Exploit SSR in Cryptromeria fortunei and application in genetic diversity analysis

Abstract

Cryptromeria fortunei is one of the main forest plantation species in the subtropical high altitude areas in China. In this paper we collected 49 C. fortunei germplasm resources and provides a study of the utility of freely available C. japonica EST resources for the development of markers necessary for genetic diversity analyses of C. fortunei. By screening 24,299 EST sequences from C. japonica with SSR Finder, we identified 2384 ESTs carrying 2783 SSR motifs. We successfully obtained 364 (15 %) primers from 2419 putative SSR loci. Of the 80 candidate SSR markers tested, 70 (87.5 %) yielded stable and clear PCR products. With those primers, the genetic diversity of 49 C. fortunei we collected was studied. The results showed that 18 primers yield polymorphism within these accessions. These 18 primers generated 48 scorable SSR loci and the average number of polymorphic SSR loci per primer was 2.7. The PIC value varied from 0.375 to 0.8101, with the average of 0.4780. The Shannon index is 0.5718, and the value of the observed number of alleles and effective number of alleles are 1.9167 and 1.7289, respectively. The genetic coefficient of these 49 accessions varied from 0.28 to 0.87. According to the genetic distances, a cluster tree was constructed. At genetic coefficient of 0.60, these 49 accessions can group into 3: group I contains only FJ-laizhou accession, and group II contains 2 accessions from FJ-layang, and the other one group contains mixed accessions. At genetic coefficient of 0.68, the former group II was constructed into 7 subgroups, with the first 3 subgroups contain 16 accessions in which 11 (69 %) are from Fujian province, and the later 4 subgroup contain 31 accessions in which 20 (65 %) were from Zhejiang province.

Keywords

Cryptromeria fortunei / EST–SSR / Germplasm collection / Genetic diversity

References

[1] Adams MD, Kelley JM, Gocayne JD, Dubnick M, Polymeropoulos MH, Xiao H, Merril CR, Wu A, Olde B, Moreno RF, Kerlavage AR, McCombie WR, Venter JC. Complementary DNA sequencing: expressed sequence tag and human genome project. Science, 1991, 252: 1651-1656.

[2] Enorki H, Sato H, Koinuma K. SSR analysis of genetic diversity among maize inbred lines adapted to cold regions of Japan. Theor Appl Genet, 2002, 104: 1270-1277.

[3] Felsenstein J. PHILIP-Phylogeny inference package. Cladistics, 1989, 5: 164-166.

[4] Futamura N, Totoki Y, Toyoda A, Igasaki T, Nanjo T, Seki M, Sakaki Y, Mari A, Shinozaki K, Shinohara K. Characterization of expressed sequence tags from a full-length enriched cDNA library of Cryptomeria japonica male strobili. BMC Genomics, 2008, 9: 383-396.

[5] Lu Y, Jia Q, Tong Z. Development of amplified consensus genetic markers in Taxodiaceae based on Cryptomeria japonica ESTs data. J For Res, 2013, 24: 503-508.

[6] Moriguchi Y, Iwata H, Ujino-Ihara T. Development and characterization of microsatellite markers for Cryptomeria japonica D. Don. Theor Appl Genet, 2003, 106: 751-758.

[7] Moriguchi Y, Ueno S, Ujino-Ihara T, Futamura N, Matsumoto A, Shinohara K, Tsumura Y. Characterization of EST–SSRs from Cryptomeria japonica. Conserv Genet Resour, 2009, 1: 373-376.

[8] Murray MG, Thompson WF. Rapid isolation of high molecular-weight plant DNA. Nucl Acids Res, 1980, 8: 4321-4325.

[9] Nei M, Li W. Mathematical model for study the genetic variation in terms of restriction endonucleases. Proc Natl Acad Sci, 1979, 74: 5267-5273.

[10] Riar DS, Rustgi S, Burke IC, Gill KS, Yenish JP. EST–SSR development from 5 Lactuca species and their use in studying genetic diversity among L. serriola biotypes. J Hered, 2011, 102: 17-28.

[11] Rozen S, Skaletsky H. Primer3 on the WWW for general users and for biologist programmers. Methods Mol Biol, 2000, 132: 365-386.

[12] Sehlarbaum SE, Tsuehiya T. Cytotaxonomy and phylogeny in certain species of Taxodiaceae. Syst, 1984, 147: 29-54.

[13] Takahashi T, Tani N, Taira H, Tsumura Y. Microsatellite markers reveal high allelic variation in natural populations of Cryptomeria japonica near refugial areas of the last glacial period. J Plant Res, 2005, 118: 83-90.

[14] Tani N, Takahashi T, Iwata H, Mukai Y, Ujino-Ihara T, Matsumoto A, Yoshimura K, Yoshimaru H, Tsumura Y. A consensus linkage map for Sugi (Cryptomeria japonica) from two pedigrees, based on microsatellites and expressed sequence tags. Genetics, 2003, 165: 1551-1568.

[15] Tani N, Takahashi T, Ujino-Ihara Y, Iwata H, Yoshimura K, Tsumura Y. Development and characteristics of microsatellite markers for sugi (Cryptomeria japonica D. Don) derived from microsatellite-enriched libraries. Ann For Sci, 2004, 61: 569-575.

[16] Ueno S, Moriguchi Y, Uchiyama K, Tsumura Y. A second generation framework for the analysis of microsatellites in expressed sequence tags and the development of EST–SSR markers for a conifer, Cryptomeria japonica. BMC Genomics, 2012, 13: 136-151.

[17] Wang J, Liu J, Huang Y, Yang H. The origin and natural distribution of Cryptomeria. J Sichuan For Sci Technol, 2007, 28: 92-94. (in Chinese with an English abstract)

[18] Xu S, Tao Y, Yang Z, Chu J. A simple and rapid method used for silver staining and gel preservation. Hereditas, 2002, 24: 335-336. (in Chinese with an English abstract)

[19] Xu Y, Chen J, Li Y, Hong Z, Wang Y, Zhao Y, Wang X, Shi J. Development of EST–SSR and genomic-SSR in Chinese fir. J Nanjing For Univ, 2014, 38(1): 9-14. (in Chinese with an English abstract)

[20] Yang L, Jin G, Zhao X, Zheng Y, Xu Z, Wu W. PIP: a database of potential intron polymorphism markers. Bioinformatics, 2007, 23: 2174-2177.

[21] Zhang Y, Peng C, Li Z, Yang Y, Hu X. Genetic diversity of genomic-SSR and EST–SSR markers in interspecies of Poplar. J Northeast For Univ, 2011, 39(12): 8-11. (in Chinese with an English abstract)