Construction Of Genetic Linkage Map For Dendrobium

Some plants of the genus Dendrobium, with great value of development and utilization and broad market prospect, are important ornamental plants for potted-flower and cut-flower. In this study, a genetic linkage map of Dendrobium was constructed by genotyping190F1progeny of the biparental cross’Zisehuoyan’x ’Fenhong No.2’with a combination analysis of SSR, ISSR and SRAP molecular markers in a double pseudo-testcross mapping strategy. The main results were reported as follows:1-. Compare the four DNA extraction methods on mapping population of Dendrobium, the results showed that the Dendrobium DNA could be effectively purified through two oscillation extractions by adding respectively the same volume of Tris-Phenol and compound of choroform and isoamyl alcohol (24:1). The values of A260/A280, A260/A230and yield of Dendrobium DNA extraction with PEX method were1.99,2.17and453.39μg/g, respectively. The Dendrobium DNA extraction using PEX method had the highest yield and better purity, of which operation was simpler. This DNA extraction method was suitable for mapping population of Dendrobium, and had obvious advantages for speed, efficiency and quality of DNA isolation on large mapping population especially.2、By using DNA of’Zisehuoyan’ and ’Fenhong No.2’as template, five main factors of template DNA, Mg2+, dNTPs, primer and Taq DNA polymerase which could affect the SSR, ISSR and SRAP amplification of Dendrobium were studied respectively. The results showed that the optimal SSR, ISSR and SRAP reaction systems were as follows:the15μl SSR reaction system contained template DNA30ng, Mg2+1.5mmol/L, dNTPs2.0mmol/L, primer0.4μmol/L, and Taq DNA polymerase1.0U; the20μl ISSR reaction system contained template DNA50ng, Mg2+1.5mmol/L, dNTPs2.0mmol/L, primer0.75μmol/L, and Taq DNA polymerase1.0U; the20μl SRAP reaction system contained template DNA80ng, Mg2+2.0mmol/L, dNTPs2.0mmol/L, primer0.6μmol/L, and Taq DNA polymerase1.0U.3、In all142pairs or strips polymorphism primers were screened from three molecular markers.A total of10pairs SSR,9ISSR and67pairs SRAP polymorphism primes were selected and used for genetic trait separation analysis in F1mapping population of Dendrobium eventually, which amplified599polymorphism marker loci in F1population. Among of39loci were amplified by SSR primers,65loci were amplified by ISSR primers and495loci were amplified by SRAP primers. Among these markers,273loci were only from female parent,172loci were only from male parent, and154loci were from parents. By way of Chi-Square Test under the P=0.1significant level,246polymorphic loci expected1:1separation ratio with accounting for41.07%,73polymorphic loci expected3:1separation ratio with accounting for12.35%, while297polymorphic loci departed from Mendelian segregation laws with accounting for46.58%. None of anomaly separation marker loci was detected.4、The genetic map was constructed by utilizing JoinMap4.0(?) software’s CP model based on the linkage analysis of421polymorphic marker loci data tested from the F1segregation population, including320Mendelian separation loci and101slight Mendelian separation distortion loci. We set a minimum value of LOD as4.0. The map included26linkage groups which contained230loci (8SSR markers,28ISSR markers,194SRAP markers) and covered genome1548.9cM with9.91cM average density between markers. Each linkage group contained a minimum of four markers. In addition, a total of76loci were distributed to10triplets and22doublets.