| Sinonovacula constricta has very high economic value, and it has a long history of breeding. Compared to the traditional cultivation pattern, molecular marker-assisted breeding can greatly improve the efficiency of S.constricta breeding. The establishment of high density genetic linkage map of S.constricta effectively helps its molecular assisted breeding. This experiment, based on the development of the micro satellite markers and SLAF markers, established S.constricta family paternity test technology, and constructed the genetic linkage map of S.constricta. Following are the main researches:1. Using a magnetic beads enrichment to establish the Microsatellite Library, design and develop 400 pairs of primers, screened and synthesized 30 pairs of microsatellite primers, and conducted polymorphic analysis of 18 microsatellite primers by using 12 S.constricta individuals.The allele of 18 microsatellite primers in 12 individuals is 2 to 6. The average allele of each pair is 3.00. G_ScSSR110 has the most allele, in the amount of 6, it has an observed heterozygosity of 0.000 to 1.000, an average value of 0.5813, an expected heterozygosity of 0.290 to 0.779, with a mean value of 0.5284; its polymorphism information content is 0.239 to 0.703 and average value 0.4322. Eight pairs of primers with high polymorphism were selected to identify the family. When the two parental genotypes are unknown, the single parent exclusion rate(E-1P) is 0.583 to 0.916, with a mean value of 0.7900; in the condition of a known parental genotype, the exclusion rate(E-2P) of the other parent is 0.406 to 0.830, with an average value of 0.6539. Eliminating the individuals that have more than a pair of mismatched-primers, this research identified that 154 individuals belong to the same family.2. This experiment developed 400 pairs of primers by magnetic beads enrichment, acquired more than 30 polymorphic markers by screening, obtained 2010 microsatellite primer sequences by searching the EST Library, 429 makers by screening pentanucleotide, tetranucleotide and trinucleotide sequence, 144 polymorphic markers by analyzing their genomic loci. The research acquired 174 pairs of microsatellite primers from the EST Library and the integration of magnesphere. Excluding 129 pure microsatellite and mapping markers, 70 markers in the genetic linkage map, the genetic map length is 340.29 cM, length range 0.91 ~ 39.72 cM on linkage group; the average distance is 10.97 cM, with a spectral coverage rate of 25%.3. The SLAF library was established by using SLAF-seq technology. In this research, I developed 315533 SLAF markers, 144920 polymorphic SLAF markers, the proportion reached 45.93%. Based on the genotype encoding rules, I encoded gene type for 144920 polymorphic SLAF markers, 96655 of which were successful. 7516 out of the screened 8212 SLAF markers, which is 91.52%, were put the genetic linkage map and formed a general genetic map with a distance of 2383.85 cM. The average genetic distance of markers on each linkage group was 0.27 cM.Among the 19 linkage groups of the male genetic linkage map, 4189 markers were made, each linkage group contains an average of 220 markers, which range from 61 to 389; and the total length of the map is 1815.64 cM. The average genetic distance of markers on linkage group was 0.44 cM. The female map includes 4180 markers, each linkage group contains an average of 220 markers, with a marking range of 55 ~ 427; the total length of the map is 2731.31 cM, with a length range of 90.30 ~ 207.10 cM on linkage group. The average genetic distance of markers on linkage group was 0.78 cM.Integrate SSR marker with SNP marker into the genetic linkage map, 7637 markers in the genetic linkage map, the genetic map length is 2345.50 cM, length range 65.53 ~ 167.41 cM on linkage group, the average distance is 0.31 cM. Among the 19 linkage groups of the male genetic linkage map, 4281 markers were made, the total length of the map is 1693.33 cM, with a length range of 82.68 ~ 211.15 cM on linkage group.The average genetic distance of markers on linkage group was 0.40 cM. The female map includes 4266 markers, the total length of the map is 2884.64 cM, with a length range of 82.68 ~ 211.15 cM on linkage group. The average genetic distance of markers on linkage group was 0.68 cM. |
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