QTL Mapping And RNA-seq Analysis Of Stem And Seed Traits In Wild And Cultivated Soybean

Lodging resistance and seed related traits are important agronomic traits of soybean.Lodging resistance is closely related to mechanical characteristics and chemical composition of stem.Cultivated soybean（Glycine Max L.Merr.）is domesticated from wild soybean（Glycine soja Sieb.and Zucc.）.Different from the robust and upright growth characteristics of cultivated soybean,one of important growth characteristics of wild soybeanis weak and creeping growth of stem.And the seed weight and oil contents between cultivated and wild soybean are also significantly different.To elucidate the genes controlling these differences and further learn more about the process of soybean domestication,we developed a RIL mapping population derived from a cross between the wild soybean ZYD00463 and the cultivated soybean WDD01514.With the help of SLAF-seq technology,we constructed a high density genetic map.We analyzed the stem anatomical structures of the parents and performed QTL mapping for chemical composition of stem,seed oil and composition,seed weight,and seed shape.Some candidate genes were further identified by the ananlysis of RNA-seq.Our results lay a foundation for further verification and mechanism analysis for these gene functions.The main results of this study are as following:1.We compared the anatomical structures,cell wall thickness and components between wild soybean ZYD00463（G.soja）and cultivated WDD01514（G.max）with the help of chemical,histochemical,and immunochemical methods.At tissues level,a more developed vascular tissue,especially for xylem,sclerenchyma（xylem fibers,and vessels wall）were presented in cultivated soybean WDD01514 stem.In comparison,a more developed epidermis tissue,collenchyma（cortex collenchyma）,sclerenchyma（phloem fibers）,parenchyma（cortex,phloem,xylem,and pith parenchyma）was observed in wild soybean ZYD00463.Besides,vessel-assiciated parameter,D,V_S,F_pf,and VI,in ZYD00463,are smaller than that of WDD01514.Oppositely,ratio of narrow vessels,V_G,and（t/b_h）² are larger than that of WDD01514.At cellular level,the thickness of secondary wall（phloem fiber cell,xylem fiber cell,and vessels wall）in WDD01514 was thicker than ZYD00463.At molecular level,a more higher level of lignin,cellulose,hemicelluloses were presented in WDD01514,and higher level of pectin were observed in ZYD00463.Besides,the distribution of mannan and degree of methyl-esterification pectin were clearly different between two accessions.No detectable mannan in phloem fibers cell wall of ZYD00463,however,appeared in WDD01514.The degree of methyl-esterification pectin in WDD01514 was obviously low than that of ZYD00463.This difference might be the new emerging characteristics of cultivated soybean acquired in the process of domestication.Those differences of anatomical structures and cell wall components make cultivated soybean to enhance mechanical strength of stem and more resistance biotic and abiotic stresses in wild.Our results could be helpful for scientists to learn the stem differentiation during soybean domestication.2.A total of 11,398 SNP markers were obtained using SLAF-seq sequencing technology.A high-density genetic linkage map covering 2913.78 c M was constructed,which was used to map QTLs for lignin and cellulose content in stem.The mean genetic distance between markers was 0.26 c M.The heritability of lignin and cellulose content in single environment is higher,however,lower in multiple environments,suggesting that environment has great influence on the heredity of lignin and cellulose.Eight QTLs including q LC1-1,q LC6-1,q LC11-1,q LC12-1,q LC12-2,q LC12-3,q LC13-1 and q LC14-1related to lignin content were identified.In addition,eight QTLs including q CC1-1,q CC1-2,q CC18-1,q CC19-1,q CC5-1,q CC5-2,q CC9-1 and q CC19-2 related to cellulose content were identified.These QTL locus were detected only in a single environment,and no stable QTLs were detected in multiple environments,suggesting that those are less stable in different environments.3.ANOVA showed that the heritability of oil content and fatty acid composition was higher under multiple environments.In this study,24 stable QTLs were identified for oil content and fatty acid composition.Seven QTLs related to oil content including q OC2＿1,q OC8＿1,q OC8＿2,q OC15＿1,q OC15＿2,q OC20＿1 and q OC20＿2 were identified.QTLs related to palmitic acid were identified including q PA10＿1,q PA13＿1,q PA15＿1 and q PA16＿1.Three QTLs associated with stearic acid including q SA14＿1,q SA14＿2 and q SA14＿3 were identified.The QTLs related to oleic acid are q OA11＿1 and q OA15＿1.Four QTLs related to linoleic acid including q LA5＿1,q LA5＿2,q LA11＿1 and q LA11＿2 were identified.Finally,four QTLs related to linolenic acid including q LNA5＿1,q LNA14＿1,q LNA15＿1 and q LNA15＿2.Among those stable QTLs,q PA10＿1 is a new QTL,which has not been reported so far.Sixteen QTLs were major QTL,including q OC15＿1,q OC15＿2,q OC20＿1,q OC20＿2,q PA10＿1,q SA14＿1,q SA14＿2,q SA14＿3,q OA11＿1,q OA15＿1,q LA11＿1,q LA11＿2,q LNA5＿1,q LNA14＿1,q LNA15＿1 and q LNA15＿2.Among these QTLs,the additive effects of q PA15＿1,q LA11＿1,q LA11＿2,q LNA5＿1,q LNA14＿1,q LNA15＿1 and q LNA15＿2 are positive,while the additive effects of other QTLs are negative.Six QTLs including q OC15＿1,q OC15＿2,q PA15＿1,q OA15＿1,q LNA15＿1 and q LNA15＿2 were detected in the genetic interval of 2.37 Mb-5.63 Mb on chromosome 15,which were regarded as hotspots of oil and fatty acid inheritance.Some candidate genes involved in lipid metabolism were obtained from the 24 stable QTLs,including 96 enzyme genes and250 transcription factor genes.4.ANOVA showed that the heritability of seed weight and seed shape were higher under multiple environments,except for seed length/seed width（LW）and seed width/seed height（WH）.Twenty-four stable QTLs were located.There were five QTLs related to seed length（SL）,namely q SL4＿1,q SL8＿1,q SL12＿1,q SL19＿1 and q SL19＿2.There were three QTLs related to seed width（SW）,including q SW5＿1,q SW7＿1 and q SW19＿1.Four QTLs including q SH5＿1,q SH7＿1,q SH15＿1 and q SH19＿1 related to seed height（SH）were identified.Four QTLs related to seed length/seed width（LW）,q LW2＿1,q LW2＿2,q LW5＿1and q LW19＿1,were identified.There was only one QTL q WH5＿1 associated with seed width/seed height（WH）.Three QTL,q LH2＿1,q LH2＿2 and q LH2＿3 were associated with seed length/seed height（LH）.There are four QTLs related to hundred-grain weight,including q HGW4＿1,q HGW19＿1,q HGW19＿2 and q HGW19＿3.Eleven of these stable QTLs have not been reported before,including q SL8＿1,q SL12＿1,q SH15＿1,q LW2＿1,q LW2＿2,q LW5＿1,q LW19＿1,q LH2＿1,q LH2＿2,q LH2＿3 and q WH5＿1.It is suggest that those may be novel QTL.Eleven QTLs were major QTL,including q SL19＿1、q SL19＿2、q SW19＿1、q SH19＿1、q LW2＿1、q LW2＿2、q LW5＿1、q LH2＿2、q LH2＿3、q WH5＿1和q HGW19＿3.Among these 24 QTLs,q LW2＿1,q LW2＿2,q LW5＿1,q LW19＿1,q LH2＿1,q LH2＿2 and q LH2＿3 have positive additive effects,while other QTLs have negative additive effects.Hotspots of seed weight and shape QTL in the interval of 39.42 Mb-44.99Mb were detected on chromosome 19,containing q SL19＿1,q SL19＿2,q SW19＿1,q SH19＿1,q HGW19＿1,q HGW19＿2 and q HGW19＿3.Some candidate genes involved in seed development and control seed size were selected from the 24 stable QTLs,including 136protein genes and 240 transcription factor genes.5.RNA-seq was conducted for parents and two extreme progeny（large grain with high oil vs small seed with low oil）at two developmental stages（20DAF vs 30DAF）.A total of 180.49 Gb of raw data was obtained.The percentage of Q30 bases was above93.06%,and the mapped ratio was from 92.18%to 96.40%.Under false discovery rate（FDR<0.01）and FC（Fold changes）≥2（i.e.,Log₂（FC）≥1）,14,698 differentially expressed genes（DEGs）were obtained from the comparative transcriptomes,and 2,217 were new genes.Some biological processes were significantly enriched involving lipid metabolism,such as regulation of lipid metabolism,acylglycerol metabolism,oil body biosynthesis and coenzyme metabolism.The biological processes involving in seed size are transcriptional and post-transcriptional regulation,hormone signaling pathway,ubiquitin-protease pathway and cell proliferation and expansion.In addition,some pathways related to carbon metabolism are also enriched,including photosynthesis,carbon fixation,glycolysis,pyruvate metabolism.Some candidate genes including 121 and 189 genes controlling oil content and seed size were obtained,respectively.Among of them,15 DEGs were located in QTLs of oil content and fatty acid,and 14 DEGs were located in QTLs of seed weight and seed shape.The WGCNA was further used to identify six modules related to seed oil content and seed weight.According to the k ME value and connectivity degree of genes in the modules,69 and 76 candidate genes were selected from modules and network,respectively.Among of them,26 candidate genes were located in QTL intervals.6.q RT-PCR analysis showed that eight genes may be involved in lipid metabolism and regulation of seed size among of the sixteen candidate genes.Glyma.06g068800 and Glyma.09g098300 encode a ERF and b HLH transcription factor,respectively.Glyma.10g119900 and Glyma.13g287600 encode a glycerol-3-phosphate acyltransferas（GPAT6）and gibberellin 2-oxidase（GA2OX8）,respectively.The function of Glyma.14g184900 is unknown,and located in the interval of q LNA14＿1.Glyma.17g154500 encodes a ras-group-related LRR protein.The above six genes were expressed in seeds,and the expression level of cultivated soybean was higher than that of wild soybean,and the expression level was significantly positively correlated with oil content and seed weight.In addition,Glyma.07g037700 encodes a MYB transcription factor,which is specifically expressed in seed.It is located in the q SW7＿1 and q SH7＿1interval,and its expression level is significantly positively correlated with oil content and seed weight.Glyma.09g111900 encodes a fatty acid desaturase（FAD2）,which is specifically expressed in seed and flower,and its expression level is significantly positively correlated with linoleic acid content and negatively correlated with oleic acid content.