Analysis Of Alfalfa Hybrids And Construction Of Genetic Map By SNP Markers

Alfalfa is an important perennial leguminous forage.Medicago sativa has the advantages of high yield,strong regeneration,good palatability and soil and water conservation.Medicago falcata has the characteristics of drought resistance,cold resistance,poor soil resistance.At present,there are relatively few studies on the hybrid progeny of these two species.In this experiment,M.sativa and M.falcata were used to hybridized firstly and then obtained the hybrids.Seccondly,using the SSR molecular markers identified the true or false of the hybrids,and analyzed the agronomic characters.Finally,using Super GBS technology analyzed the bioinformatics of the hybrids and parents,and constructed the genetic map by SNP markers.Which lay a foundation for genetic variation analysis,quantitative trait QTL mapping and molecular marker-assisted breeding of alfalfa.It is of great value for functional gene mining of target traits and breeding of new lines and varieties of alfalfa.The main findings are as follow:1?Hybridization of M.sativa and M.falcata had obtained a large number of hybrids.The pod-setting rate of combination I was 88.68%and 65.45%,respectively,in the cross of P1×P2 and its reciprocal.The ratio of P1×P2 was higher than the P2×P1.Hybrids obtained from which were 460 and202,among them the health hybrids were 371 and 188.The percentage of health hybrids were 80.65%and 93.07%,respectively.The pod setting rates of P3×P4 and the reverse crosses in combination II were73.87%and 52.22%,279 and 174 hybrid seeds,228 and 154 health hybrids were obtained,and the health hybrid rates were 81.72%and 88.51%,respectively.2?In the two hybrid combinations,the seedling emergence rates of the positive and reverse crosses F₁ of combination I were 58.76%and 82.98%,respectively,and the reverse crosses were higher than the positive ones.After overwintering in greenhouse,80 and 48 F₁ plants were obtained from the positive and reverse crossing of combination I,and the overwintering rates of hybrids were 36.70%and 30.77%,respectively,which were between parents.The seedling emergence rates of the positive and reverse crosses F₁ of combination II were 67.11%and 49.35%,respectively,the positive were higher than those of the reverse crosses.Fifteen and six F₁ generations plants were obtained after overwintering,and the overwintering rates of the hybrids were 9.80%and 7.89%,respectively,slightly higher than those of their parents.3?Identification of true and false hybrids of the hybrid offspring of the two combinations,the results show that there are 3 pairs of SSR primers that can be used to identify the authenticity of the hybrid of the two combinations.The identification of SSR molecular markers indicated that the hybrids of the two combinations were true.4?The growth speed of hybrids and their parents showed the same growth trend with time.The growth speed in the early branching stage increased slowly,and the growth speed decreased after branching,but it showed a rapid upward trend in the early budding stage,and then slowed down after entering the reproductive growth stage.The results of cluster analysis on the plant height,branch number,stem diameter,plant type,inflorescence number and seed number of the hybrids of M.sativa and M.falcata showed that the hybrids had great variation compared with their parents,and some hybrid plants had super parental advantages in the above different phenotypic indicators.The correlation analysis of the phenotypic traits of the two hybrid combinations showed that the dry weight had a very significant correlation with plant height,and the number of main stem nodes,and the correlation with plant height was the strongest.Plant height was significantly correlated with the number of main stem nodes and plant type.5?By using Super GBS sequencing technology,the hybrids and parents of M.sativa and M.falcata were genotyped.A large number of GBS molecular markers were obtained.Through SNP calling and filtration,15221 SNP sites,and 478 indel sites were obtained,which included 205 insertion sites and273 deletion site.6?The Super GBS genotyping of the parents and hybrids of alfalfa was studied in this paper.According to the results,Super GBS sequencing technique can identify the genetic differences between the hybrids and their parents well.It can be used as an important method for molecular identification of alfalfa hybrids and parents.The phylogenetic tree of hybrids and parents based on SNP variation showed that the genetic distance between the two M.falcata parent P1 and P3 was very long,and the genetic basis was very different,but the genetic basis difference between the two M.sativa parents P2 and P4was small and the genetic distance was close.This is very consistent with the actual situation,fully confirmed the reliability of Super GBS sequencing technology in alfalfa hybrid analysis application.The hybrids of each combination and their parents were basically grouped into the same cluster.But in the same combination,hybrids and reciprocal hybrids of F₁ plants interspersed in the cluster without obvious regularity.7?The genetic map of parents and F₁ hybrids based on SNP molecular markers was constructed for the Combination I Genetic map containing 32 linkage groups were obtained,and the number of SNP marked above the linkage groups is 460,and the number of marked positions is 394.Total length of the Linkage group is 1192.067 cM,coverage 89.74%.Average spacing between markers is 2.59 cM.The results provide important genetic basis for phenotypic difference and genetic analysis of complex characters of hybrids and parents of alfalfa.