| To explore and use existing jackfruit germplasm efficiently, a powerful analytical tool for jackfruit germplasm research is necessary. In this thesis, SSR marker for jackfruit was developed, with the intention to provide an efficient mean for jackfruit germplasm research.This study was composed of two parts: one is the development of Jackfruit chloroplast SSR primers and analysis of genetic diversity using cpSSRs; another is the development of the nuclear genome SSR primers and analysis of genetic diversity using nSSRs. Chloroplast SSRs were developed by screening the existing chloroplast SSR primers in other plant species. Nuclear genomic SSR primer were developed by a manetic enrichment procedure, in which, jackfruit genomic DNA was digested and ligated into the AFLP DNA fragment, and then biotin-labeled simple sequence repeat (SSR) were used as a probe to hybrid with it, then hybrided complex were fixed to a streptavidin-coated magnetic beads, and after a series of washing process, the AFLP fragments containing the SSRs were adsorbed on the surface of magnetic beads. These fragments were eluted off, and cloned with pGEM-T Easy Vector System, and finally sequenced.The major findings were listed as follows:1. The reaction system involved in cpSSR analysis was optimized for jackfruit. The total volume of 20μL contained 2.5 ng/μl template DNA, 1 U Taq DNA polymerase, 0.2μmol/L primer, 0.2 mmol/L dNTPs, 1.5 mmol/L Mg2+ and 2.0μl 10×buffer. The program for the PCR amplification was that, denatured the template DNA at 94℃for 4min; denatured the DNA at 94℃for 1min; annealed 50s (the annealing temperature was decided on primer); extended at 72℃for 1min; 35cycles; extended at 72℃for 10 min.2. Totally, 23 primers were screened, and nine SSR primers showing stable amplifications were used for further analysis, In this analysis, twenty-four jackfruit germplasms were used, however, none of these cpSSR primer pairs have amplified polymorphic bands anong these 24 jackfruit germplasm.3. A total of 83 picked clones were sent for sequencing, and four clones turned out to be repeat sequences resulting in 79 sequences. In these 79 sequences, 88.6%(70/79) contained at least one micro-satellite type. 39.2% contained microsatellite of at least 3 repeat units of two nucleic acid bases.4. In the successfully sequenced 83 microsatellite sequences, 31 were effective loci, four were repeat clones. Except for those microsatellite sequences with undesired structure or too short in their flanking sequence, all the other microsatellite sequences were used to develop SSR markers using Primer 5.0 software, and finally, a total of 19 pairs of primer were obstained.5. The reaction system for SSR analysis was optimized for jackfruit. The total volume of 20μL contained 2 ng/μl template DNA, 1 U Taq DNA polymerase, 0.2μmol/L primer, 0.2 mmol/L dNTPs, 2.0 mmol/L Mg2+ and 2.0μl 10×buffer. The program for the PCR amplification was that, denatured the template DNA at 94℃for 4min; denatured the DNA at 94℃for 1min; annealed 50s (the annealing temperature was decided on primer); extended at 72℃for 1min; 35 cycles; extended at 72℃for 10 min.6. Among the 68 germplasm used for the genetic analysis using nuclear genomic SSRs, 18 SSR primers amplified 50 bands. In which, polymorphic bands were 44, polymorphic loci were 88%. Primer JSR6 amplified the most bands, which amplified 5 bands.7. Of the 44 loci, the average PIC (polymorphism information content, polymorphism information content) were 0.24, and PIC value of 13 loci ranged from 0.40 to 0.50 and 0.00 to 0.10, PIC value of four loci ranged from 0.10 to 0.20. Primers JSR17 showed the highest PIC value (0.4341); the lowest PIC value was 0.0938.8. Genetic similarity coefficient of the 68 germplasm was between 0.5763 and 0.9655, with an average of 0.8140.9. UPGMA clustering based on SSR data revealed that, the jackfruit germplasm used in this study were completely divided into two groups. The first group included all germplasm collected from Hainan, Yunnan and part of germplasm collected from Leizhou peninsula; the second group included jackfruit germplasm collected from Pakistan, champedak and the remained jackfruit germplasm collected from Leizhou penisula. Germplasm collected from Hainan, Yunnan and Pakistan was separated from each other. But germplasm collected from Malaysia could not be distinguished. Accessions of soft flesh type and firm flesh type could not be discriminated. |
