| Bamboo (Bambusoideae), the largest subfamily of the grasses (Poaceae), contains about 70 genera and 1200 species. Bamboo, the fastest growing and most widely-used woody species in the world, which is of economic significance, furthermore it plays an important role in the function of water conservation and soil conservation. It is difficult to classify and identify bamboos by using the traditional taxonomy method, which is based on the forms of flowers and fruit, because they seldom flowering and once it happens the plant or even the whole bamboo forest become dead. At present, the current bamboo classification is always in dispute in the academic field, and the application of molecular marker technology provide the basis for bamboo taxonomy. Simple Sequence Repeat or Microsatellite marker is an ideal tool for germplasm identification, genetic diversity and pedigree analysis, because they are abundant in genomic DNA, high polymorphism, easy to detect and uninfluenced by environment and gene expression. However, seldom bamboo microsatellite marker have been reported.This research developed 31 pairs of bamboo SSR primers by using the method of microsatellite enrichment of magnetic beads and analyzed 62 samples in 4 genera. The results are as follows:1. Three microsatelite enriched libraries (GT, AG, CCA) were constructed from Ph. heterocycla var. pubescens (Mazel) Ohwi genomic DNA, 2330 clones were screened by colony-PCR, with 1080, 620, 630 clones respectively, 219, 113, 119 positive clones were sequenced, the ratiowas 19.4% (451/2330).2. 350 non-redundancy sequences were obtained, which were between 61 bp and 831bp. The average length was 288bp, and the total length was 100 704bp, about 1/20 000 the size of Ph. heterocycla var. pubescens (Mazel) Ohwi genomic DNA. 641 SSR sites were found, the number of repeat motifs in each site were between 3~54, with 415, 86, 37, 14, 19, 8, 12, 11, 39 sites of 3, 4, 5, 6, 7, 8, 9, 10, >10 repeat unit. The sites of dinucleotide composed the most, with 521 sites, trinucleotide, tetronucleotide were 108, 35 sites respectively. 3. Blast analysis of obtained sequences showed that: 63 sequences producing significant alignments to Ph. edulis sequences in GenBank, composed the most, and 28, 11, 3, 3 sequences producing significant alignments to rice, sweet sorghum, wheat and corn respectively, which consistent with the phylogenetic position of Ph. heterocycla var. pubescens (Mazel) Ohwi.4. 53 pairs of SSR primers were designed, 34 of which amplified target fragment from Ph.heterocycla var. pubescens (Mazel ) Ohwi genomic DNA and the annealing temperature were determined by using temperature gradient PCR.5. FAM labeled forward primer of 34 pairs of primers, capillary electrophoresis analyzed the length of SSR-PCR product of 9 samples of intraspecies of moso bamboo, loci 27, 36, 40 amplified specific fragments, clone and sequenced several fragments of these three primers' PCR amplification product in Ph. heterocycla cv. Luteosulcata. The result showed that, these loci manifested gene duplication. The other 31 loci analysis of 9 species showed that 31 loci amplified expected alleles at all 8 samples of moso bamboo except Ph. heterocycla cv. Luteosulcata. Ph. heterocycla cv. Luteosulcata manifested difference to the other 8 samples. Loci 2, 17, 38, 42, 50 showed null allele, loci 8, 13, 15, 18, 21, 24, 28, 31, 32, 33, 37, 41, 43, 46, 49, 51 ampified specific alleles to other 8 species, these loci takes the rate at 61.8% (5+16/34).6. 31 paris of developed SSR markers were used to assess the genetic diversity among the 8 moso bamboo, 18 loci were amplified only one band, 13 loci were polymorphism, at the rate of 41.94 %, the observed number of alleles were between 1~3, at an average of 1.548, and the effective number of alleles were between 1~2.6, at an average of 1.375; the Observed heterozygosities and Expeeted heterozygosities were between 0~1, 0~0.67, at an average of 0.309, 0.201. The PIC (Polymophism Information Content) is between 0~0.575, at an average of 0.147, showed the low level of mutation in these 8 moso bamboo.7. Capillary electrophoresis analyzed 31 SSR loci in 11 species belongs to 3 genera Semiarundinaria,Sinobambusa,Indosasa, Ntsys software calculated Nei's genetic distance and clustering analyzed by unweighted pair-group method with arithmetic means, the result showed that these 31 loci could distinguish these species 3 genera. 8. Capillary electrophoresis analyzed 31 SSR loci in 51 species (varieties or types) of genus Phyllostachys, the result showed that: On the whole, the clustering result consistent with the traditional taxonomy, shows that these SSR loci could be used to classify the genetic distance of Phyllostachys; the genetic distance between intraspecies is larger than 0, shows the presence of genetic variation and genetic polymorphism between them, so these loci could developed for species identification markers; the result also shows the genetic distance between species is larger than intraspecies, so these loci also could used for defining species which is in disputing.
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