| Cowpea [Vigna unguiculata(Linn.)Walp.] is a dicotyledonous legume that originated in the African continent.Our country is a secondary center of origin of cowpea.Globally cowpea is an important legume crop.In this study,271 cowpea germplasm were used as material for genetic diversity and relatedness analysis of this genetic population using a combination of morphological and SNP and InDel molecular marker technologies developed based on genome resequencing;GWAS analysis using SNP markers and phenotypic data to explore candidate genes controlling days to flowering(DTF)and pod length(PL)traits in cowpea;Molecular fingerprinting of 271 cowpea germplasm was constructed using InDel markers,and techniques for the identification of cowpea germplasm were discussed.The aim was to provide the scientific basis for the effective utilization of cowpea resources and the selection of new varieties in China.The main results are as follows:(1)The results of the spring and fall in 2021 field trial studies showed that the mean values of DTF of cowpea variety populations were 52 and 37 d,with an average of 15.6 d earlier in fall than in spring,and coefficients of variation of 6.59% and 11.13%for the two seasons of cultivation,respectively.The distribution range of PL in spring and autumn was basically the same,with mean values of 53.36 and 54.22 cm,respectively,and coefficients of variation close to 25%.The mean values of pod width were 7.16 and 7.51 mm,with coefficients of variation of 16.33% and 10.48%,respectively;the mean values of pod thickness were 6.80 and 7.66 mm,with coefficients of variation of 22.68% and 13.91%.PW and PT had less variation and were more influenced by environmental factors.Analysis of variance yielded highly significant differences in PL,PW,PT and DTF phenotypic data between breeds and between quarters for the two quarters of 2021,and calculated heritabilities of 92.62%,27.32%,43.54% and 87.27% respectively,with PL and DTF mainly influenced by breeds and PW and PT mainly influenced by environmental factors.The data distribution characteristics of DTF,PL,PW and PT for both seasons were visualized using the violin plot method.Correlation analysis showed that PL was positively correlated with PT,negatively correlated with DTF,and PW was positively correlated with PT.(2)Based on the morphological traits of 251 cowpeas,they can be divided into three various groups at Euclidean distance 6.6.Variety group I contains 219 entries,which can be subdivided into two subgroups;variety group II contains 31 entries.Variety group III contained only one copy,an early half-moon bean from Hubei.Traits such as PL and DTF showed large differences among the various groups,but the clustering results did not correlate significantly with the geographical origin of the cowpea material.And 46 germplasm were difficult to distinguish,39 of which could be distinguished using InDel marker technology,and 7 varieties could not be discriminated,which may be related to synonymy.(3)A total of 2237892 high-quality SNP markers were obtained from 200 cowpea materials by second-generation genome resequencing,with an average of 1SNP marker per 0.22 Kb distributed throughout the chromosome.A total of 23,390 InDel markers with insertions and deletions in the range of 20-50 bp were obtained,with an average of 1 marker per 21.1 Kb of the entire chromosome,which can provide information for the identification and discrimination of cowpea germplasm resources.The MLM model detected four SNP loci associated with DTF,and a total of 17 genes were searched for using the LIS(Legume Information System)database;the four SNP loci detected for possible association with PL were all located in Chr10,and a total of six genes were searched for,and the genes near the SNP loci associated with DTF overlapped.These can be used as candidate genes to control DTF and PL traits.(4)The results of InDel primer development were as follows: 44 pairs of polymorphic InDel primers were screened from 111 pairs of primers that could amplify the target bands,and the ratio of polymorphic InDel primers was 40.45%.The 20 pairs of polymorphic InDel primers with good repeatability and clear bands were further screened and used to genotype 271 cowpea materials,with genetic diversity values ranging from 0.064 to 0.496 with a mean value of 0.371;polymorphic information content values ranging from 0.069 to 0.375 with a mean value of 0.260;Shannon’s index The results indicated that the 271 cowpea materials were rich in genetic diversity at the molecular level,and a useful cowpea gene pool was constructed to provide a material basis for variety improvement.(5)Based on the InDel electrophoresis profiles of each variety,0 and 1 matrices were constructed,and the clustering map of 271 cowpea materials was produced using the UPGMA method,and the cowpea materials could be classified into nine variety groups at Jaccard similarity coefficient 0.488,reflecting the differences in the molecular genetic structure of the various genomes,especially the differences in insertions and deletions on the genomes.This molecular genetic sequence difference was correlated with the phenotypes of the varieties,as shown by the fact that variety group II and variety group IV were mostly long-pod varieties,dwarf varieties were mostly concentrated in variety group II,and white flowering varieties were mostly concentrated in variety group IV;variety groups VII,VIII and IX were mainly shortpod varieties,and varieties with longer DTF were mostly in variety group VII;varieties with shorter DTF were mostly in variety group VI.The varieties with short DTF are mostly found in variety group VI.(6)A total of 139 molecular fingerprint types were obtained for 271 cowpea varieties constructed by InDel primers.They can be divided into two categories,the first category containing 108 distinctness fingerprints and the second category containing 31 non-distinctness fingerprints.This method is simple and feasible,but not all germplasm have distinctness fingerprints,and more InDel markers need to be screened for additional information or combined with other markers for discrimination.The combined analysis showed that the combination of morphological traits and InDel molecular markers could better identify and discriminate cowpea germplasm. |
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