| Low temperature chilling injury is the main influencing factor of late autumn cultivation and early spring cultivation of asparagus bean in China.Screening and breeding cold-tolerant asparagus bean germplasm is an effective way to reduce chilling injury.The screening for cold tolerance germplasm and the research of genetic theory of asparagus bean have important theoretical and practical significance.In this study 305 asparagus bean germplasm resources were preliminarily evaluated for cold tolerance at pod-setting stage.After further comprehensive cold tolerance evaluation of the preliminarily selected 50 materials at germination stage,seedling stage and adult stage,The cold-resistant parent,Dubai cowpea,and the cold-sensitive parent,‘Ningjiang 3',were selected.Using two parents and hybrids F1,F2,F2: 3 generations,We analyzed the main gene +polygenes of cold-tolerance related traits,constructed the high-density genetic map by Specific-Locus amplified fragment sequencing(SLAF-seq),and carrid out QTL mapping for cold tolerance at pod-setting stage of asparagus bean.The results showed that:1.The cold tolerance of 305 asparagus bean germplasm resources cultivated in late autumn was preliminarily evaluated by natural low temperature condition..305 asparagus bean germplasm resources were preliminarily subdivided into 6 types,and one highly cold-tolerant material,26 cold-tolerant materials and 23 cold-sensitive materials were selected.It was found that the cold tolerance of the main cultivated asparagus bean varieties was correlated with their yield under low temperature stress.2.Principal component analysis,membership function analysis,cluster analysis and regression analysis were used to evaluate the cold tolerance of 50 germplasm resources of asparagus bean at different growth stages(budding stage,seedling stage,adult stage).Three mathematical model for comprehensive evaluation of cold tolerance of asparagus bean in three periods were established.The model of cold tolerance evaluation of budding stage of asparagus bean was D=-0.357+1.032*X2+0.404* X3+0.555* X6.Three indexes which include relative germination index(RGI),relative germination activity index(RVI)and relative total fresh weight(RFW),were determined to be the main evaluation indexes of cold-tolerance in budding stage.7 cold-tolerant and 9 cold-sensitive materials were selected.The evaluation model of cold tolerance in seedling stage of asparagus bean was D=-1.853 +1.145* X2+0.545* X3+0.537* X6+0.173* X7+0.113* X8+0.224* X9-0.099* X10-0.134* X12.The relative root number(RRN),relative root length(RRL),relative total chlorophyll content(CT),relative soluble protein content(SP),relative soluble sugar(SS),relative cata-lase activiety(CAT),relative peroxidase activity(POD)and relative ascorbate peroxidase activity(APX)were screened as the main morphological and physiological index of cold tolerance evaluation in seedling stage.2 accessions of asparagus bean germplasm with strong chilling tolerance and 5 accessions of cold-sensitive asparagus bean germplasm were obtained.The evaluation model of cold tolerance asparagus bean at adult stage was D=-0.481+0.210* X1+0.157* X3+0.494* X5+0.128*X10+0.178*X11.The cold tolerance index(CRI),the relative yield per plant(RYP),the relative pod length(RPL),the relative superoxide dismutase activity(SOD)and the relative peroxidase activity(POD)were obtained as an effective index for evaluating cold tolerance of asparagus bean in adult stage.1 asparagus bean germplasm with extremely strong cold tolerance and 12 asparagus bean germplasm with strong cold-tolerance and 3 cold-sensitive germplasm were screened out.3.The Study on the correlation of Cold tolerance in different growth and development stages of asparagus bean showed that the cold tolerance of asparagus bean at adult stage was significantly positive correlation with that at germination stage,but there was no significant correlation between cold tolerance at adult stage and that at seedling stage.The most cold-tolerant variety at adult stage,‘Dubai cowpea',also showed cold-tolerance at germination stage,but it was extremely senstilve-tolerant at seedling stage.The cold-sensitive cultivar at adult stage ‘Ningjiang3' was also cold-sensitive at germinating stage,but cold-tolerant at seedling stage.4.Genetic analysis of chilling tolerance related traits in asparagus bean during pod setting using main gene+polygene model showed that the chilling damage index of asparagus bean was controlled by two pairs of additive main genes + additive-dominant polygene(MX2-A-AD),and the number of pods per plant under cold stress was controlled by 2 pairs of additive-dominant-epistatic main gene(2MG-ADI).Under cold stress,pod length and pod diameter were controlled by a pair of additive-dominant gene additive-dominant-epistatic polygene(MX1-AD-ADI).Main genes were detected in all of the four cold-tolerant related traits.The heritability of main genes in the F2:3 generation is higher than polygenes in low temperature tolerance traits.5.The SLAF-seq technology was firstly used to construct a high density genetic map of asparagus bean,which was the first map based on NGS(high-throughput sequencing technology).The map consisted of 11 linkage groups,including 5225 SNP markers,spanning 1,850.81 c M,and the average distance between the markers is 0.35 c M.The genetic map of cowpea is so far the second longest and the density is far higher than the other three SNP maps based on Golden Gate platform.6.Comparing asparagus bean with other 4 kinds of beans(soybean,common bean,mung bean and adzuki bean),asparagus bean was genetically more closely related to adzuki bean.7.QTL associated with cold tolerance in pod stage of asparagus bean was identified for the first time and the QTL loci: q CTRS2 and q CTRS10,which were located on linkage group 2 and linkage group 10,respectively.q CTRS2 explained 10.74% of the phenotypic variation and there were two SNP markers in this chromosome region.q CTRS10 explained 13.67% of the phenotypic variation and there were six SNP markers in this chromosome region.Both QTL had positive additive effect. |
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