Study On The Gene Introgression Between Pinus Massoniana And P. Hwangshanensis

Using pine ESTs deposited in the public database, we performed analyses on microsatellites search, primer design and primer screen. SSR primer pairs that generated clear band profile were selected with megagametophytes from a single tree in the hybrid zone, and these primers were employed to track the gene introgression between P. massoniana and P. hwangshanensis. Needle samples of 270 trees were collected according to different elevations from Huang Mountian in Anhui province. These samples were divided into 8 groups based on the elevation gradients. By analyzing the allele frequency, gene introgression trends varying with elevations were revealed. The main studies and the derived results were as follows:1.The seed germination ratio, percentage of plump seeds, and the effective seed germination ratio of cones collected from different elevation were experimentally tested and statistically analyzed. It is noteworthy that germination ratio drops sharply for seeds collected from trees in hybrid zone (about 900-1100m). We proposed it may relate to the reproduction barriers between P. massoniana and P. hwangshanensis.2.Molecular markers were developed to investigate the genetic mechanism underlying speciation of P. massoniana and P. hwangshanensis. In order to obtain bioinformatics reference, we carried out in-silicon analyses on comparison of microsatellites in the EST sequences from different tree species, including pine, poplar and eucalyptus. Microsatellite sequences are important characteristics of eukaryotic genomes, and their feature is shaped by the evolution process of the focal organism. This part of work provides desirable bioinformatics guidance for developing SSRs to study the speciation of P. massoniana and P. hwangshanensis.3.SSR primers were designed following searching the microsatellites in pine ESTs. Thereafter,301 pairs of SSR primers were synthesized, and 218 primer pairs succeeded in PCR amplification. Then 150 primer pairs were tested for their transferability in Pinaceae. Results showed that the tested SSR primers were highly transferable in Pinaceae, with percentage greater than 90%. Base on primer amplification test, the 150 primer pairs were further tested with 8 megagametophytes of seeds from a single tree located in the hybrid zone.4.Based on the amplification experiment with the haploid pedigree materials,14 pairs of primers that yielded clear band profile were chosen to amplify allelic variation for the collected samples to reveal the introgression trends varying with elevation between P. massoniana and P. hwangshanensis. Totally,56 alleles were obtained from this experiment. The number of alleles amplified by each primer was between 2 and 6. Most alleles (about 70%) were found to have no obvious introgression trends with changes in elevations. By contrast, frequencies of 17 alleles were found to vary distinctly with elevation changes,12 of them decrease in frequency with an increase in elevation, and the other 5 increase in frequency with an increase in elevation. The above results implied that locus from different part of the genome varied in their introgression ability, and introgression occurred in two-way direction between two species, Introgression from P. massoniana to P. hwangshanensis was dominant. The different introgression ability indicated that genetic materials from different regions of the genome played different role in maintain the speciation of the two species. Our results provide direct evidence for our better understanding of the genetic mechanism triggering the speciation of P. massoniana and P. hwangshanensis.