Phyllostachys edulis (Carrière) J. Houz. (Poaceae) is the most ecologically and economically important bamboo species in China, and accounts for more than 70% of commercially planted bamboo (Fu, 2001). Because of its wide commercial value, this bamboo was widely cultivated in past decades, which unavoidably decreased the range of natural bamboo stands. Therefore, currently the investigation of gene diversity and preservation of genetic resources are crucial issues. Microsatellite markers are increasingly used for understanding population genetics and evolution (Thomson et al., 2010). A few simple sequence repeat (SSR) markers have been reported for this bamboo in recent years; however, they were only applied for interspecies identification (Tang et al., 2010). To date, studies on the population genetics of P. edulis are still rare because of limited codominant markers. The 20 polymorphic SSRs presented in our study will be valuable for determining the molecular ecology and population genetic structure in P. edulis.
METHODS AND RESULTS
In total, 10,608 cDNAs of P. edulis were downloaded from the National Center for Biotechnology Information (NCBI) database. A total of 425 SSRs ≥20 nucleotides in length (unit/minimum number of repeats: 2/10, 3/7, 4/5, 5/4) were identified from nonredundant P. edulis cDNAs using the Simple Sequence Repeat Identification Tool (SSRIT; http://www.gramene.org/db/markers/ssrtool; Temnykh et al., 2001). Primer Premier 5 software (PREMIER Biosoft International, Palo Alto, California, USA) was used to successfully design 191 primer pairs against the sequences flanking each SSR according to these criteria: optimum annealing temperature ranging from 52°C to 62°C; maximum of 3°C difference in annealing temperature between primer pairs; GC content of 40–60%; and PCR product size of 100–380 bp.
To characterize microsatellite loci polymorphisms, we genotyped 71 individuals from three natural populations in China, including Renhua (RH), Guizhou (25°7′–21′N, 113°48′–58′E); Jianou (JO), Fujian (26°58′-27°9′N, 118°13′–18′E); and Xianning (XN), Hubei (29°37′–48′N, 114°10′–18′E). The interval between samples, to avoid being from the same genet, was at least 1 km in every population. Voucher specimens were deposited at the Herbarium of Nanjing Forestry University (Appendix 1). Total genomic DNA was extracted from silica gel–dried young leaves using the cetyltrimethylammonium bromide (CTAB) method (Doyle and Doyle, 1990) with minor modifications. PCR was performed in a 20-µL reaction volume containing 50–70 ng of template DNA, 2 µL of 10× PCR buffer, 0.1 mM dNTPs, 0.87 mM MgCl2, 0.48 µM of each primer, and 1 unit of Taq DNA polymerase (TaKaRa Bio Inc., Otsu, Shiga, Japan). All PCR reactions were performed in an Eppendorf Mastercycler gradient PCR thermal cycler (Eppendorf, Hamburg, Germany) using a modified touchdown protocol (Don et al., 1991): 94°C for 5 min; 12 cycles of 94°C for 30 s, 62°C decreasing to 50°C at 1°C per cycle for 30 s, 72°C for 30 s; 20 cycles of 94°C for 30 s, 52°C for 30 s, 72°C for 30 s; and a final extension at 72°C for 5 min. After prescreening 12 individuals (randomly sampled from the three populations), 20 of 191 microsatellite loci were identified based on their PCR results and obviously variable bands detected in an 8% denaturing Polyacrylamide gel with silver nitrate.
The final 20 loci were 5′ end-labeled using a forward primer with 6-FAM or 6-HEX and genotyped for three populations (Tables 1 and 2). All PCR products were separated with GeneScan 500 ROX Size Standard on an ABI 3730xL DNA analyzer (Applied Biosystems, Carlsbad, California, USA), and fragment sizes were estimated with GeneMapper version 4.0 (Applied Biosystems). Number of observed alleles (A), observed and expected heterozygosity (Ho and He), Shannon's information index (I), deviations from Hardy–Weinberg equilibrium (HWE), and linkage disequilibrium (LD) between loci were estimated using GenAlEx version 6.5 (Peakall and Smouse, 2012).
All loci displayed polymorphisms when compared across populations, with the total A ranging from two to 10 alleles per locus and an average of five alleles. Excluding monomorphic loci, Ho and He were from 0 to 1 and from 0.041 to 0.676, respectively, while I ranged from 0.101 to 1.443 (Table 2). A total of 14 loci significantly (P < 0.005) deviated from HWE in all three populations, and there was no significant LD among all pairs of loci. Similar results were reported in other bamboo species (Kaneko et al., 2008; Miyazaki et al., 2009). The deviation at 14 loci was possibly caused by nonrandom mating within populations due to the unique biological characteristics of bamboo species, such as their highly clonal propagation, monocarpic nature with gregarious flowering, and long flowering intervals (67–120 yr) (Janzen, 1976; Watanabe et al., 1982), as well as the decreasing size of wild populations.
TABLE 1.
Characteristics of 20 polymorphic microsatellite loci for Phyllostachys edulis. a
CONCLUSIONS
Twenty novel microsatellite loci showed a useful degree of polymorphism at the population level and will be helpful for molecular ecological studies of P. edulis, such as clonal identification, genetic structure, the evolution of gregarious flowering behavior, as well as for elucidating the biogeographic history of this bamboo species.
TABLE 2.
Polymorphism analyses using 20 markers in three geographically disparate populations of Phyllostachys edulis. a
