Development And Application Of SSR Markers From Pineapple (Ananas Comosus L., Merr.)

Pineapple is evergreen perennial herbs,tropical fruit, one of the forth most important tropical fruit with banana, coconut and mango. It has been many varieties, varieties and types by long-term process of the natural evolution and artificial domestication. Over the years, the classification of pineapple are still have some problems, such as the Standard is difficult to unity, results homonym and synonym phenomena exist in the regional and international frequent communication of germplasm; This is not only hinders the rational utilization of pineapple germplasm resources, but also affected the cultivar improvement and breeding.The experimental development of pineapple simple sequence repeat (Simple Sequence Repeat, SSR) markers. Lay the foundation of pineapple cultivar identification, population genetics, genetic linkage mapping and molecular maker assisted breeding and so on. The major results are as follows:From the NCBI database searched pineapple EST sequence 5660 (as of December 1, 2008), which contains the sequence of SSR repeat loci 1110, developed the 190 pairs EST-SSR primers. Pineapple transcript group non-redundant EST-SSRs in the distribution of frequencies is about 1SSR/3.73kb, most are smaller repeating units, the dominant dinucleotide repeat, in which AG/CT (83.5%) is the dinucleotide the most abundant repeat loci; randomly selected 30 pairs of primers, got an effective amplification of 22 SSR markers. Based on cluster analysis and pedigree analysis consistent with the results show that the development of EST-SSR markers can be used to evaluate genetic diversity of Pineapple.Using SAM method development genomic SSR markers, chosen 99 clones to sequence and find SSR loci, we found 86 sequences containing SSR loci. Using Cluster analysis stackPACK v2.2, got 68 single sequence repeats and 8 groups reapte sequence, indicating that only 76 sequences can be used to design primers. Selection of the design of primers for DNA sequence of 36 to 36 pair of primers; these,24 pairs of primers were effective amplification, 13 pairs of primers in 16 different pineapple germplasm showed polymorphism. PstI SAM primers and 5’anchored primer PAC/PGT’s portfolio was developed with 44 sequences, PstI SAM primers and 5’anchored primer PCT/PGA combination was developed with 55 sequences, indicate that the pineapple genome, CT/GA ratio AC/GT-rich.Using the development of the 18 SSR markers to analysis the genetic diversity of 48 accessions, clustering results show that the similarity coefficient of 0.66 can be divided into four groups:the first group is Riply Queen as represented by 25 species, the second, there is no thorn lidocaine groups on behalf of the 20 species, the third group only 2000sh2 of two varieties of golden pineapple, the fourth groups belonging to the red spain of the Spanish class.The results consistent with morphological classification. Selection of the five markers were amplified by 10 materials, recycling and sequencing results showed that different markers are amplified to the corresponding sequence repeat loci, Loci mainly by the SSR repeat number and flanking sequences mutations.Preliminary construction of 31 fingerprints of pinapple germplasm by using 5 SSR makers. It also was on the foundation the next system of SSR fingerprints construction.