QTL Mapping And Candidate Gene Analysis Of Three Seed-related Traits In Soybean

Soybean is one of the most economically important crops being rich in edible oil and protein.The economic value and utilization of soybean is determined by seed and its related traits such as seed weight(SW),seed protein content(SPC),seed oil content(SOC)and others.These important agronomic traits are quantitative traits controlled by polygenes/quantitative trait loci(QTL)with major/minor effects,and influenced by the environment.Identification of QTL/genes underlying these agronomic traits is the foundation for molecular breeding.The genetic architecture of a trait of interest significantly influences which breeding strategy that will be most effective.Therefore,the present studies were conducted to elucidate genetic architecture of SW,SPC and SOC in diverse soybean germplasm populations.1 Genetic Architecture of Seed Weight Revealed by Linkage and Association MappingThe linkage mapping was conducted with an inter-specific RIL population(NJIR4P)derived from a cross between Nannong493-1(G.max)and PI342618B(G.soja).SW mainQTL mapping with NJIR4P RIL population was conducted in WinQTLCart 2.5 with composite interval mapping(CIM)model.A total of 19 SW QTL distributed on 12 chromosomes were identified in all individual environments and combined environment by linkage mapping in NJIR4P.Out of these,six were novel whereas eight were stable,thus,identified in more than one environments.Chromosome(Chr)17 harbored greatest number of 4 QTL(qSW-17-1,qSW-17-2,qSW-17-3&qSW-17-4)with phenotypic variation(PV/R2)and logarithm of odd(LOD)of 7.07-13.20%and 3.05-7.28,respectively.These major QTL coincided with soybean domestication related genomic loci including seed weight The remaining 15 QTL were minors distributed across 8 different chromosomes.Thirty-one probable candidate genes were predicted including Glyma02g13210 which has been demonstrated to regulate SW with its paralog genes in soybean.On other hand,multiple models of genome-wide association study(GWAS)mapping were conducted with Yangtze-Huai Soybean Breeding Line Population(YHSBLP)with 573 lines and 61166 single nucleotide polymorphisms.In all,two hundred and thirtyone quantitative trait nucleotides(QTNs)were identified to regulate SW in YHSBLP with varied effect.Out of these,seventeen SNPs were concurrently detected by both single-locus models and multi-locus models on 8 chromosomes.Ninety-four QTNs co-localized with previously detected QTL/QTN by linkage/association mapping studies.A total of 36 candidate genes were predicted.Out of the predicted candidate genes,4 hub-genes(Glyma06g44510,Glyma08g06420,Glyma12g33280 and Glyma19g28070)were identified by the integration of co-expression network2 Linkage and Association Mapping Strategies Revealed Complex Genetic Architecture of Seed Protein and Oil Contents in SoybeanA total of 44 main-effect QTL for SPC and SOC were found on 17 chromosomes by CIM model in LM6 RIL population.Out of these QTL,four were major and stable,viz.,qPro-7-l,qOil-8-3,qOil-10-2 and qOil-10-4,detected in at least two environments plus combined environment with average R2 values>10%.Three protein QTL(qPro-10-2,qPro-16-1&qPro-20-1)were mapped within the same genomic and physical positions of three oil QTL(qOil-10-4,qOil-16-1&qOil-20-1,respectively)exhibiting pleiotropic effect/linkage drag on both traits.Based on RNA sequencing(RNA-seq)data analyses,fifteen genes were highly expressed during seed development stage and root nodules,hence,they may be potential candidate genes.Application of the 2 models of GWAS(MLM and mrMLM)detected 96 and 118 QTNs for SPC and SOC,respectively,across the 20 chromosomes in YHSBLP.Of the total QTNs,forty-nine exhibited pleotropic effect on both traits on Chr11,Chr12,Chr16 and Chr20.The first three chromosomes contain 1 QTN each whereas Chr20 contain 46.Most of the genomic regions identified for SPC and SOC on Chr20 were detected consistently across the 4 environments.The SNPs detected for both traits simultaneously indicate the possibility of marker-assisted selection(MAS)aimed at decreasing the strong negative correlation between the two economically important traits.A number of hub-genes on consistently detected loci:Chr20:26596099-32075119 bp for both traits and Chr05:37632148-3971054 bp for SOC were identified for further screening and functional validation.3 Genome-wide Analysis of Phospholipases A Superfamily and Function of Three Members in Soybean.Genomic analysis is an efective means of transferring knowledge from one taxonomy to another.In-silico identification of Phospholipase A(PLA)superfamily genes were conducted.One hundred and twelve genes PLA superfamily genes were identified consisting of 78,29 and 5 genes in PLA1,pPLA and sPLA2 sub-class.Thirty seven(37)representing 33.04%of the 112 genes identified had undergone tandem duplication events on Chr01,Chr02,Chr06,Chr09,Chr14,Chr17 and Chr18.With the exception of genes identified on Chr12 and Chr13,slightly above 56%of the genes on the other chromosomes had also undergone segmental duplication events.Nine hormone-related Cis-acting regulatory elements(CAREs)(ABRE,AuxRR-core,TGA-element,CGTCA and TGACG motifs,ERE,GARE-motif,P-box,TATC-box and TCA-element)plus 4 stress-related CAREs(TC-rich repeats,ARE,MBS and LTR)and several light and seed-specific elements were detected within 1.5kb upstream of ATG of the identified genes.CRISPR/Cas9 technology revealed potential roles of GmpPLA-??,GmpPLA-?? and GmpPLA-?? in regulating soybean response in flooding,phosphorus(P)and iron(Fe)deficient conditions.Six mutants(dbt-66-2-2,dbt-55-1-2,dbt-76-1-2,dbt-36-2-1,dbt-72-51 and dbt-60-2-4)obtained from GmpPLA-?? and GmpPLA-?? plus 2 genotypes from GmpPLA-??(B58 and B59)would be useful in unraveling molecular mechanism underlying the abiotic stresses and seed-related traits in soybean.The overall results indicate complex architecture of three seed quality traits which buttress the need to molecular breeding strategies such as MAS.This could be achieved by pyramiding/stacking of the stable QTL(qPro-7-1,qOil-10-2,qSW-17-1&qSW-17-4)plus numerous stable QTNs into an elite cultivar to improve these traits.Also,the homozygotes mutants obtained from this study are recommended for future study to evaluate effect of the knock-out on seed-related traits as well as other agronomic traits of economic importance.