- Email: [email protected]
Research of Genetic Diversity in Seven Kobresia by AFLP in Tibetan Plateau
Agricultural Sciences in China
August 2009
2009, 8(8): 994-999
Research of Genetic Diversity in Seven Kobresia by AFLP in Tibetan Plateau ZHENG Hong-mei1, HU Tian-ming2, WANG Quan-zhen2, ZHANG Guo-yun3 and SONG Jiang-hu2 College of Resources and Environment, Northwest A&F University, Yangling 712100, P.R.China College of Animal Science and Technology, Northwest A&F University, Yangling 712100, P.R.China 3 Key Laboratory of Agricultural Molecular Biology of Shaanxi Province, Yangling 712100, P.R.China 1 2
Abstract This work analyzed the genetic diversity of Kobresia accessions at the molecular level, and further obtained the necessary information for breeding and germplasm evaluation. Genomic DNA of Kobresia was amplified with four E+3 and M+3 primer combinations with AFLP (amplified fragment length polymorphism). AFLP analysis produced 164 scorable bands, of which 154 (93.96%) were polymorphic. The mean Nei’s gene diversity index (H) was 0.2430, and the Shannon’s information index (I) was 0.4012, indicating the abundant genetic diversity of Kobresia. The 11 Kobresia accessions from Tibetan Plateau, China, can be classified into five groups after cluster analysis based on the UPGMA (unweighted pair group method arithmetic average) method. In general, there was abundant genetic diversity among Kobresia accessions resources, and the genetic coefficient was unrelated to their geographic latitude. Natural habitats influenced genetic differentiation of Kobresia. Key words: Kobresia, genetic diversity, AFLP
INTRODUCTION Kobresia Willd., a perennial grass, belongs to the sedge family and carex subfamily; most members of the sedge family are constructive species on the alpine meadow. Kobresia was enjoyed by cattle for its softness and rich quality, and is also an important plant for controlling soil erosion and maintaining ecology balance (Zhou et al. 1982). This study will promote ecological construction of alpine rangeland, improve natural grassland, and develop the grassland-animal husbandry industry. The method of AFLP (amplified fragment length polymorphism) has been applied to study the biodiversity (Barrett et al. 1998; Bohn et al. 1999), map construction (Caicedo et al. 1999; Zhong et al. 2004), cultivar identification (Renganayaki et al. 2001; Lombard et al.
2000), locating, isolating genes (Julio et al. 2006; Dong et al. 2000), heterosis prediction (He et al. 2002), molecular marker assisted breeding (Lokko et al. 2005), and so on. Great progress has been made in the research of many plants, such as wild cocksfoot, lupin, leymus chinensis, tobacco, corn, and so on (Peng et al. 2006; Yang et al. 2006; Liu et al. 2002; Hai et al. 2002; Hao et al. 2003). Although some preliminary researches have also been done on Kobresia germplasm resources by RAPD (random amplified polymorphic DNA) molecular markers (Li et al. 2006; Zhao et al. 2006), the study on Kobresia by AFLP has not been reported in literature. The main objective of the present study is to analyze Kobresia genetic diversity in different habitats by AFLP molecular marker and provide evidence for comprehensive evaluation of Kobresia germplasm.
This paper is translated from its Chinese version in Scientia Agricultura Sinica. Correspondence HU Tian-ming, Professor, E-mail: [email protected]
© 2009, CAAS. All rights reserved. Published by Elsevier Ltd. doi:10.1016/S1671-2927(08)60305-3
Research of Genetic Diversity in Seven Kobresia by AFLP in Tibetan Plateau
995
MATERIALS AND METHODS
separation production (Bassam et al. 1991).
Materials
Data statistic and analysis method
Eleven plant accessions representing seven different species were sampled from Tibet alpine on September 2006. Details on these genotypes including geographic origin and geographic information system (GIS) data, wherever possible, are provided in Table 1. Sampling vouchers were deposited at the herbarium of Northwest A&F University, China.
Data analysis By AFLP polymorphic bands statistical method, each allele was scored as present (1) or absent (0) for each loci. Each band was scored which is clear and repeated, and the size ranged from 100-500 bp in length. Analysis method The percentage of polymorphic loci, Nei’s gene diversity index (Nei 1973), Shannon’s information index (Lewontin 1972), and gene flow (Nm) were calculated using POPGENE1. 31 (Yeh et al. 1999). The numerical taxonomy and multivariate analysis system (NTSYS-pc 2.1) was constructed using unweighted pair group method arithmetic average (UPGMA) dendrograms of AFLP, the Mantel (1967) test statistic (Z) infered correlation genetic coefficient based on the geography distance matrix using the Mxcomp procedure of NTSYS-pc (Nei and Li 1979). Principal coordinate analysis was carried out by EIGEN.
Methods Genomic DNA of each accession was extracted from a pool of 30 plants using the modified CTAB method (Chen and Ronald 1999). DNA concentration was measured using a UV spectrophotometer, and concentrations were adjusted to 50 ng L-1 for PCR analysis. AFLP optimized procedure refers to Vos’s method and other previous research (Vos et al. 1995; Yan et al. 2004). PAG electrophoresis and silver staining detect electrophoretic
Table 1 The environmental data for the different accessions of Kobresia Species
Code
Origion
K. royleana (N.) B. K. macrentha Boeck. K. littledalei C. B. Clarke K. royleana (N.) B K. prattii C. B. Clarke K. caillifolia Decne K. littledalei C. B. Clarke K. littledalei C. B. Clarke K. humilis C. A. Mey K. humilis C. A. Mey K. pygmaea C. B.
C1 C2 C3 C4 C5 C6 C7 C8 C9 C10 C11
Langkazi Langkazi Langkazi Naqu Dangxiong Dangxiong Naqu Dangxiong Naqu Dangxiong Naqu
Altitude (m) 4 455 4 455 4 455 4 456 4 278 4 278 4 456 4 278 4 456 4 278 4 456
Latitude and longitude 28°59´11N 28°59´11N 28°59´11N 31°26´34N 30°29´35N 30°29´35N 31°26´34N 30°29´35N 31°26´34N 30°29´35N 31°26´34N
90°26´04E 90°26´04E 90°26´04E 92°16´32E 91°05´58E 91°05´58E 92°16´32E 91°05´58E 92°16´32E 91°05´58E 92°16´32E
Habitat Alpine steppe Alpine meadow Alpine meadow Alpine steppe Alpine steppe Alpine meadow Marshy meadow Marshy meadow Alpine steppe Alpine steppe Alpine meadow
RESULTS Genetic diversity of Kobresia Different primers were screened and all were found to be polymorphic. The band numbers and the fragment length were different in each primer. Four primers were identified out of the 20 primers (Fig.1). AFLP markers, which amplified 154 polymorphic bands, showed high levels of polymorphism by the PIC value ranging from 87.80% (primer pair E-ACC + M-CAA) to
Fig. 1 AFLP fingerprint of 11 Kobresia accessions.
© 2009, CAAS. All rights reserved. Published by Elsevier Ltd.
996
ZHENG Hong-mei et al.
96.97% (primer pair E-ACA + M - CTG) with an average of 93.96% across the germplasm assayed (Table 2). The mean Nei’s gene diversity index (H) of all accessions was 0.2430, the Shannon’s information index (I) was 0.4012, indicating that the genetic diversity of Kobresia is very rich. E-ACC+M-CAG, E- ACA+MCAG, E-ACA+M-CTG, E-ACC+M-CAA detected effective number of alleles (Ne) ranged from 1.2393 to 1.2838; gene diversity ranged from 0.1586 to 0.1894 (Table 3). The above indicators suggested that the genetic germplasm of Kobresia showed great variability within species, high polymorphic, and abundant genetic diversity.
Analysis of Kobresia germplasm genetic distance and cluster analysis
Table 2 The AFLP amplified result of Kobresia accessions
Table 3 Estimates of genetic diversity among Kobresia (mean ± SD)
Primers
Total loci
E-ACC+M-CAG EACA+M-CAG E-ACA+M-CTG E-ACC+M-CAA Total Average
47 43 33 41 164 41
Proportion of polymorphic loci
Polymorphic loci 45 41 32 36 154 38.5
95.74 95.34 96.97 87.8
A matrix of the Jaccard’s coefficients of similarity based on the data of AFLP markers was calculated between accessions as shown in Table 3. Similarity coefficient of pairwise comparisons among different accessions yielded values ranging from 0.1159 to 0.4390, its mean value for all possible pairwise comparisons was 0.2774. Maximal genetic distance came from accession C1 and accession C10, minimum genetic distance came from accession C3 and accession C4, genetic identity among accession of Kobresia reached 0.56098 to 0.88415 (Table 4).
Primer combinations
Effective number of alleles
Nei’s gene diversity
Shannon’s information index
E-ACC+M-CAG E-ACA+M-CAG E-ACA+M-CTG E-ACC+M-CAA
1.2838 ± 0.2883 1.2781 ± 0.2770 1.2393 ± 0.2735 1.2439 ± 0.3028
0.1894 ± 0.1456 0.1871 ± 0.1457 0.1602 ± 0.1553 0.1586 ± 0.1595
0.3176 ± 0.1892 0.3122 ± 0.1958 0.2614 ± 0.2274 0.2622 ± 0.2222
93.96
Table 4 Coefficient in the accessions of Kobresia ID C1 C2 C3 C4 C5 C6 C7 C8 C9 C10 C11
C1
C2
C3
C4
C5
C6
C7
C8
C9
C10
C11
1.0000 0.2379 0.2379 0.2317 0.2744 0.3110 0.2805 0.2805 0.2866 0.3720 0.3598
0.7621 1.0000 0.2439 0.2988 0.3171 0.3293 0.3232 0.3232 0.2927 0.4146 0.4390
0.7621 0.7561 1.0000 0.1890 0.2317 0.2073 0.3232 0.2988 0.2927 0.3415 0.3780
0.7683 0.7012 0.8110 1.0000 0.1402 0.2378 0.2561 0.2195 0.2134 0.3232 0.3475
0.7256 0.6829 0.7683 0.8598 1.0000 0.3049 0.2256 0.2378 0.2073 0.3049 0.3536
0.6890 0.6707 0.7927 0.7622 0.6951 1.0000 0.3598 0.3720 0.3659 0.4024 0.4268
0.7195 0.6768 0.6768 0.7439 0.7744 0.6402 1.0000 0.1829 0.2134 0.2988 0.4085
0.7195 0.6768 0.7012 0.7805 0.7622 0.6280 0.8171 1.0000 0.1159 0.2866 0.3963
0.7134 0.7073 0.7073 0.7866 0.7927 0.6341 0.7866 0.8841 1.0000 0.2805 0.3415
0.6280 0.5854 0.6585 0.6768 0.6951 0.5976 0.7012 0.7134 0.7195 1.0000 0.3902
0.6402 0.5610 0.6220 0.6524 0.6463 0.5732 0.5915 0.6037 0.6585 0.6098 1.0000
Nei’s genetic identity (above diagonal); genetic distance (below diagonal).
The eleven accessions can be classified into five major genetic groups when genetic similarity is 0.74 (Fig.2). Accessions C6, C9, C10, and C11 cluster together, accessions C7 and C8 are in one group, accessions C3, C4, and C5 had a closer relationship, and accessions C1 and C2 do not group with any others.
Genetic structure of Kobrsia Supposing genetic equilibrium by means of POPGENE32 software, genetic diversity within Kobresia accessions
Fig. 2 The dendrogram generated by AFLP data using UPGMA method among 11 Kobresia accesions.
© 2009, CAAS. All rights reserved. Published by Elsevier Ltd.
Research of Genetic Diversity in Seven Kobresia by AFLP in Tibetan Plateau
997
is 0.223, the total index of genetic diversity is 0.2430; genetic differentiation level was calculated according to the total index of genetic diversity and genetic diversity within accessions (Fig. 3). The gene differentiation coefficient values (0.0932) showed that most of the genetic variability resided among individuals within accessions, whereas only 9.32% resided among accessions. The number of migrants per generation indicates genetic variance among and within populations. Mean gene flow is 4.8639, which correlates to high genetic variance among Kobresia populations of the species.
Fig. 4 Relationship between coefficient and elevation interval among accessions of Kobresia.
Fig. 3 A two-dimensinal PCO plot of 11 Kobresia accessions.
Effects of altitude on genetic similarity within accessions The relationship between genetic similarity coefficient and altitude within Kobresia accessions was analyzed using Matel test (Mantel 1967), the result is r = 0.75363, P = 1.0000 > 0.05, which suggests that there is no correlation between them (Fig.4).
DISCUSSION The high efficiency of genetic diversity for Kobresia by AFLP The results show that AFLP was stabile and repeatable which could completely reveal the hereditary feature of germplasm resources especially with close genetic relationship. For the first time, AFLP was adopted in the research of molecular markers on Kobresia
accessions. Four primer pairs were used, and produced 164 polymorphic loci, with an average of 38.5 polymorphic bands per primer pair by AFLP. Each primer amplified an average of 9.9 and 12 DNA polymorphic bands by RAPD, according to results of predecessors’ research. It can be concluded that the detection efficiency of polymorphic bands per primer is higher by AFLP than by RAPD (Zhao et al. 2006; Barrett et al. 1998). AFLP clearly detected DNA special polymorphic fragment and reflected genetic difference, deification, and classifications within Kobresia accessions. The results of the ratio between polymorphic loci, genetic similarity, and cluster analysis suggest the relatively abundant genetic diversity within Kobresia accessions by AFLP.
Genetic structure analysis The materials were grown in different habitats in this experiment, and were separately collected from discontinuous distribution places Langkazi, Naqu, Dangxiong, China, thereby creating genetic differentiation among accessions. Kobresia’s main mode of reproduction is rhizome asexual propagation, assisted by the wind-pollinated character. The special life history characteristics of Kobresia cause gene flow among accessions. The
© 2009, CAAS. All rights reserved. Published by Elsevier Ltd.
998
ZHENG Hong-mei et al.
migrant number of per generation 4.8639 (Nm) suggests that genetic drift does not lead to Kobresia’s genetic diversity. Hong et al. (2001) suggest that heritable variation of asexual plants is not less than that of the sexual reproductive; environmental heterogeneity may play an important role in clone plant genetic variation. It concludes that genetic variation of Kobresia accessions depends on different habits, which is a result of adaptation to ecological surroundings over a long period of time.
Genetic similarity and cluster analysis The relationship between genetic variation and geographical environment is an issue of universal concern. 11 Kobresia accessions were clustered into 4 groups based on the genetic similarity data using hierarchical cluster analysis. The result showed that C6, C9, C10, and C11 clustered together with close relationship; C7, C8, C3, C4, and C5 clustered together; C1 and C2 separately clustered. As for the geographic distribution, C1, C2, and C3 were collected from Langkazi; C6, C5, C8, and C10 were collected from Dangxiong; and C4, C7, C9, and C11 from Naqu. It is concluded from cluster analysis that the genetic coefficient is unrelated to their geographic latitude. Most same Kobresia species accessions are clustered together, but some accessions also are special, thus suggesting that many factors affect Kobresia genetic differentiation (Fig.2).
Acknowledgements This work was supported by the Key International Science and Technology Cooperation Program of China (2006DFA33630), and the Key Science and Technology Research Program of Tibet, China (2005011) .
References Barrett B A, Kidwell K K, Fox P N. 1998. AFLP-based genetics diversity assessment among wheat cultivars from the pacific northwest. Crop Science, 38, 1261-1271. Bassam B J, Caetano-Anollés G, Greshoff P M. 19991. Fast and sensitive silver staining of DNA in polyacrylamide gels. Analytical Biochemistry, 196, 80-83. Bohn M, Utz H F, Melchinger A E. 1999. Genetics similarities
among winter wheat cultivars determined on the basis of RFLPs, AFLPs, and SSRs and their use for predicting progeny variance. Crop Science, 39, 228- 237. Caicedo A L, Gaitán E, Duque M C, Toro Chica O, Debouck D G, Tohme J. 1999. AFLP fingerprinting of Phaseolus lunatus L. and related wild species from South America. Crop Science, 39, 1497-1507. Chen D H, Ronald P C. 1999. A rapid DNA minipreparation method suitable for AFLP and other PCR applications. Plant Molecular Biology Reporter, 17, 53-57. Dong N V, Subudhi P K, Luong P N, Quang V D, Quy T D, Zheng H G, Wang B, Nguyen H T. 2000. Molecular mapping of a rice gene conditioning thermosensitive genic male sterility using AFLP, RFLP and SSR techniques. Theoretical and Applied Genetics, 100, 727-734. Hai L, Xiao S H, Yan C S, Zhang X Y. 2002. Analysis of genetic difference between species in Lupins by AFLP. Scientia Agricultura Sinica, 35, 911-915. (in Chinese) Hao C Y, Wang L F, Dong Y S, Shang X W, Zhang X Y. 2003. Genetic diversity of wheat varieties released in northwest spring wheat region revealed by AFLP. Journal of Plant Genetic Resources, 4, 285-291. (in Chinese) He G H, Hou L, Li D M, Luo X Y, Niu G Q, Tang M, Pei Y. 2002. Predication of yield components in hybrid rice by using molecular markers. Acta Genetic Sinica, 29, 438-444. (in Chinese) Hong R M, Wang Y S, Yang J H. 2001. Analysis of phenotypic and genetic variation in colonial population of Leymus chinensis and others. Journal of Northeast Agricultural University, 32, 313-319. Julio E, Verrier J L, Dorlhac de Borne F. 2006. Development of SCAR markers linked to three disease resistances based on AFLP within Nicotiana tabacum L. Theoretical and Applied Genetics, 2006, 112, 335-346. Lewintin R C. 1972. The apportionment of human diversity. Evolutionary Biology, 6, 391-398 Li Q X, Zhao Q F, Cui Y, Ma S R. 2006. Genetic diversity of Kobresia setchwanensis along the east-southern of QinghaiTibet plateau. Journal of Northwest Normal University (Natural Science), 42, 69-73. (in Chinese) Liu J, Zhu Z Q, Liu G S, Qi D M, Li F F. 2002. AFLP variation analysis on the germplasm resources of Leymus chinensis. Acta Botanica Sinica, 44, 845-851. Lokko Y, Danquah E Y, Offei S K, Dixon A G O, Gedil M A. 2005. Molecular markers associated with a new source of resistance to the cassava mosaic disease. African Journal of Biotechnology, 4, 873-881. Lombard V, Baril C P, Dubreuil P, Blouet F, Zhang D. 2000. Genetic relationships and fingerprinting of rapeseed cultivars by AFLP: Consequences for varietal registration. Crop
© 2009, CAAS. All rights reserved. Published by Elsevier Ltd.
Research of Genetic Diversity in Seven Kobresia by AFLP in Tibetan Plateau
999
Science, 40, 1417-1425. Mantel N. 1967. The detection of disease clustering and a
analysis of Pigeon pea germplasm. Journal of Plant Genetic Resources, 5, 342-345. (in Chinese)
generalized regression approach. Cancer Research, 27, 209220.
Yang Y C, Zhou Q M, Yin H Q, Zhu L S. 2006. Studies on genetic diversity and relationship in tobacco germplasms by AFLP
Nei M, Li W H. 1979. Mathematical model for studying genetic variation in terms of restriction endonucleases. Proceedings
analysis. Scientia Agricultura Sinica, 39, 2194-2199. (in Chinese)
of the National Academy of Sciences of the USA, 76, 52695273.
Yeh F C, Yang R C. 1999. A joint project. POPGENE VERSION 1.31. University of Alberta And Tim Boyle, Centre for
Peng Y, Zhang X Q, Liu J P, Yi Y J. 2006. AFLP analysis on genetic diversity of wild Dactylis glomerata L. germplasm
International Forestry Research August. [2006-07-20]. ftp:// ftp. microsoft.com/Softlib/MSLFILES/HPGL.EXE
resources. Hereditas, 28, 845-850. (in Chinese) Renganayaki K, Read J C, Fritz A K. 2001. Genetic diversity
Zhao Q F, Li Q X, Ma S R, Cui Y, Wang G. 2006. RAPD analysis of genetic diversity of Kobresia humilis along the
among Texas bluegrass genotypes (Poa arachnifera Torr.) revealed by AFLP and RAPD markers. Theoretical and
eastern of Qinghai-Tibet Plateau of China. Acta Ecological Sinica, 26, 2494-2501. (in Chinese)
Applied Genetics, 102, 1037-1045. Vos P, Hogers R, Bleeker M. 1995. AFLP: a new technique for
Zhong D, Pai A, Yan G. 2004. AFLP-based genetic linkage map for the red flour beetle (Tribolium castaneum). Journal of
DNA fingerprinting. Nucleic Acids Research, 23, 4407- 4414. Yan L, Guan J P, Zong X X. 2004. Optimization of AFLP
Heredity, 9, 53-61. Zhou X M. 1982. Kobresia Meadow of China. Science Press,
fingerprinting and screening of primer pairs in genetic diversity
Beijing. (in Chinese) (Edited by ZHANG Yi-min)
© 2009, CAAS. All rights reserved. Published by Elsevier Ltd.
August 2009
2009, 8(8): 994-999
Research of Genetic Diversity in Seven Kobresia by AFLP in Tibetan Plateau ZHENG Hong-mei1, HU Tian-ming2, WANG Quan-zhen2, ZHANG Guo-yun3 and SONG Jiang-hu2 College of Resources and Environment, Northwest A&F University, Yangling 712100, P.R.China College of Animal Science and Technology, Northwest A&F University, Yangling 712100, P.R.China 3 Key Laboratory of Agricultural Molecular Biology of Shaanxi Province, Yangling 712100, P.R.China 1 2
Abstract This work analyzed the genetic diversity of Kobresia accessions at the molecular level, and further obtained the necessary information for breeding and germplasm evaluation. Genomic DNA of Kobresia was amplified with four E+3 and M+3 primer combinations with AFLP (amplified fragment length polymorphism). AFLP analysis produced 164 scorable bands, of which 154 (93.96%) were polymorphic. The mean Nei’s gene diversity index (H) was 0.2430, and the Shannon’s information index (I) was 0.4012, indicating the abundant genetic diversity of Kobresia. The 11 Kobresia accessions from Tibetan Plateau, China, can be classified into five groups after cluster analysis based on the UPGMA (unweighted pair group method arithmetic average) method. In general, there was abundant genetic diversity among Kobresia accessions resources, and the genetic coefficient was unrelated to their geographic latitude. Natural habitats influenced genetic differentiation of Kobresia. Key words: Kobresia, genetic diversity, AFLP
INTRODUCTION Kobresia Willd., a perennial grass, belongs to the sedge family and carex subfamily; most members of the sedge family are constructive species on the alpine meadow. Kobresia was enjoyed by cattle for its softness and rich quality, and is also an important plant for controlling soil erosion and maintaining ecology balance (Zhou et al. 1982). This study will promote ecological construction of alpine rangeland, improve natural grassland, and develop the grassland-animal husbandry industry. The method of AFLP (amplified fragment length polymorphism) has been applied to study the biodiversity (Barrett et al. 1998; Bohn et al. 1999), map construction (Caicedo et al. 1999; Zhong et al. 2004), cultivar identification (Renganayaki et al. 2001; Lombard et al.
2000), locating, isolating genes (Julio et al. 2006; Dong et al. 2000), heterosis prediction (He et al. 2002), molecular marker assisted breeding (Lokko et al. 2005), and so on. Great progress has been made in the research of many plants, such as wild cocksfoot, lupin, leymus chinensis, tobacco, corn, and so on (Peng et al. 2006; Yang et al. 2006; Liu et al. 2002; Hai et al. 2002; Hao et al. 2003). Although some preliminary researches have also been done on Kobresia germplasm resources by RAPD (random amplified polymorphic DNA) molecular markers (Li et al. 2006; Zhao et al. 2006), the study on Kobresia by AFLP has not been reported in literature. The main objective of the present study is to analyze Kobresia genetic diversity in different habitats by AFLP molecular marker and provide evidence for comprehensive evaluation of Kobresia germplasm.
This paper is translated from its Chinese version in Scientia Agricultura Sinica. Correspondence HU Tian-ming, Professor, E-mail: [email protected]
© 2009, CAAS. All rights reserved. Published by Elsevier Ltd. doi:10.1016/S1671-2927(08)60305-3
Research of Genetic Diversity in Seven Kobresia by AFLP in Tibetan Plateau
995
MATERIALS AND METHODS
separation production (Bassam et al. 1991).
Materials
Data statistic and analysis method
Eleven plant accessions representing seven different species were sampled from Tibet alpine on September 2006. Details on these genotypes including geographic origin and geographic information system (GIS) data, wherever possible, are provided in Table 1. Sampling vouchers were deposited at the herbarium of Northwest A&F University, China.
Data analysis By AFLP polymorphic bands statistical method, each allele was scored as present (1) or absent (0) for each loci. Each band was scored which is clear and repeated, and the size ranged from 100-500 bp in length. Analysis method The percentage of polymorphic loci, Nei’s gene diversity index (Nei 1973), Shannon’s information index (Lewontin 1972), and gene flow (Nm) were calculated using POPGENE1. 31 (Yeh et al. 1999). The numerical taxonomy and multivariate analysis system (NTSYS-pc 2.1) was constructed using unweighted pair group method arithmetic average (UPGMA) dendrograms of AFLP, the Mantel (1967) test statistic (Z) infered correlation genetic coefficient based on the geography distance matrix using the Mxcomp procedure of NTSYS-pc (Nei and Li 1979). Principal coordinate analysis was carried out by EIGEN.
Methods Genomic DNA of each accession was extracted from a pool of 30 plants using the modified CTAB method (Chen and Ronald 1999). DNA concentration was measured using a UV spectrophotometer, and concentrations were adjusted to 50 ng L-1 for PCR analysis. AFLP optimized procedure refers to Vos’s method and other previous research (Vos et al. 1995; Yan et al. 2004). PAG electrophoresis and silver staining detect electrophoretic
Table 1 The environmental data for the different accessions of Kobresia Species
Code
Origion
K. royleana (N.) B. K. macrentha Boeck. K. littledalei C. B. Clarke K. royleana (N.) B K. prattii C. B. Clarke K. caillifolia Decne K. littledalei C. B. Clarke K. littledalei C. B. Clarke K. humilis C. A. Mey K. humilis C. A. Mey K. pygmaea C. B.
C1 C2 C3 C4 C5 C6 C7 C8 C9 C10 C11
Langkazi Langkazi Langkazi Naqu Dangxiong Dangxiong Naqu Dangxiong Naqu Dangxiong Naqu
Altitude (m) 4 455 4 455 4 455 4 456 4 278 4 278 4 456 4 278 4 456 4 278 4 456
Latitude and longitude 28°59´11N 28°59´11N 28°59´11N 31°26´34N 30°29´35N 30°29´35N 31°26´34N 30°29´35N 31°26´34N 30°29´35N 31°26´34N
90°26´04E 90°26´04E 90°26´04E 92°16´32E 91°05´58E 91°05´58E 92°16´32E 91°05´58E 92°16´32E 91°05´58E 92°16´32E
Habitat Alpine steppe Alpine meadow Alpine meadow Alpine steppe Alpine steppe Alpine meadow Marshy meadow Marshy meadow Alpine steppe Alpine steppe Alpine meadow
RESULTS Genetic diversity of Kobresia Different primers were screened and all were found to be polymorphic. The band numbers and the fragment length were different in each primer. Four primers were identified out of the 20 primers (Fig.1). AFLP markers, which amplified 154 polymorphic bands, showed high levels of polymorphism by the PIC value ranging from 87.80% (primer pair E-ACC + M-CAA) to
Fig. 1 AFLP fingerprint of 11 Kobresia accessions.
© 2009, CAAS. All rights reserved. Published by Elsevier Ltd.
996
ZHENG Hong-mei et al.
96.97% (primer pair E-ACA + M - CTG) with an average of 93.96% across the germplasm assayed (Table 2). The mean Nei’s gene diversity index (H) of all accessions was 0.2430, the Shannon’s information index (I) was 0.4012, indicating that the genetic diversity of Kobresia is very rich. E-ACC+M-CAG, E- ACA+MCAG, E-ACA+M-CTG, E-ACC+M-CAA detected effective number of alleles (Ne) ranged from 1.2393 to 1.2838; gene diversity ranged from 0.1586 to 0.1894 (Table 3). The above indicators suggested that the genetic germplasm of Kobresia showed great variability within species, high polymorphic, and abundant genetic diversity.
Analysis of Kobresia germplasm genetic distance and cluster analysis
Table 2 The AFLP amplified result of Kobresia accessions
Table 3 Estimates of genetic diversity among Kobresia (mean ± SD)
Primers
Total loci
E-ACC+M-CAG EACA+M-CAG E-ACA+M-CTG E-ACC+M-CAA Total Average
47 43 33 41 164 41
Proportion of polymorphic loci
Polymorphic loci 45 41 32 36 154 38.5
95.74 95.34 96.97 87.8
A matrix of the Jaccard’s coefficients of similarity based on the data of AFLP markers was calculated between accessions as shown in Table 3. Similarity coefficient of pairwise comparisons among different accessions yielded values ranging from 0.1159 to 0.4390, its mean value for all possible pairwise comparisons was 0.2774. Maximal genetic distance came from accession C1 and accession C10, minimum genetic distance came from accession C3 and accession C4, genetic identity among accession of Kobresia reached 0.56098 to 0.88415 (Table 4).
Primer combinations
Effective number of alleles
Nei’s gene diversity
Shannon’s information index
E-ACC+M-CAG E-ACA+M-CAG E-ACA+M-CTG E-ACC+M-CAA
1.2838 ± 0.2883 1.2781 ± 0.2770 1.2393 ± 0.2735 1.2439 ± 0.3028
0.1894 ± 0.1456 0.1871 ± 0.1457 0.1602 ± 0.1553 0.1586 ± 0.1595
0.3176 ± 0.1892 0.3122 ± 0.1958 0.2614 ± 0.2274 0.2622 ± 0.2222
93.96
Table 4 Coefficient in the accessions of Kobresia ID C1 C2 C3 C4 C5 C6 C7 C8 C9 C10 C11
C1
C2
C3
C4
C5
C6
C7
C8
C9
C10
C11
1.0000 0.2379 0.2379 0.2317 0.2744 0.3110 0.2805 0.2805 0.2866 0.3720 0.3598
0.7621 1.0000 0.2439 0.2988 0.3171 0.3293 0.3232 0.3232 0.2927 0.4146 0.4390
0.7621 0.7561 1.0000 0.1890 0.2317 0.2073 0.3232 0.2988 0.2927 0.3415 0.3780
0.7683 0.7012 0.8110 1.0000 0.1402 0.2378 0.2561 0.2195 0.2134 0.3232 0.3475
0.7256 0.6829 0.7683 0.8598 1.0000 0.3049 0.2256 0.2378 0.2073 0.3049 0.3536
0.6890 0.6707 0.7927 0.7622 0.6951 1.0000 0.3598 0.3720 0.3659 0.4024 0.4268
0.7195 0.6768 0.6768 0.7439 0.7744 0.6402 1.0000 0.1829 0.2134 0.2988 0.4085
0.7195 0.6768 0.7012 0.7805 0.7622 0.6280 0.8171 1.0000 0.1159 0.2866 0.3963
0.7134 0.7073 0.7073 0.7866 0.7927 0.6341 0.7866 0.8841 1.0000 0.2805 0.3415
0.6280 0.5854 0.6585 0.6768 0.6951 0.5976 0.7012 0.7134 0.7195 1.0000 0.3902
0.6402 0.5610 0.6220 0.6524 0.6463 0.5732 0.5915 0.6037 0.6585 0.6098 1.0000
Nei’s genetic identity (above diagonal); genetic distance (below diagonal).
The eleven accessions can be classified into five major genetic groups when genetic similarity is 0.74 (Fig.2). Accessions C6, C9, C10, and C11 cluster together, accessions C7 and C8 are in one group, accessions C3, C4, and C5 had a closer relationship, and accessions C1 and C2 do not group with any others.
Genetic structure of Kobrsia Supposing genetic equilibrium by means of POPGENE32 software, genetic diversity within Kobresia accessions
Fig. 2 The dendrogram generated by AFLP data using UPGMA method among 11 Kobresia accesions.
© 2009, CAAS. All rights reserved. Published by Elsevier Ltd.
Research of Genetic Diversity in Seven Kobresia by AFLP in Tibetan Plateau
997
is 0.223, the total index of genetic diversity is 0.2430; genetic differentiation level was calculated according to the total index of genetic diversity and genetic diversity within accessions (Fig. 3). The gene differentiation coefficient values (0.0932) showed that most of the genetic variability resided among individuals within accessions, whereas only 9.32% resided among accessions. The number of migrants per generation indicates genetic variance among and within populations. Mean gene flow is 4.8639, which correlates to high genetic variance among Kobresia populations of the species.
Fig. 4 Relationship between coefficient and elevation interval among accessions of Kobresia.
Fig. 3 A two-dimensinal PCO plot of 11 Kobresia accessions.
Effects of altitude on genetic similarity within accessions The relationship between genetic similarity coefficient and altitude within Kobresia accessions was analyzed using Matel test (Mantel 1967), the result is r = 0.75363, P = 1.0000 > 0.05, which suggests that there is no correlation between them (Fig.4).
DISCUSSION The high efficiency of genetic diversity for Kobresia by AFLP The results show that AFLP was stabile and repeatable which could completely reveal the hereditary feature of germplasm resources especially with close genetic relationship. For the first time, AFLP was adopted in the research of molecular markers on Kobresia
accessions. Four primer pairs were used, and produced 164 polymorphic loci, with an average of 38.5 polymorphic bands per primer pair by AFLP. Each primer amplified an average of 9.9 and 12 DNA polymorphic bands by RAPD, according to results of predecessors’ research. It can be concluded that the detection efficiency of polymorphic bands per primer is higher by AFLP than by RAPD (Zhao et al. 2006; Barrett et al. 1998). AFLP clearly detected DNA special polymorphic fragment and reflected genetic difference, deification, and classifications within Kobresia accessions. The results of the ratio between polymorphic loci, genetic similarity, and cluster analysis suggest the relatively abundant genetic diversity within Kobresia accessions by AFLP.
Genetic structure analysis The materials were grown in different habitats in this experiment, and were separately collected from discontinuous distribution places Langkazi, Naqu, Dangxiong, China, thereby creating genetic differentiation among accessions. Kobresia’s main mode of reproduction is rhizome asexual propagation, assisted by the wind-pollinated character. The special life history characteristics of Kobresia cause gene flow among accessions. The
© 2009, CAAS. All rights reserved. Published by Elsevier Ltd.
998
ZHENG Hong-mei et al.
migrant number of per generation 4.8639 (Nm) suggests that genetic drift does not lead to Kobresia’s genetic diversity. Hong et al. (2001) suggest that heritable variation of asexual plants is not less than that of the sexual reproductive; environmental heterogeneity may play an important role in clone plant genetic variation. It concludes that genetic variation of Kobresia accessions depends on different habits, which is a result of adaptation to ecological surroundings over a long period of time.
Genetic similarity and cluster analysis The relationship between genetic variation and geographical environment is an issue of universal concern. 11 Kobresia accessions were clustered into 4 groups based on the genetic similarity data using hierarchical cluster analysis. The result showed that C6, C9, C10, and C11 clustered together with close relationship; C7, C8, C3, C4, and C5 clustered together; C1 and C2 separately clustered. As for the geographic distribution, C1, C2, and C3 were collected from Langkazi; C6, C5, C8, and C10 were collected from Dangxiong; and C4, C7, C9, and C11 from Naqu. It is concluded from cluster analysis that the genetic coefficient is unrelated to their geographic latitude. Most same Kobresia species accessions are clustered together, but some accessions also are special, thus suggesting that many factors affect Kobresia genetic differentiation (Fig.2).
Acknowledgements This work was supported by the Key International Science and Technology Cooperation Program of China (2006DFA33630), and the Key Science and Technology Research Program of Tibet, China (2005011) .
References Barrett B A, Kidwell K K, Fox P N. 1998. AFLP-based genetics diversity assessment among wheat cultivars from the pacific northwest. Crop Science, 38, 1261-1271. Bassam B J, Caetano-Anollés G, Greshoff P M. 19991. Fast and sensitive silver staining of DNA in polyacrylamide gels. Analytical Biochemistry, 196, 80-83. Bohn M, Utz H F, Melchinger A E. 1999. Genetics similarities
among winter wheat cultivars determined on the basis of RFLPs, AFLPs, and SSRs and their use for predicting progeny variance. Crop Science, 39, 228- 237. Caicedo A L, Gaitán E, Duque M C, Toro Chica O, Debouck D G, Tohme J. 1999. AFLP fingerprinting of Phaseolus lunatus L. and related wild species from South America. Crop Science, 39, 1497-1507. Chen D H, Ronald P C. 1999. A rapid DNA minipreparation method suitable for AFLP and other PCR applications. Plant Molecular Biology Reporter, 17, 53-57. Dong N V, Subudhi P K, Luong P N, Quang V D, Quy T D, Zheng H G, Wang B, Nguyen H T. 2000. Molecular mapping of a rice gene conditioning thermosensitive genic male sterility using AFLP, RFLP and SSR techniques. Theoretical and Applied Genetics, 100, 727-734. Hai L, Xiao S H, Yan C S, Zhang X Y. 2002. Analysis of genetic difference between species in Lupins by AFLP. Scientia Agricultura Sinica, 35, 911-915. (in Chinese) Hao C Y, Wang L F, Dong Y S, Shang X W, Zhang X Y. 2003. Genetic diversity of wheat varieties released in northwest spring wheat region revealed by AFLP. Journal of Plant Genetic Resources, 4, 285-291. (in Chinese) He G H, Hou L, Li D M, Luo X Y, Niu G Q, Tang M, Pei Y. 2002. Predication of yield components in hybrid rice by using molecular markers. Acta Genetic Sinica, 29, 438-444. (in Chinese) Hong R M, Wang Y S, Yang J H. 2001. Analysis of phenotypic and genetic variation in colonial population of Leymus chinensis and others. Journal of Northeast Agricultural University, 32, 313-319. Julio E, Verrier J L, Dorlhac de Borne F. 2006. Development of SCAR markers linked to three disease resistances based on AFLP within Nicotiana tabacum L. Theoretical and Applied Genetics, 2006, 112, 335-346. Lewintin R C. 1972. The apportionment of human diversity. Evolutionary Biology, 6, 391-398 Li Q X, Zhao Q F, Cui Y, Ma S R. 2006. Genetic diversity of Kobresia setchwanensis along the east-southern of QinghaiTibet plateau. Journal of Northwest Normal University (Natural Science), 42, 69-73. (in Chinese) Liu J, Zhu Z Q, Liu G S, Qi D M, Li F F. 2002. AFLP variation analysis on the germplasm resources of Leymus chinensis. Acta Botanica Sinica, 44, 845-851. Lokko Y, Danquah E Y, Offei S K, Dixon A G O, Gedil M A. 2005. Molecular markers associated with a new source of resistance to the cassava mosaic disease. African Journal of Biotechnology, 4, 873-881. Lombard V, Baril C P, Dubreuil P, Blouet F, Zhang D. 2000. Genetic relationships and fingerprinting of rapeseed cultivars by AFLP: Consequences for varietal registration. Crop
© 2009, CAAS. All rights reserved. Published by Elsevier Ltd.
Research of Genetic Diversity in Seven Kobresia by AFLP in Tibetan Plateau
999
Science, 40, 1417-1425. Mantel N. 1967. The detection of disease clustering and a
analysis of Pigeon pea germplasm. Journal of Plant Genetic Resources, 5, 342-345. (in Chinese)
generalized regression approach. Cancer Research, 27, 209220.
Yang Y C, Zhou Q M, Yin H Q, Zhu L S. 2006. Studies on genetic diversity and relationship in tobacco germplasms by AFLP
Nei M, Li W H. 1979. Mathematical model for studying genetic variation in terms of restriction endonucleases. Proceedings
analysis. Scientia Agricultura Sinica, 39, 2194-2199. (in Chinese)
of the National Academy of Sciences of the USA, 76, 52695273.
Yeh F C, Yang R C. 1999. A joint project. POPGENE VERSION 1.31. University of Alberta And Tim Boyle, Centre for
Peng Y, Zhang X Q, Liu J P, Yi Y J. 2006. AFLP analysis on genetic diversity of wild Dactylis glomerata L. germplasm
International Forestry Research August. [2006-07-20]. ftp:// ftp. microsoft.com/Softlib/MSLFILES/HPGL.EXE
resources. Hereditas, 28, 845-850. (in Chinese) Renganayaki K, Read J C, Fritz A K. 2001. Genetic diversity
Zhao Q F, Li Q X, Ma S R, Cui Y, Wang G. 2006. RAPD analysis of genetic diversity of Kobresia humilis along the
among Texas bluegrass genotypes (Poa arachnifera Torr.) revealed by AFLP and RAPD markers. Theoretical and
eastern of Qinghai-Tibet Plateau of China. Acta Ecological Sinica, 26, 2494-2501. (in Chinese)
Applied Genetics, 102, 1037-1045. Vos P, Hogers R, Bleeker M. 1995. AFLP: a new technique for
Zhong D, Pai A, Yan G. 2004. AFLP-based genetic linkage map for the red flour beetle (Tribolium castaneum). Journal of
DNA fingerprinting. Nucleic Acids Research, 23, 4407- 4414. Yan L, Guan J P, Zong X X. 2004. Optimization of AFLP
Heredity, 9, 53-61. Zhou X M. 1982. Kobresia Meadow of China. Science Press,
fingerprinting and screening of primer pairs in genetic diversity
Beijing. (in Chinese) (Edited by ZHANG Yi-min)
© 2009, CAAS. All rights reserved. Published by Elsevier Ltd.