Istvan Rajcan

Identification of loci governing eight agronomic traits using a GBS-GWAS approach and validation by QTL mapping in soya bean

Soya bean is a major source of edible oil and protein for human consumption as well as animal feed. Understanding the genetic basis of different traits in soya bean will provide important insights for improving breeding strategies for this crop. A genome-wide association study (GWAS) was conducted to accelerate molecular breeding for the improvement of agronomic traits in soya bean. A genotyping-by-sequencing (GBS) approach was used to provide dense genome-wide marker coverage (>47,000 SNPs) for a panel of 304 short-season soya bean lines. A subset of 139 lines, representative of the diversity among these, was characterized phenotypically for eight traits under six environments (3 sites × 2 years). Marker coverage proved sufficient to ensure highly significant associations between the genes known to control simple traits (flower, hilum and pubescence colour) and flanking SNPs. Between one and eight genomic loci associated with more complex traits (maturity, plant height, seed weight, seed oil and protein) were also identified. Importantly, most of these GWAS loci were located within genomic regions identified by previously reported quantitative trait locus (QTL) for these traits. In some cases, the reported QTLs were also successfully validated by additional QTL mapping in a biparental population. This study demonstrates that integrating GBS and GWAS can be used as a powerful complementary approach to classical biparental mapping for dissecting complex traits in soya bean.

Prediction of cultivar performance based on single- versus multiple-year tests in soybean

based on multiple-location trials across multiple years. Surprisingly, the hypothesis that multiple-year data give Because of the omnipresent genotype year or genotype better prediction of the next year’s performance has location year interactions in crop performance trials, it is commonly been tested by only a few researchers (Cross and Helm, believed that multiple-year data should be used in selecting cultivars for the next year. An implicated but rarely tested hypothesis is that 1986; Gellner, 1989; Bowman, 1998). On the other hand, multiple-year data are more predictive than single-year data of cultivar it is not feasible for researchers to make decisions on performance in the next year. Yield data of the 1991 to 2000 Ontario the basis of many years of testing because few genotypes Soybean Variety Trials in the 2800 Crop Heat Unit (CHU) area were (except the checks) are tested in many years and many used to study the power of single-year, multiple-location trials in genotypes are withdrawn from the trials if they do not predicting cultivar performances in the following year, and to see if perform well in the first year. data from multiple-year trials are more predictive. Mixed models were This project was set up to address the following quesused to estimate best linear unbiased predictions (BLUP) of tested tions: (i) what is the predictive power of single-year genotypes on the basis of singleor multiple-year trials, and the t-stamultiple-location trials in cultivar selection; (ii) are data tistic of BLUP (tBLUP) was used as a measure of cultivar perforfrom multiple-year trials more predictive than those mance. Results indicated that a single-year, multiple-location trial had sufficient power for identifying genotypes that would perform well from a single-year trial; and (iii) what is the merit of or poorly in the next year. Two to four years’ data gave only slightly using multiple-year data? better predictions of next-year performances than single-year data but allowed more genotypes to be evaluated conclusively. The tBLUP MATERIALS AND METHODS of genotype effects based on 2 yr of multiple-location trials should be used as a basis for soybean cultivar selection and recommendation Data Source in the 2800 CHU area of Ontario. Yield data from the 1991 to 2000 Ontario Soybean Variety Test (OSVT) in the 2800 crop heat unit (CHU) (OMAFRA, 1993) area of Ontario, Canada, were used in this study. The R performance trials are conducted annuOSVT is an official annual test conducted by the Ontario Oil ally for all major crops throughout the world to and Protein Seed Crop Committee (OOPSCC) and supported help growers select cultivars for the next year. Such a by Ontario Ministry of Agriculture and Food and the Ontario Soybean Growers. The OSVT assumes the functions of both decision would be straightforward were there no soybean [Glycine max (L.) Merr.] registration trials and pergenotype environment (GE) interaction (Gauch and formance trials and is conducted across Ontario covering cultiZobel, 1996). GE interaction is, however, almost omnivars from 2300 to 3400 CHU. The cultivars in the 2800 CHU present, which complicates the decision making. test belong approximately to relative maturity groups from GE interaction in multiple-location and multiple-year 0.4 to 1.5. The 2800 CHU trials included four test locations, trials can be dissected into genotype location interacnamely, Exeter, St. Pauls, Woodstock, and Winchester. tion (GL), genotype year interaction (GY), and genoGeographically, the first three locations are in southwestern type location year three-way interaction (GLY) Ontario, whereas Winchester is in eastern Ontario. Although (Comstock and Moll, 1963; Annicchiarico and Perenzin, there were strong crossover GL interactions each year, the 1994). Presence of GL within a single year necessitates interaction pattern varied considerably across years (Yan and Rajcan, 2002). Each year, 60 to 113 adapted cultivars or breedmultiple-location trials; presence of GY warrants multiing lines from public and/or private breeding programs were ple-year trials; and presence of GLY requires both multested. Many more entries were tested in recent years, as tiple-year and multiple-location trials. Since GE intercompared with the earlier years (Table 1). Although the same action is almost omnipresent, and as yearly variation sets of genotypes were tested at all locations within a single is typically the largest source of yield variation, it is year, the genotypes varied greatly with the year. In general, commonly believed that the greater the number of years about 50% or more of the entries were removed each year; a genotype is tested, the more reliable its evaluation and only one cultivar was tested in all 10 yr (Table 1). A total will be. An extended belief is that results based on of 526 genotypes were tested during the 10-yr period. Except more years of performance trials are more predictive for the check cultivars, which were determined by the of cultivar performance in the next year, which becomes OOPSCC, cultivar sponsors were solely responsible for the entering of their cultivars into the tests. Also, in some years, a dogma for cultivar recommendation. Thus, it is recomas a result of poor germination, data from only three locations mended in regional performance trial reports, almost without exception, that cultivar selection should be Abbreviations: BLUP, best linear unbiased prediction; CHU, crop heat units; E, environment main effect; G, genotype main effect; GE, genotype environment interaction; GL, genotype location Dep. of Plant Agriculture, Crop Sci. Bldg., Univ. of Guelph, Guelph, interaction; GLY, genotype location year interaction; GY, ON, N1G 2W1, Canada. Received 25 Feb. 2002. *Corresponding author genotype year interaction; L, location main effect; tBLUP, t-statis(irajcan@uoguelph.ca). tics of BLUP; Y, year main effect; OOPSCC, Ontario Oil and Protein Seed Crop Committee; OSVT, Ontario Soybean Variety Test. Published in Crop Sci. 43:549–555 (2003).

Detection of molecular markers associated with linolenic and erucic acid levels in spring rapeseed (Brassica napus L.)

Undesirable characteristic of rapeseed oil is a relatively high level of linolenic acid (18:3), which is easily oxidized leading to rancidity and a shortened shelf life of the oil. Previous attempts to reduce linolenic acid levels in rapeseed oil through breeding have been impaired by complex genetics and strong environmental sensitivity of this trait. Therefore, our objective was to develop molecular markers for low linolenic acid that could facilitate the breeding of low linolenic rapeseed. Bulked segregant analysis was employed to identify two RAPD markers associated with 18:3 in a doubled haploid population segregating for linolenic and erucic acid levels. Based on analysis of individual DH lines, the markers RM350 and RM574, representing two independent loci, accounted for a total of 39% of the genetic variability in this population. This marker RM350 alone accounted for 25% genetic variation for this trait with no evidence of recombination. Significant interlocus interaction found between the markers RM350 and RM574 suggested that epistasis was involved in the genetic control of 18:3 level in this population. Another marker designated as RM322, which was independent of the other two, was found significantly associated with the erucic acid level and oil content. RAPD markers identified in this study should be a useful tool for the early detection of low linolenic, or low or high erucic acid genotypes in rapeseed breeding programs based on doubled haploids.

Genetic control of soybean seed oil: II. QTL and genes that increase oil concentration without decreasing protein or with increased seed yield

Soybean [Glycine max (L.) Merrill] seed oil is the primary global source of edible oil and a major renewable and sustainable feedstock for biodiesel production. Therefore, increasing the relative oil concentration in soybean is desirable; however, that goal is complex due to the quantitative nature of the oil concentration trait and possible effects on major agronomic traits such as seed yield or protein concentration. The objectives of the present study were to study the relationship between seed oil concentration and important agronomic and seed quality traits, including seed yield, 100-seed weight, protein concentration, plant height, and days to maturity, and to identify oil quantitative trait loci (QTL) that are co-localized with the traits evaluated. A population of 203 F4:6 recombinant inbred lines, derived from a cross between moderately high oil soybean genotypes OAC Wallace and OAC Glencoe, was developed and grown across multiple environments in Ontario, Canada, in 2009 and 2010. Among the 11 QTL associated with seed oil concentration in the population, which were detected using either single-factor ANOVA or multiple QTL mapping methods, the number of QTL that were co-localized with other important traits QTL were six for protein concentration, four for seed yield, two for 100-seed weight, one for days to maturity, and one for plant height. The oil-beneficial allele of the QTL tagged by marker Sat_020 was positively associated with seed protein concentration. The oil favorable alleles of markers Satt001 and GmDGAT2B were positively correlated with seed yield. In addition, significant two-way epistatic interactions, where one of the interacting markers was solely associated with seed oil concentration, were identified for the selected traits in this study. The number of significant epistatic interactions was seven for yield, four for days to maturity, two for 100-seed weight, one for protein concentration, and one for plant height. The identified molecular markers associated with oil-related QTL in this study, which also have positive effects on other important traits such as seed yield and protein concentration, could be used in the soybean marker breeding programs aimed at developing either higher seed yield and oil concentration or higher seed protein and oil concentration per hectare. Alternatively, selecting complementary parents with greater breeding values due to positive epistatic interactions could lead to the development of higher oil soybean cultivars.

Multiple allelic forms of acetohydroxyacid synthase are responsible for herbicide resistance in Setaria viridis

In weed species, resistance to herbicides inhibiting acetohydroxyacid synthase (AHAS) is often conferred by genetic mutations at one of six codons in the AHAS gene. These mutations provide plants with various levels of resistance to different chemical classes of AHAS inhibitors. Five green foxtail [Setaria viridis (L.) Beauv.] populations were reported in Ontario with potential resistance to the AHAS-inhibiting herbicide imazethapyr. The objectives of this study were to confirm resistance, establish the resistance spectrum for each of the five populations, and determine its genetic basis. Dose response curves were generated for whole plant growth and enzyme activity, and the AHAS gene was sequenced. Resistance was confirmed by determining the resistance factor to imazethapyr in the five resistant green foxtail populations for whole plant dose response experiments (21- to 182-fold) and enzyme assays (15- to 260-fold). All five imazethapyr-resistant populations showed cross-resistance to nicosulfuron and flucarbazone while only three populations had cross-resistance to pyrithiobac. Sequence analyses revealed single base-pair mutations in the resistant populations of green foxtail. These mutations were coded for Thr, Asn, or Ile substitution at Ser653. In addition, a new mutation was found in one population that coded for an Asp substitution at Gly654. There is an agreement between the spectra of resistance observed and the type of resistance known to be conferred by these substitutions. Moreover, it indicates that, under similar selection pressure (imazethapyr), a variety of mutations can be selected for different populations, making the resistance pattern difficult to predict from herbicide exposure history.

Validation of mega-environment universal and specific QTL associated with seed yield and agronomic traits in soybeans.

The value of quantitative trait loci (QTL) is dependant on the strength of association with the traits of interest, allelic diversity at the QTL and the effect of the genetic background on the expression of the QTL. A number of recent studies have identified QTL associated with traits of interest that appear to be independent of the environment but dependant on the genetic background in which they are found. Therefore, the objective of this study was to validate universal and/or mega-environment-specific seed yield QTL that have been previously reported in an independent recombinant inbred line (RIL) population derived from the cross between an elite Chinese and Canadian parent. The population was evaluated at two field environments in China and in five environments in Canada in 2005 and 2006. Of the seven markers linked to seed yield QTL reported by our group in a previous study, four were polymorphic between the two parents. No association between seed yield and QTL was observed. The result could imply that seed yield QTL were either not stable in this particular genetic background or harboured different alleles than the ones in the original mapping population. QTLU Satt162 was associated with several agronomic traits of which lodging was validated. Both the non-adapted and adapted parent contributed favourable alleles to the progeny. Therefore, plant introductions have been validated as a source of favourable alleles that could increase the genetic variability of the soybean germplasm pool and lead to further improvements in seed yield and other agronomic traits.

QTL in mega-environments: II. Agronomic trait QTL co-localized with seed yield QTL detected in a population derived from a cross of high-yielding adapted × high-yielding exotic soybean lines

Seed yield mega-environment-universal and specific QTL (QTLU and QTLSP, respectively) linked to Satt100, Satt130, Satt162, Satt194, Satt259 Satt277 and Sat_126, have been identified in a population derived from a cross between a Chinese and a Canadian soybean [Glycine max (L.) Merrill] elite line. The variation observed in yield could be the consequence of the variation of agronomic traits. Yield-component traits have been reported in the literature, but a better understanding of their impact at the molecular level is still lacking. Therefore, the objectives of this study were to identify traits correlated with yield and to determine if the yield QTLU and QTLSP were co-localized with QTLU and QTLSP associated with an agronomic trait. A recombinant inbred line (RIL) population was developed from a cross between a high-yielding adapted Canadian and a high-yielding exotic Chinese soybean elite line. The RIL were evaluated in multiple environments in China and Canada during the period from 2004 to 2006. Four yield QTLU, tagged by markers Satt100, Satt277, Satt162 and Sat_126, were co-localized with a QTL associated with an agronomic trait, behaving as either QTLU or QTLSP for the agronomic trait. For example, the yield QTLU, tagged by marker Satt100 was associated also with 100 seed weight, pods per plant, pods per node, plant height, R1, R5, R8, oil content and protein content in all Canadian environments, but only with pods per plant, pods per node, plant height, R1, R5, R8 and oil content in two or more Chinese environments. No agronomic traits QTL were co-localized with the yield QTLU tagged by the marker Satt139 or the yield QTLSP tagged by Satt259, suggesting a physiological basis of the yield in these QTL. The results suggest that a successful introgression of crop productivity alleles from plant introductions into an adapted germplasm could be facilitated by the use of both the QTLU and QTLSP because each type of QTL contributed either directly or indirectly through yield-component traits to seed yield of RILs.

Advances in Breeding of Seed-Quality Traits in Soybean

SUMMARY Soybean is the leading oil and protein crop of the world, which is used as a source of high quality edible oil, protein, and livestock feed. Soybean seeds also contain carbohydrates, ash and a number of minor compounds with potential nutraceutical properties. For several decades, soybean breeders have focused on improving the oil quality and on increasing the protein content for both feed and human food, such as tofu. Mutation breeding and, more recently, biotechnology, have been used. Newly emerging areas of research and an improved understanding of the genetic control of nutraceutical compounds such as isoflavones, should facilitate the breeding and development of new soybean cultivars with enhanced healthy properties.

Soybean cyst nematode: Challenges and opportunities

Soybean cyst nematode (SCN) is the primary pest responsible for yield losses of Glycine max. Management of SCN remains difficult in commercial soybean production due to the length of its biological cycle, frequent changes in population virulence, and ease of spread via infested soil. Effective management relies on crop rotation in combination with resistant cultivars, which have been derived from a limited germplasm base. Breeding for SCN resistance in soybean is difficult due to the quantitative nature of the trait, genetic variation within SCN populations, time required for phenotyping experimental soybean lines, and environmental factors affecting SCN reproduction. Quantitative trait loci associated with SCN resistance have been identified on 17 of the 20 soybean linkage groups, explaining 1–91% of the total phenotypic variation. Two major resistance genes, rhg 1 and Rhg 4, have been identified on linkage groups G and A2, respectively. Several minor resistance genes have been identified, but their impo...

Using the candidate gene approach for detecting genes underlying seed oil concentration and yield in soybean

Increasing the oil concentration in soybean seeds has been given more attention in recent years because of demand for both edible oil and biodiesel production. Oil concentration in soybean is a complex quantitative trait regulated by many genes as well as environmental conditions. To identify genes governing seed oil concentration in soybean, 16 putative candidate genes of three important gene families (GPAT: acyl-CoA:sn-glycerol-3-phosphate acyltransferase, DGAT: acyl-CoA:diacylglycerol acyltransferase, and PDAT: phospholipid:diacylglycerol acyltransferase) involved in triacylglycerol (TAG) biosynthesis pathways were selected and their sequences retrieved from the soybean database (http://www.phytozome.net/soybean). Three sequence mutations were discovered in either coding or noncoding regions of three DGAT soybean isoforms when comparing the parents of a 203 recombinant inbreed line (RIL) population; OAC Wallace and OAC Glencoe. The RIL population was used to study the effects of these mutations on seed oil concentration and other important agronomic and seed composition traits, including seed yield and protein concentration across three field locations in Ontario, Canada, in 2009 and 2010. An insertion/deletion (indel) mutation in the GmDGAT2B gene in OAC Wallace was significantly associated with reduced seed oil concentration across three environments and reduced seed yield at Woodstock in 2010. A mutation in the 3′ untranslated (3′UTR) region of GmDGAT2C was associated with seed yield at Woodstock in 2009. A mutation in the intronic region of GmDGAR1B was associated with seed yield and protein concentration at Ottawa in 2010. The genes identified in this study had minor effects on either seed yield or oil concentration, which was in agreement with the quantitative nature of the traits. However, the novel gene-specific markers designed in the present study can be used in soybean breeding for marker-assisted selection aimed at increasing seed yield and oil concentration with no significant impact on seed protein concentration.