| Dragon fruit(Hylocereus spp.),a kind of rising tropical and subtropical fruit belonging to the Hylocereus genus or Seleniereus genus of the Cactaceae family.Based on the RNA-Seq of Hylocereus polyrhizuse,the pairs of EST-SSR primers were designed and synthesized,and the optimal system EST-SSR amplification was established.As the same time genomic DNA of 38 pitaya germplasms which distinguished obviously in phenotype was used to carry out PCR and preliminary election,in order to provide a reliable marker system for germplasm identification,genetic relationship elucidation,as well as genetic mapping of pitaya.1)A total of 7,622 SSR loci were identified from 108,127 unigenes in the transcriptomic sequence of Hylocereus polyrhizus using MISA software.The frequency of these SSRs was 6.02%,and the mean distance per locus was 9.00 kb.The type of single nucleotide SSR was the most abundant,which counted for 56.59%of the total loci,followed by dinucleotide and trinucleotide,accounting for 28.4% and14.4% of the total,respectively.The other types of SSR accounted for less than 1%.Dinucleotide repeat motifs of AG/CT and AC/GT were the predominant repeat types,accounting for 25.37% and 2.02% of the total,respectively.The trinucleotide repeat motifs of AAG/CTT was the predominant repeat type that counted for 3.31% of the total.2)Based on the RNA-Seq of Hylocereus polyrhizuse,EST-SSR markers were investigated.SSR primers were designed and synthesized,and genomic DNA of 11 pitaya germplasms which distinguished obviously in phenotype were used to carry out PCR.To optimize the experimental system: effects degree of three factors including2×Taq PCR MasterMix,primers and DNA template concentration was analyzed respectively through an orthogonal test.Furthermore,the suitable loading quantityand concentration of de-naturing polyacrylamide gel electrophoresis were determined.The results showed that the optimal amplification system was 10?L mixtures containing 30 ng DNA template,1.2×Taq PCR MasterMix,and 0.6 ?M of each EST-SSR primer.The optimal concentration for de-naturing polyacrylamide gel electrophoresis was 10%,and the loading quantity was 2.5 ?L.3)Totally,125 pairs of EST-SSR primers were designed and synthesized,and genomic DNA of eight pitaya germplasms which distinguished obviously in phenotype was used to carry out PCR.Agarose gel electrophoresis and 10% of de-naturing polyacrylamide gel electrophoresis were performed for the preliminary election.The results showed that bands of 32 primer pairs were clear and sharp,among which 16 pairs showed polymorphic bands among the 38 accessions of pitaya.A total of 47 SSR markers were scored from 38 accessions of pitaya amplified by the screened EST-SSR primers.The polymorphism information content(PIC)was0.243~0.667 with the average of 0.459.The similarity coefficient of the 38 germplasms ranged from 0.32 to 0.92.And the average observed number of alleles(Na)and Nei's genetic diversity(I)in the 38 pitaya gemplasms were 3 and 0.891,respectively.4)The investigated germplasms could be completely distinguished by 8 primer pairs,e.g.C31931?C13719 and C3214.The UPGMA dendrogram demonstrated that at a genetic distance of 0.62,the 38 germplasms could be divided into three groups.The first group containing the red pulp accessions and the pink pulp accessions,the second including white pulp accessions,and the third only consisting of two accessions belonging to Seleniereus.Therefore,the unigenes generated from RNA-Seq of Hylocereus polyrhizus can be used as an effective source to develop EST-SSR markers,which provide a reliable marker system for germplasm identification,genetic relationship elucidation,as well as genetic mapping of pitaya. |
PDF Full Text Request