Development And Applications Of Molecular Markers Based On RNA-seq In Chinese Soft-shelled Turtles

Chinese Soft-shelled turtle(Pelodiscus sinensis) is one of the main aquatic products in Zhejiang Province. In this paper, the transcriptome high-throughput sequencing(RNA-seq) technology was used for developping SSR markers in Chinese Soft-shelled turtles, and successfully developed 27 pairs SSR primers. Based on these primers,population genetic diversity was analyzed in Wubie turtle population, specific SSR primers of anti-Aeromonas hydrophila and sex-related were screened seperatly.These studies can be applied in individual genetic breeding, group evolution analysis, and other SSR analysis associated with outstanding economic traits in Chinese Soft-shelled turtles.The main results are listed as followes:1. Development and validation of SSR markers soft-shelled turtleAbout 4.7 ten million reads and 73,954 unigenes were obtained by RNA-seq.. Based on the information of SSR sequences, 117 pairs of primers were designed, and 88 pairs were successfully amplified., Based on polymorphism test, 27 pairs of primers were selected, accounting for 30.68% of all primers. These 27 pairs of primers amplified 79 alleles, the average number of alleles was 2.9259, PIC values ranged between0.3207~0.6996, with an average of 0.4795, measured heterozygosity, expected heterozygosity were 0.1111~0.7778,0.2941~0.7908. This study of the genetic diversity of soft-shelled turtle and molecular marker-assisted breeding provides molecular markers available resources.2. Genetic diversity analysis of the Wubie Turtle use SSR markersQingxi Wubie turtle is a new breeding variety of Chinese Soft-shelled turtles. We successfully screened out 13 pairs of SSR primers suitable for Wubie turtle population analysis. The number of alleles from 3 to 5, and the average number of alleles was 3.36.Observed heterozygosity and expected heterozygosity were 0.1333~0.7333 and 0.3011 ~0.7609, respectively 0.4667,0.5673 average heterozygosity. By Hardy-Weinberg equilibrium test observed four sites a serious deviation from Hardy- Weinberg equilibrium (PHWE <0.01). Also we found three potential black turtle-specific SSR markers, primer 12138 bp allele, the primer 36 169 bp allele, primers 89 102 bp, 106 bp, 111 bp allele, which can be used as a candidate Ukrainian SSR markers specific turtle populations tags.3. Development ofanti-Aeromonas hydrophila SSR markersThe common disease of Chinese turtle mainly cause by bacterial, fungal, viral infection. While bacterial infection is the most popular, in which Aeromonas hydrophila disease hazards caused by the most serious.We use 27 pairs of polymorphic primers for finding anti-Aeromonas hydrophila molecular marker and genetic diversity of population.It was found that there are three pairs of potential anti-Aeromonas hydrophila SSR primer:primer 12 of 186 bp allele, primer 35 of 150 bp allele, primer 36 of 186 bp allele. These SSR markers can be used as a candidate anti-Aeromonas hydrophila population mark.4. Development of s gender-related SSR markersIn terms of shape characteristics,it's unable to identify the sex of newly hatched larval,you can find the gonads with the careful observation of dissected young turtle under microscope, but can not distinguish male and female. By means of molecular markers,you can distinguish sex during juvenile soft-shelled. The results showed that, the genetic diversity difference between femaland male did not reach significant levels. the average number of alleles of 27 pairs of primers between male and female were 2.7037,3.1111,mean observed heterozygosity were 0.3956,0.3131.The average expected heterozygosity were 0.4324,0.4504. Paired sample T test showed that,female, Na and Ho did not reach significant levels between male and femal population.