| Asparagus officinalis L. is an edible plants with high economic value, it’s also dioecious plant. On the production of cultivations, the production output of Male seedlings is significantly higher than female. But there are also the mixed issue of the existing germplasm resources and the issue of unclear genetic background. Secondly, In basic research, Asparagus officinalis one of the model materials to Sex chromosome evolution and sex determination mechanism research. Therefore, it will contribute to breeding of fine varieties by research the genetic diversity of species at the molecular level, it’s also provide basic information for the study of sex determination and differentiation mechanism. Simple sequence repeat(SSR) molecular markers widely exists in the genome, they have some advantages of high polymorphic, rich sites, high repeatability. That can be used to study genetic diversity, molecular marker-assisted breeding, gene mapping, germplasm resources evaluation. Therefore, this study analysis the SSR distribution of asparagus genome the first time using repeated sequencing technology, and on this basis for analysis the genetic diversity and sex-linked markers of asparagus mainstream hybrids. The main findings are as follows:1. SSR types of Asparagus officinalis genome is very rich, to excavate the convergence SSR scaffolds, which were found 13 497 SSR loci. Repeat bases include a total of nine types of repetition, they are two, three, four, five, six, seven, eight, nine and ten nucleotide repeats respectively. But the frequency of various types are quite different. Dinucleotide and trinucleotide at most in the repeat types of Asparagus officinalis genomic SSR, 87% of the total, and the number of dinucleotide repeat obviously significantly greater than trinucleotide, they including 7315 and 4427, accounting for 54.2% and 32.8% of SSR total, respectively. Followed by four nucleotide repeats which has 1321, accounting for 9.79%. Penta-nucleotide and hexa-nucleotides has fewer 238 and 165, accounting for 1.76% and 1.22% respectively. The seven nucleotides, eight nucleotides, nine nucleotides and ten nucleotide repeats has 24,4,1,2 at least, respectively. All of them accounted for 0.23%. The SSR repetitions of Asparagus are mainly concentrated in the low repetitions of 5-11, which have a total of 9656 SSRs, accounting for 71.54% of the total and being the main part in Asparagus officinalis genomic SSRs. The repetitions of 12-23 times have a total of 3407 SSRs and occupied 25.24% of the total. The remaining repetitions altogether have 434 sequences, only occupied 3.22% of the total.2. Using 22 pairs of SSR primers amplify 40 different male and female plant of asparagus. 175 loci were amplified, of which there are 150 polymorphic loci,the percentage of polymorphic bands(PPB) were 85.71%. The primer of 22 pairs which amplified polymorphic bands most is the ssr-41, 13 bands were amplified. In the development of 22 molecular markers, the average observed number of alleles(Na) per locus was 1.2758. The average effective number of alleles(Ne) was 1.1912. Nei’s genetic diversity(He) with an average of 0.1082. Shannon information index(I) with an average of 0.1588. The trends of Nei’s genetic diversity(He) and Shannon information index(I) in the cultivars generally are consistent. And the trends of Shannon information index(I) and the percentage of polymorphic bands(PPB) in cultivars are broadly consistent. Genetic differentiation coefficient between cultivars is 0.5324, indicating that the 53.24% variation of total variation occurs between the cultivars, while the variation within different plant varieties is 46.76%. Gene flow between the cultivars Nm = 0.4392 <1, indicating that genetic drift is the dominant factor affecting the genetic structure between the ten asparagus species. The genetic distance between asparagus cultivars is from 0.078 to 0.183 by using the program popgene32. UPGMA cluster analysis was performed based on the Nei’s genetic distance, UPGMA cluster analysis in the computer program MEGA version 6.0 and Cluster analysis tree is established, found that the tree can be divided into four branch : UC309 and UC157 is each branch respectively. TD818, Grande and UC308 is a branch. Jersey Giant, Apollo, UC800, U88, MLJZ is the other branch. This shows that the asparagus germplasm resources have the same genetic background, but there are some differences between the varieties.3. By UPGMA cluster analysis for Male and female plant in the different varieties of asparagus found that the 39 plants showed more specific clustering model, the main features as follow: 1)the male and female have the gender differences in the clustering pattern of different cultivars, as UC309 and UC157 plant’s male and female is a clade, the other cultivars are not cluster together. 2) The clustering results appears that male and female-specific clustering pattern, namely there are 10 male plants of most cultivars are cluster together, nine female plants are cluster together. This phenomenon indicates that the genetic background of asparagus cultivars is more complicated.4. Preliminary analysis of SSR markers were also found that ssr-56 primers amplified 4 sex-linked markers linked with male or female in different cultivars. After repeated verification found three markers can’t appeared stablely and exhibit distinct species specificity, further explain that asparagus germplasm resources have complex genetic background. |
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