| Soybean[Glycine max(L.)Merr.]is one of the most important oilseed crops worldwide which is rich in oil,protein,and nutraceutical compounds such as isoflavones and saponins,and is important for human food,animal feed,and industrial products.But alkalinity is a major abiotic constraint factor that limits soybean productivity and quality.Different from salinity,alkalinity is mainly caused by sodium bicarbonate(NaHCO3)or sodium carbonate(Na2CO3),which affects by the presence of excess Na+,but combined with HCO3-,CO32-,high pH(>8.5),and poor soil structure.The Song-Nen Plain,a major soybean cultivation region in northeastern China,is one of the top three major contiguous saline-alkaline affected areas in the world,which has an estimated area of 3.73 million ha alkaline soils,and the size of which is increasing by 1.4%annually.The alkalinity-tolerance related traits in soybean are complex quantitative traits and controlled by multiple genes.Comapring with salinity,less progress has been made on the study of candidate genes and mechanism of alkalinity-tolerance in plants.Digging the candidate genes and exploring the molecular mechanisms of soybean response to alkaline stress is important for breeding alkalinity-tolerant soybean variety,improving soybean yield on saline-alkaline land,and essential for food security.Therefore,the objective of this research was to dig the candidate genes/QTL for soybean alkalinity tolerance.The alkaline tolerant of 129 soybean germplasms originate from different regions were screened and 12 alkalinity-tolerant indices were evaluted.Based on the screening result,one alkalinity tolerant wild soybean var.N24952 was chosen for RNA-sequncing.Differentially expressed genes(DEGs)between alkaline stress and control were identified and the functional classification and enrichment of DEGs were analyzed to explore the molecular mechanism of alkalinity-tolerance in soybean.In addition,two soybean recombination inbred line(RIL)populations were used to map alkalinity-tolerance related QTL.The main results of this research were as follows:1.A total of 129 soybean accessions originated from different regions were screened for the alkalinity tolerance.After 16 days of NaHCO3 treatment,12 alkalinity-tolerance related traits,including sodic(alkaline)tolerance rating(STR),leaf chlorophyll content(SPAD),shoot dry weigth(SDW),Na+concentration,K+ concentration,Ca2+ concentration,Mg2+ concentration,P concentration,Fe concentration,Na+/K+,Na+/Ca2+ and Na+/(K++Ca2+),were investigated.Na+ concentration showed a significantly negetitive correlation(r =-0.67,P<0.01)with STR.And Na+/K+?Na+/Ca2+?Na+/(K++Ca2+)also showed a significantly negetitive correlation(P<0.01)with STR.It means that the more the soybean tolerant to alkaline stress,the fewer Na+ concentration,and lower Na+/K+,Na+/Ca2,Na+/(K++Ca2+)in soybean leaves.The phenotypic coefficient variation of STR,Na+ concentration,Na+/K+,Na+/Ca2+,Na+/(K++Ca2+)ranged from 30.67%to 44.13%,and the broad sense heritability ranged from 60.32%to 85.60%.We suggest that using STR together with Na+concentration,Na+/K+,Na+/Ca2+,Na+/(K++Ca2+)as indicators would help accurately evaluate the alkalinity-tolerance of soybean.2.Based on the alkalinity-tolerance indices of STR,Na+ concentration,Na+/K+,Na+/Ca2 and Na+/(K++Ca2+),a total of 129 soybean accessions were screened.Eight soybean varieties showed strong alkaline-tolerance with STR ? 4.5 and lower Na+concentration,Na+/K+,Na+/Ca2+,and Na+/(K++Ca2+),including Meng8206,N24852,Qianxiqiyuedou,Heibiqing,Mengzixiqingdou,Jidandou,Baihua,and Yuxihuangdou.Eighteen soybean varieties were sensitive to alkalinity with STR<1.5 and higher Na+concentration,Na+/K+,Na+/Ca2+,Na+/(K++Ca2+),such as Zhengyang148(ZY148)and Linhedafengqing(LH).3.The transcriptome profile in the roots of an alkalinity tolerant wild soybean variety N24852 was analyzed.The soybean plants at V3 stage(approximately 14 d after planting)received a treatment solution containing 90 mM NaHCO3.Root tips(3 cm)were harvested after 0.5 and 1 day after alkaline stress for transcriptome analysis.DEGs were identified by comparing the NaHCO3 treated and control samples at the same time point using the R package DESeq.The significant DEGs were identified using the false discovery rate(FDR)<0.01 and |log2FoldChange| ? 1.A total of 449 DEGs were identified,including 95 and 140 up-regulated genes,and 108 and 135 down-regulated genes at 0.5 and 1 day after NaHCO3 treatment,respectively.Quantitative RT-PCR analysis of 14 DEGs showed a high consistency with the expression profiles of RNA-sequencing with a determination coefficient of 0.9763(P<0.01).Ten and 38 Gene Ontology(GO)terms were enriched at 0.5 and 1 day after alkalinity stress,respectively.GO terms related to transcription factors(TF)and transporters were significantly enriched in the up-regulated genes at 0.5 and 1 day after alkaline stress,respectively.Transcription factors enrichment analysis showed that NF-YA TF family was enriched at 0.5 day after NaHCO3 stress.Nine and 27 genes related to ion transporters,such as ABC transporter,aluminium activated malate transporter(ALMT),glutamate receptor(GLR),nitrate transporter(NRT)/proton dependent oligopeptide(POT)family,and S-type anion channel(SLAH),were differentially expressed at 0.5 day and 1 day after NaHCO3 treatment compared with control,respectively,implying their important roles in maintaining ion homeostasis in soybean roots under alkaline stress.KEGG pathway enrichment analysis showed "phenylpropanoid biosynthesis" and "phenylalanine metabolism" pathways might participate in soybean response to alkalinity.4.Two RIL populations,NJRIZM6 and NJRILM6,derived from the crosses of ZY148 x M8206,LH x M8206,respectively,were used to map the QTL for soybean tolerance to alkalinity.The alkalinity tolerant parent was M8206.The NJRIZM6 population included 126 F2:9 lines,and the NJRILM6 population is consisted of 104 F2:9 lines.Soybean alkaline tolerance was evaluated based on STR,concentration of Na+,K+,and Ca2+,as well as the ratio of Na+/K+,Na+/Ca2+,and Na+/(K++Ca2+),after 16 days of NaHCO3 treatment in greenhouse.QTL mapping was performed with composite interval mapping(CIM)method of the Windows QTL Catographer V2.5 software.In NJRIZM6 population,22 QTL within 17 QTL cluster regions were detected,which were distributed on 12 different chromosomes(Chr),including Chr 1,3,4,6,9,10,12,13,14,16,17,and 18.In NJRILM6 population,19 QTL within 14 QTL cluster regions were detected,which were distributed on 11 different chromosomes,including Chr 1,2,3,5,7,8,9,12,13,16,and 20.Three QTL clusters were detected in both populations.The QTL cluster on Chr.9 with the phiscal position of 0.32-1.60 Mb,Gm09G0.32-1.60,associated with four traits,Na+ concentration,Na+/K+,Na+/Ca2+,and Na+/(K++Ca2+),included 3 QTLs that accounted for 10.14-15.26%of the phenotypic variation of.The second QTL cluster Gm01G47.71-51.44 was detected on Chr.1 with the physical position of 47.71-51.44 Mb,including 3 QTL that explained 6.27%,11.59%,and 19.79%of the phenotypic variation of STR.The third QTL cluster Gml3G31.16-32.94 was detected on the Chr.13 with the phiscal position of 31.16-32.94 Mb,including 3 QTL that explained 6.51%?8.30%of the phenotypic variation for Ca2+ concentration and Na+/Ca2+,which is an sugestative QTL with a LOD value greater than 2.5 but lower than the LOD threashold obtained by 1000 permutations at 0.05 level. |
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