Discovery Of A New Cytoplasmic Male-Sterile Source,Detection Of A Nuclear Male-Sterile Gene And QTL-Allele Constitutions Of Heterosis In Soybean[Glycine Max(L.)Merr.]

Soybean[Glycine max（L.）Merr.]is an important protein and oil crop,and the utilization of heterosis is an important way to increase the yield of soybean.Two kinds of male-sterile materials,cytoplasmic male sterility（CMS）and genic male sterility（GMS）,are the basic for heterosis utilization.The success of wild-abortive cytoplasm in rice hybrid production shows that the elite male-sterile cytoplasm could be a breakthrough for crop utilization of heterosis.The male-sterile cytoplasm sources of soybean CMS lines were mainly from four soybean accessions.Therefore,discovery of new male-sterile cytoplasm source and development of male-sterile line were important for soybean.Soybean GMS line maybe also was a potential way for heterosis utilization because of the successful application of GMS line in rape and rice,and the research on the soybean hybrid seed production using GMS lines.A genic male sterile mutant with higher seed set rate was discovered in 2001.The study of this GMS mutant was necessary for its application in the future.South-Eastern Huang-Huai Valley was a part of the soybean production areas in China.Selection of strong heterotic combinations and the genetic constitutions of heterosis were essential for this area.In this study,3 group combinations according to a North Carolina II mating design were developed using the elite cultivars.The genetic constitutions of heterosis and its improvement were analyzed using high-resolution markers in the 3 NCII designs.1 Discovery of a male-sterile cytoplasm source N23661 and development of a cytoplasmic male-sterile line NJCMS4AFor screening new male-sterile cytoplasm sources,143 crosses were made using a group of 44 cultivated and 43 wild soybean accessions from six eco-regions.45 BCiFi crosses were obtained in 2012.Only two BC1Fi crosses indicated partial male sterility,i.e.,[（N23239xN04631）F1xN04631]and[（N23661xN23658）F1xN23658].The two BC1F1 crosses were further backcrossed to their respective recurrent parent;Only the latter was successful.The BCiF1 of the cross（N23661xN23658）was further backcrossed to obtain BC4F1 with a pollen germination rate of 0%.This continuously backcrossed line were defined as a male-sterile line,NJCMS4A.The cytoplasm of NJCMS4A came from Dabu,Guangdong province,Eco-region Ⅵ,and was different from the previously reported CMS sources based on the geographic source.The mitochondrial genomes of NJCMS4A,NJCMS1A,NJCMS3A,and Zhongdou-19 were analyzed using mitochondrial SSR,ORF and SCAR markers.The results showed that the cytoplasm of NJCMS4A was not identical to that of NJCMS1A,Zhongdou-19 and Runantianedan.The cytoplasm of NJCMS4A was also not identical to that of NJCMS1A and NJCMS3A by comparing the mitochondrial genome sequences.So,the cytoplasm of NJCMS4A is a new cytoplasm source.But the CMS-related gene of cytoplasm was not confirmed as a new one.2 Molecular mapping and candidate gene analysis of a recessive male-sterility,female-fertility gene ms NJ in soybeanNJS-13H was a spontaneous male-sterile mutant,and the sterility was controlled by a recessive sterile gene.The male-sterile locus msNJ was rough mapped into the region between SSR markers Satt550 and Satt094 on Chromosome 10（molecular linkage group[MLG]O）.Using three different mapping populations with total 1075 male-sterile plants,msNJ was mapped to a new location on chromosome 10 flanking two SSR markers BARCSOYSSR₁0₇94 and BARCSOYSSR₁0₈19.The physical distance between these two markers was approximately 1.32Mb on Gm10:29078678..30401139.This target region harbors 27 annotated genes,and four genes of these genes may be was related male sterility.Based the sequence analysis of four genes,there was a mutation which caused the change of amino acid in the code sequence in Glyma.10G117000.According to the sequence and expression analysis,the Glyma.10G11 7000 may be was the male-sterile gene.3 The QTL genetic constitutions of yield heterosis and its improvement in soybean from South-Eastern Huang-Huai Valley of ChinaSouth-Eastern Huang-Huai Valley was a part of the soybean production areas in China.In this present study,34 elite soybean cultivars（lines）mainly from South-Eastern Huang-Huai Valley and US,were used to develop 3 group combinations according to North CarolinaⅡ mating design in 2010-2013.2 group combinations（5 female parents×10 male parents and 5 female parents×5 male parents）were made in Huai’ an experimental point,and 1 group combinations（5 female parents×10 male parents）were made in Linyi experimental point under two years.The yield,protein,oil of combinations were investigated.Then the elite combinations was selected and the F1 heterosis and were analyzed.The QTLs related hybrid yield were detected and its improvement was designed.（1）The heterosis of soybean yield in South-Eastern Huang-Huai ValleyHeterosis of yield,was relatively larger,while no obvious heterosis in protein and oil contents.The heterobeltiosis of combinations in 3 NCII designs were average of 10.12%,5.01%,16.42%respectively.The highest yield of combinations in 3 NCII designs were Huaidou-9×NN88-31（3077.12 kg/hm2）,Xudou-13xBedford（4386.57 kg/hm2）and Jidou-12×Lindou-9（3900.56 kg/hm2）,with its heterobeltiosis 33.57%,30.84%,95.77%,respectively.7,5 and 7 elite yield combinations were identified from 3 NCII designs.（2）Genetic analysis of QTL and its alleles related Fi hybrid yield in soybean47 loci in total of hybrid yield were detected based on the software PLSRCGA 1.0（Partial least square regression combined with genetic algorithm）in the three NCII designs.QTL qHyb yld-15-4 was detected in all 3 NCII designs,qHyb yld-2-3,qHyb yld-2-8,qHyb yld-5-3,qHyb yld-9-3,qHyb yld-15-2 and qHyb yld-15-5 were detected in two NCII designs.All the 47 QTLs were corresponded to 41 candidate genes.The values of the additive and dominance alleles of each QTL were estimated.The values of the additive alleles qHyb yld-2-2-Al,qHyb yld-3-5-A2/qHyb yld-15-5-A3 and qHyb yld-2-7-Al were the highest in 3 NCII designs,respectively.The parents,NN88-31,Yudou-22,Forrest,Jidou-17,Bedford,Xudou-13,Jindou-34,Zhoudou-11 and Jidou-12 carry more positive additive loci.The values of the dominance allele qHyb yld-2-4-A1/A6,qHybyld-15-1-A1/A2,qHyb yld-16-1-A3/A4 were the highest in 3 NCII designs,respectively.（3）Genetic basis of QTLs related hybrid yield in superior combinationsThe top 5 hybrid combinations of yield in the three groups of NCII were selected respectively for calculate the QTL-alleles related hybrid yield.The results showed that the overdominance allele was the main genetic composition of the high yield combinations,and the overdominance effect was the basis of the heterosis in soybean.（4）Hybrid combinations improvementThe improvement strategies were put forward for the parents of soybean hybrid combinations according to the effect of QTL-alleles in this study.The improvement of the parent was completed through changing the low effect value QTL-allele to high effect value QTL-allele in the hybrid combination.Because each QTL contains several alleles,the parent of each hybrid combination may have many improvement strategies.Here,we only used the QTL-alleles with the highest effect value alleles to improve hybrid combinations.We described the improvement strategy for the highest yield combination in each NCII design as an example.（5）Analysis on combining ability of yield based on the effects of QTL-allelesThe genotypic combining ability was proposed and calculated based on the effects of QTL-alleles.The genotypic combining ability was significantly correlated with the heterosis ratio and phenotypic combining ability.The heterosis could be predicted using the genotypic combining ability.