| Yardlong bean(Vigna unguiculata ssp.sesquipedalis)has considerable nutritional value and is widely cultivated in China.With the development of protected cultivation of yardlong bean,salt accumulation in soil has become a serious problem and secondary salinization of soil severely affects the yield and quality of yardlong bean.On the other hand,coastal regions are main areas of yardlong bean production in China.These areas have extremely severe salinity and alkalinity problems due to the high rate of moisture evaporation from soil.The mining of salt-tolerant germplasm resources and research on the molecular mechanisms of salt stress response in yardlong bean have important significance for genetic improvements to salt tolerance in yardlong bean.In this study,a method for identification of salt tolerance on yardlong bean seedlings was established.This method was used to screen the germplasm resources of yardlong bean,and the salt-tolerant yardlong bean germplasm resources were excavated.Transcriptome sequencing was used to analyze the differentially expressed genes of salt-tolerant and salt-sensitive yardlong bean varieties in response to salt stress,and to explore the molecular mechanism of salt-tolerance on yardlong bean.InDel markers were developed based on transcriptome data,and genetic diversity analysis and fingerprinting of yardlong bean resources were carried out.A F2 mapping population was constructed using Suzi 41 and Sujiang 1419 as female parent and male parent respectively.The F2 population was used to QTL mapping analysis of salt tolerance.The main findings are as follows:1.Establishment of the method and evaluation for salt tolerance of yardlong bean varieties at seedling stageThis work was performed on twelve varieties of yardlong bean that have been cultivated across large areas in Jiangsu Province for more than 10 years.Vermiculite was used as a cultivation matrix.During the cotyledon expansion stage,50,100,150,200,and 250 mmol/L of NaCl solution was added to 1/2 Hoagland nutrient solution for treatment and the 1/2 Hoagland nutrient solution served as a control.After 4 weeks of treatment,the effects of salt-tolerant traits of yardlong bean under different concentration of salt stresses were studied.With the increase of salt concentration,the salt tolerance rating(STR)of 12 varieties increased,and the coefficient of variation was the highest under 150 mmol/L NaCl stress.Thus,we choose it was as the optimum concentration of salt tolerance of yardlong bean.Under the stress of 150 mmol/L NaCl,the salt tolerance of 12 materials was evaluated by membership function value(F_i).This method provides a reference for identification of salt tolerance of yardlong bean seedlings and selection of salt-tolerant varieties on a large scale.2.Evaluation on salt tolerance of yardlong bean germplasm resources at seedling stageBased on the above method,we assessed salt-tolerance germplasm resources in 84 representative yardlong bean germplams from 11 Chinese provinces at the seedling stage.Results showed the following:(1)There was a large variation in F_i,0.06-0.88,for yardlong bean germplasm resources,indicating abundant genetic variability of salt tolerance in yardlong bean.(2)The cluster analysis was carried out according to Fj.Suzijiang No.1,Zijinroudoujiao,Lvyuantiaojiangdou etc.as the salt-tolerance materials from the 84 germplasm resources,which can be used for breeding of salt-tolerant yardlong bean varieties.3.Transcriptomics analysis of yardlong bean seedlings in response to salt stressThe differential transcriptomics analysis between the roots of Suzi 41(salt-tolerant)and Sujiang 1419(salt-sensitive)plants in yardlong bean under salt stress for 3 days was conducted using Illumina sequencing technology(RNA-seq).FDR≤0.01 and log2 fold change≥1 were set as threshold values to compare differences in transcription level and differentially expressed genes(DEGs)in the treated and control samples of the two varieties.The differentially expressed genes(DEGs)were compared in the treated and control samples of the two varieties.There were 32 upregulated DEGs and 2 downregulated DEGs that were specific to the Suzi 41 variety,respectively.Gene Ontology(GO)annotation showed that 42 DEGs(79.2%)in Suzi 41 were annotated to 24 functional categories,and 289 DEGs(75.5%)in Sujiang 1419 to 36 functional categories.The KEGG pathway enrichment results showed that only 53 of the 53 DEGs in Suzi 41 were enriched in the "cysteine and methionine metabolism" pathway,while 383 DEGs in Sujiang 1419 were enriched in 28 metabolic pathways.qRT-PCR analysis validated that the gene expression pattern in RNA-seq was reliable.4.Analysis of genetic diversity and construction of fingerprint map of yardlong beanThe number of 208 InDel markers were developed based on the transcriptome data using Phytozome v.12.1 of cowpea genome data as the reference genome,of which 54 InDel markers were polymorphic in Suzi 41 and Sujiang 1419.The 54 InDel markers were used for genetic diversity analysis in 84 yardlong bean germplasm resources.A total of 124 alleles were detected.The genetic diversity index ranged from 0.0887 to 0.5907,and polymorphic information content(PIC)ranged from 0.0848 to 0.5103 with a mean of 0.3479.Furthermore,14 of 54 InDels were used to construct DNA molecular fingerprint.The DNA molecular fingerprint provides technical support for the identification of yardlong bean varieties.5.QTL analysis of salt tolerance-related traits in yardlong bean seedlingsThe F2 mapping population that included 143 individuals were constructed from the hybridization of Suzi 41 and Sujiang 1419.A genetic linkage map of yardlong bean was constructed with the F2 population and 398 InDel markers developed earlier in our laboratory.This map included 103 InDel markers,covered 9 linkage groups,and had a total length of 975.7 cM.The mean map distance was 9.5 cM.Four major QTLs were detected combined by the genetic map and the phenotypic data of salt tolerance-related traits from the F2:3 pedigrees.Three major QTLs were detected in the interval between VUIn584 and VUIn724 in Chr.09,which explains 10.5～48.7%of phenotypic variability.One QTL was detected in VUIn675～VUIn578 on Chr.08,which explains 12.6～18.2%of phenotypic variability.By comparison with the reference genome of yardlong bean,we found that the VUIn584～VUIn724 interval in Chr.09(29,347,612 bp～30,139,092 bp)included 69 candidate genes,including the NAC transcription factor(Vigun09g139400),cytochrome P450(Vigun09g136000),ion channel regulating protein UNC-93(Vigun09g137700),and GNAT family acetyltransferase(Vigun09g135900),which may be associated with salt tolerance.By integrating transcriptome data and QTL mapping analysis,we found the VUIn584～VUIn724 interval on Chr.09 containing one DEG,UNC-93 ion channel regulating protein gene(Vigun09g137600),which was up-regulated in Suzi 41 that of Sujiang 1419 under salt stress.This gene may be a candidate gene that explains genetic differences in salt stress. |
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