QTL Mapping And Candidate Gene Analysis For Seed Shape And Size In Soybean

Seed shape and size is an economically important trait determining the yield and quality in soybeans.Hence,developing soybean cultivars with improved seed shapes and sizes is considered as a critical objective of soybean breeding programs.However,to develop improved cultivars,it is a prerequisite to have a detailed understanding of genetic architecture,as well as a mechanism underlying the trait of interest.Both seed shape and size are complex quantitative traits,governed by multiple genes and are highly environmentally sensitive.Although,over the past decades,many QTLs related to soybean seed shape/size have been reported but not stable and confirmed,due to small-sized mapping populations and low-density genetic maps,and,hence,not implied for breeding improved seed shapes and sizes in soybean.Thus,this study aimed to evaluate variation of seed shape and size of two related recombinant inbred lines(RIL)populations(ZY and K3N)in three different environments,to identify the stable significant main-effect QTLs,"QTL Hotspots",and epistatic-effect QTLs,as well as their interactions with the environment by utilizing high-density intraspecific linkage maps;additionally,to mine possible candidate genes within the genomic region of "QTL Hotspots" and their genome wide characterization,evolutionary analysis and functional validation for soybean seed size and shape traits.1.QTL Mapping for Soybean Seed Shape and Size Traits by Using Two Related Recombinant Inbred Line PopulationsTwo related recombinant inbred lines(RIL)populations(ZY and K3N)derived through a single seed descent(SSD)method by crossing a common higher seed size parent Nannong1138-2(N)with two cultivated soybean varieties viz.,Zhengxiaodou(Z)and KeFeng 35(K3),having smaller seed size were evaluated for six seed size and shape traits.A total of 88 and 48 QTLs were detected through composite interval mapping(CIM)and mixed-model-based composite interval mapping(MCIM),respectively,and 15 QTLs were common among both methods;two of them were major(R2>10%)and novel QTLs(viz.,qSW-1-1ZN and qSLT-20-1K3N).Additionally,51 and 27 QTLs were identified for the first time through CIM and MCIM methods,respectively.Colocalization of QTLs occurred in four major QTL hotspots/clusters,viz.,"QTL Hotspot A","QTL Hotspot B","QTL Hotspot C",and "QTL Hotspot D" located on Chr06,Chr10,Chr13,and Chr20,respectively.Based on gene annotation,gene ontology(GO)enrichment,and RNA-Seq analysis,23 genes within four "QTL Hotspots" were predicted as possible candidates,regulating soybean seed size and shape.Network analyses demonstrated that 15 QTLs showed significant additive x environment(AE)effects,and 16 pairs of QTLs showing epistatic effects were also detected.However,except for three epistatic QTLs,viz.,qSL-13-3ZY,qSL-13-4ZY,and qSW-13-4ZY,all the remaining QTLs depicted no main effects.2.Genome-wide Characterization of Soybean GmFBXL Gene Family and Overexpression of GmFBXL12 GeneF-box proteins have a large family in eukaryotes and being a component of the Skp1pcullin-F-box complex,considered as critical for the measured degradation of cellular proteins.Genome-wide analysis of GmFBXLs identified 45 genes with similarity in gene structure.An analysis of a complete set of GmFBXL proteins in soybean is presented,including classification,chromosomal location,conserved motifs,and phylogenetic relationship.The phylogenetic and evolutionary analysis showed that FBXLs could be subdivided into twelve subgroups and exhibited close relationship with Arabidopsis,chickpea and Medicago.Based on the phylogenetic relationship within GmFBXLs genes variation exists in intron numbers and conserved motifs.The subcellular predictions for most of the members of GmFBXLs were primarily located in different organelles(chloroplast,nuclear region,mitochondria and cytoplasm).Besides,identification of common cis-regulatory elements showed that hormones,stress and development also influence the expression of several GmFBXLs protein-encoding genes.Moreover,we overexpressed GmFBXL12,leading to selective protein degradation and hence altering the outcome of the cellular process involved.The transcript level of wild type(WT)and two overexpression lines(OE1&OE2)were recorded with the help of qRT-PCR during the different stages of days after flowering(DAF)and leaf tissue.Results demonstrated that transcript level of GmFBXL12 increased and peaked on 15 DAF;after that,as the seeds matured it declined slightly on 42 DAF,and comparatively showed the highest expression in leaf tissue.The overexpression of GmFBXL12 in soybean plants showed early maturity with a significant difference in seed size,a higher number of pods and number of seeds per plant resulted in a yield-increased phenotype.The function and structure of GmFBXLs protein in plants are discussed in light of these results and the published information.This information could be useful for prioritization of GmFBXLs proteins for further functional validation in soybean.3.Soybean GmIAA8 Gene Regulates the Seed Size and Stress ToleranceConsidering that GmIAA8 is developmentally regulated and regulate seed size,we performed CRISPR/Cas9 genome editing in GmIAA8 mutant,trying to integrate seed size and abiotic stress tolerance traits in soybean,and also to examine whether GmIAA8 may regulate other aspects of plant growth and development.Analysis of the phenotypes and frequencies of edited genes in the first generation of transformed plants(T0)showed that the CRISPR/Cas9 system was low in inducing targeted gene editing,with the desired gene being edited in 3.5%of the transformed plants.The T2 generation of the GmIAA8 mutants featured 39.6%enhanced seed number,up to 12.6%more number of pods with slight reduction in plant height and 100-seed weight.We found that the loss-of-function mutants of GmIAA8 produced smaller seeds and showed enhanced tolerance to drought than does the wild type(WT).Whereas,for seed thickness and seed shape traits we found no significant difference among knockout mutant and wild type.Drought stress in combination with GmIAA8-knockout increased net photosynthesis rate,stomatal conductance,transpiration rate and maintained plant growth,suggesting that GmIAA8-knockout transgenic plants were less stressed than TN1 plants.Our results proved that the seed size and abiotic stress tolerance traits by CRISPR/Cas9 editing resulted in a loss of function mutation,and thus facilitate the dissection of complex gene regulatory network of GmIAA8 and the stacking of important traits in cultivated soybean.