Elroy R. Cober

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.

Regulation of seed yield and agronomic characters by photoperiod sensitivity and growth habit genes in soybean

Soybean genotypes are adapted to narrow bands of latitude due to photoperiod sensitivity. There are several photoperiod-sensitive loci (E1, E2, E3, E4, E5, E6, E7, E8). Determinate and indeterminate growth habits are controlled by a single locus. The objective of our research was to examine the effects of photoperiod sensitivity and growth habit alleles on seed yield and other agronomic characters using isogenic lines. Twenty ‘Harosoy’ isolines with 11 photoperiod-sensitive genotypes many with both indeterminate and determinate growth habits were grown in the field at Ottawa, ON, from 2003 to 2007. Maturity ranged from 97 to 127 days, and seed yield increased linearly with maturity until about 112 days when it plateaued. Determinate lines were always shorter than indeterminate lines of equivalent maturity. Seed yield was associated with plant height, maturity, seed sugar concentration, seed weight and lodging. Effects of alleles at individual loci, and additive and epistatic effects across multiple loci were examined. At a single locus, photoperiod-insensitive alleles produced isolines that matured 8–11 days earlier, yielded less, and had shorter plants with reduced lodging. In multiple loci analyses, additive effects explained most of the variation in agronomic characters since additive models with E1, E3, E4, E7 and Dt1 loci included compared well to additive plus epistatic models and genotype-based models. Variation in photoperiod sensitivity and growth habit alleles results in a range of maturity, with pleiotropic effects on seed yield and agronomic characteristics, and play an important role in providing adaptation across latitudes.

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.

Relationship between asparagine metabolism and protein concentration in soybean seed

The relationship between asparagine metabolism and protein concentration was investigated in soybean seed. Phenotyping of a population of recombinant inbred lines adapted to Illinois confirmed a positive correlation between free asparagine levels in developing seeds and protein concentration at maturity. Analysis of a second population of recombinant inbred lines adapted to Ontario associated the elevated free asparagine trait with two of four quantitative trait loci determining population variation for protein concentration, including a major one on chromosome 20 (linkage group I) which has been reported in multiple populations. In the seed coat, levels of asparagine synthetase were high at 50 mg and progressively declined until 150 mg seed weight, suggesting that nitrogenous assimilates are pre-conditioned at early developmental stages to enable a high concentration of asparagine in the embryo. The levels of asparaginase B1 showed an opposite pattern, being low at 50 mg and progressively increased until 150 mg, coinciding with an active phase of storage reserve accumulation. In a pair of genetically related cultivars, ∼2-fold higher levels of asparaginase B1 protein and activity in seed coat, were associated with high protein concentration, reflecting enhanced flux of nitrogen. Transcript expression analyses attributed this difference to a specific asparaginase gene, ASPGB1a. These results contribute to our understanding of the processes determining protein concentration in soybean seed.

Natural variation at the soybean J locus improves adaptation to the tropics and enhances yield

Soybean is a major legume crop originating in temperate regions, and photoperiod responsiveness is a key factor in its latitudinal adaptation. Varieties from temperate regions introduced to lower latitudes mature early and have extremely low grain yields. Introduction of the long-juvenile (LJ) trait extends the vegetative phase and improves yield under short-day conditions, thereby enabling expansion of cultivation in tropical regions. Here we report the cloning and characterization of J, the major classical locus conferring the LJ trait, and identify J as the ortholog of Arabidopsis thaliana EARLY FLOWERING 3 (ELF3). J depends genetically on the legume-specific flowering repressor E1, and J protein physically associates with the E1 promoter to downregulate its transcription, relieving repression of two important FLOWERING LOCUS T (FT) genes and promoting flowering under short days. Our findings identify an important new component in flowering-time control in soybean and provide new insight into soybean adaptation to tropical regions.

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.

Molecular identification of genes controlling flowering time, maturity, and photoperiod response in soybean

Most plants activate the developmental transition from the vegetative to the reproductive phase in response to photoperiod length, temperature, and other environmental stimuli. Successful identification of major genes underlying flowering time and maturity in soybean is a prerequisite for understanding of the regulation of flowering time. Recent progress has been made toward molecular bases of soybean maturity loci by using both candidate gene and positional cloning approaches. In particular, successful identification of the molecular identity of the soybean maturity locus E1 is a remarkable achievement, because this gene is essential for understanding the regulation of flowering time and maturity in soybean. The E1 gene has a putative bipartite nuclear localization signal, and a domain distantly related to B3. Transcriptional profiling showed the E1 gene is under photoperiodic regulation. The E2 gene in soybean encodes GmGIa, a homolog of Arabidopsis GIGANTEA that has multiple functions involved in the circadian clock and flowering. Both of the E3 and E4 genes encode copies of PHYTOCHROME A proteins, and both genes response differentially to light with different red to far-red quantum (R:FR) ratios. In addition, two homologs (GmFT2a and GmFT5a) of FLOWERING LOCUS T coordinately promote photoperiodic flowering in soybean. Public availability of the soybean genome sequence to the research community will greatly facilitate fine mapping and cloning of more genes underlying flowering time and photoperiodic response. Further research on identified genes will help us to understand the exquisite regulatory network of parallel and intertwining pathways controlling flowering time and photoperiodic response in soybean.

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.

Evaluation of the pathogenicity of Fusarium graminearum and Fusarium pseudograminearum on soybean seedlings under controlled conditions

Fusarium graminearum, the cause of fusarium head blight of small-grain cereals and of gibberella ear rot of corn, has recently been reported to attack soybean, causing root rot and pod blight. A morphologically similar species, Fusarium pseudograminearum, is also an important pathogen of the roots and crown of cereals, but its pathogenicity against soybean has not been tested yet. Pathogenicity tests were conducted under controlled conditions to compare aggressiveness within and between isolates of F. graminearum and F. pseudograminearum in causing root rot of soybean. In each experiment, agar plugs (2 mm in diameter) from 7-day-old fungal cultures were placed on the primary root at the VE (vegetative emergence) growth stage. Twenty seedlings of each of three soybean cultivars were inoculated separately with six isolates each of the two Fusarium spp. All isolates caused visible infection 10 d after inoculation, but F. graminearum isolates were significantly more aggressive than those of F. pseudograminearum. The two species caused average disease severities of 2.3 and 1.1, reduced shoot lengths by 28.9% and 6.9%, and reduced plant dry masses by 12.8% and 7.9%, respectively. Significant differences (P < 0.05) were found among cultivars for all parameters and among isolates of F. graminearum in disease severity and shoot length, but there were no significant cultivar × isolate interactions. Further research is needed to verify, in soybean, genotypic difference and possible resistance to the two species.

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.