grounds under the U.S. Constitution. These opponents have non-frivolous grounds upon which to pursue these U.S. constitutional challenges. Whether these opponents can add a claim challenging Proposition 37 based on alleged violations of the SPS Agreement or the TBT Agreement is much less clear. TBT Agreement Article 14.4 highlights that the opponents will have difficulty in bringing a WTO-based challenge. TBT Article 14.4 makes clear is that Member States have the legal status to bring WTO-based complaints.Proponents of Proposition 37 will challenge the standing of those opponents who seek to challenge Proposition 37. Proponents will seek to have this WTO-based claim dismissed because the opponents do not have a right to make a legal claim based on the WTO. Proponents will argue that standing to bring a WTO-based claim resides solely in exporting Member States or the United States. By contrast, opponents bringing the immediate challenge containing a WTO-based claim will argue that they are not invoking the WTO Agreements directly. Opponents will argue that they are challenging Proposition 37 to enforce the supreme law of the United States. By invoking the supreme law of the United States, opponents will hope to blunt the standing issue and to avoid dismissal of the WTO-based claim.Assuming that the United States does not file a lawsuit against California and that other opponents are blocked, by the doctrine of standing, from raising WTO-based challenges, Proposition 37, if adopted in November 2012, would become California law. Thus, the first lawsuits related to Proposition 37 would come through either administrative action or a consumer lawsuit against food companies and grocery stores alleging failure to label or misbranding. When facing administrative actions or consumer lawsuits, food companies and grocery stores will want to respond with all possible legal challenges to Proposition 37. Food companies and grocery stores will want to raise the issues of whether Proposition 37 complies with the SPS Agreement and the TBT Agreement as defenses to being found liable for administrative penalties or consumer damages. The agency or consumer bringing the lawsuit against the food company or grocery store will argue that the food company or grocery store does not have standing to raise the WTO-based challenges. The plaintiff likely has to concede that the defendant faces an actual injury. However, the plaintiff will contest vigorously that the defendant is not within the zone of interests that the WTO Agreements mean to protect. In other words, the plaintiff will argue that the WTO Agreements only mean to protect sovereign interests and not private commercial interests.