珍稀濒危植物硬叶兜兰的遗传多样性及遗传结构研究

doi:10.7677/ynzwyj201413085

摘要/Abstract

摘要：

由于人为采集、走私贩卖以及生境的破坏，分布于中国西南石灰岩地区的野生硬叶兜兰居群受到严重的干扰与威胁。为有效地保护这种珍稀野生植物，本研究采用ISSR和SRAP两种分子标记对15个硬叶兜兰野生居群进行遗传多样性及遗传结构的研究。结果表明，硬叶兜兰在物种水平上具有较高的遗传多样性（ISSR: PPB=9166%, He=03839; SRAP: PPB=9929%, He=02806）。硬叶兜兰居群间存在一定程度的遗传分化 (ISSR: Gst=02577; SRAP: Gst=02383)，可能由于较低的基因流（ISSR: Nm=07201; SRAP: Nm=07991）所致。UPGMA聚类分析以及主成分分析均把15个居群分成2个主要分支。居群间的地理距离和海拔差距是引起居群遗传分化的自然因素。

关键词: 资源保护, 分子标记, 遗传多样性, 遗传分化, 硬叶兜兰

Abstract:

Paphiopedilum micranthum is an endangered pink slipper orchid mainly distributed in the limestone areas of southwestern China. Wild populations of this species have been seriously threatened by excessive collections, rampant smuggling for export, and habitat destruction. We used 15 ISSR markers and 11 SRAP markers to investigate the genetic diversity and structure of 15 natural populations. A high degree of diversity was observed at the species level (ISSR: PPB=9166%, He=03839; SRAP: PPB=9929%, He=02806). Certain degree of genetic differentiation among populations (ISSR: Gst=02577; SRAP: Gst=02383) was detected maybe caused by low gene flow (ISSR: Nm=07201; SRAP: Nm=07991). Consistent with the results of Principal Coordinate Analysis, the UPGMA dendrogram analysis divided the 15 populations into two main clades. In addition to geographic distance, the difference in elevation was another natural factor contributing to this differentiation. Knowledge about genetic diversity and structure gained from our study will be beneficial for the development of reasonable and efficient strategies to conserve this endangered species.

Key words: Conservation, DNA markers, Genetic diversity, Genetic differentiation, Paphiopedilum micranthum

中图分类号:

Q 16

黄家林, 李树云, 胡虹. 珍稀濒危植物硬叶兜兰的遗传多样性及遗传结构研究[J]. Plant Diversity, 2014, 36(02): 209-218.

HUANG Jia-Lin, LI Shu-Yun, HU Hong. ISSR and SRAP Markers Reveal Genetic Diversity and Population Structure of an Endangered Slipper Orchid, Paphiopedilum micranthum (Orchidaceae)[J]. Plant Diversity, 2014, 36(02): 209-218.

参考文献

Bassam BJ, CaetanoAnolle G, Gresshoff PM, 1991. Fast and sensitive silver staining of DNA in polyacrylamide gels［J］. Analytical Biochemistry, 196: 81—84
Bayer RJ, 1992. Allozyme variation, genecology, and phytogeography of Antennaria arcuata (Asteraceace), a rare species from the great Basin and Red Desert with small disjunct populations［J］. American Journal of Botany, 79: 872—881
Brzosko E, Blewska AW, Taaaj I et al., 2011. Genetic diversity of Cypripedium calceolus in Poland［J］. Plant Systematics and Evolution, 295: 83—96
Cai NF, Yan N, Hu H et al., 2008. Genetic structure and clonal diversity of Cypripedium flavum (Orchidaceae) populations from SouthWest China［J］. Acta Botanica Yunnanica, 30: 69—75
Cai XY, Feng ZY, Zhang XX, 2011. Genetic diversity and population structure of an endangered orchid (Dendrobium loddigesii Rolfe) from China revealed by SRAP markers［J］. Scientia Horticulturae, 129: 877—881
Chang W, Zhang SB, Li SY, 2011. Ecophysiological significance of leaf traits in Cypripedium and Paphiopedilum［J］. Physiologia Plantarum, 141: 30—39
Chen TY, Chen JT, Chang WC, 2004. Plant regeneration through direct shoot bud formation from leaf cultures of Paphiopedilum orchids［J］. Plant Cell, Tissue, and Organ Culture, 76: 11—15
Chung SY, Choi SH, 2012. Genetic variability and relationships among interspecific hybrid cultivars and parental species of Paphiopedilum via ribosomal DNA sequence analysis［J］. Plant Systematics and Evolution, 298: 1897—1907
Cribb PJ, 1998. The Genus Paphiopedilum［M］. Borneo, Kota Kinabalu, & Royal Botanical Garden, Kew, London, UK
Cribb PJ, McGough N, 1997. The Thin DivideSlipper Orchid Distributions in China［M］. Proceedings of the European Orchid Conference, Geneva, Switzerland
Ding G, Zhang DZ, Ding XY, 2008. Genetic variation and conservation of the endangered Chinese endemic herb Dendrobium officinale based on SRAP analysis［J］. Plant Systematics and Evolution, 276: 149—156
Dirlewanger E, Pronier V, Parvery C et al., 1998. Genetic linkage map of peach (Prunus persica L.) using morphological and molecular markers［J］. Theoretical and Applied Genetics, 97: 888—895
Doyle JJ, Doyle JL, 1987. A rapid DNA isolation procedure for small quantities of fresh leaf tissue［J］. Phytochemical Bulletin, 19: 11—15
Ehlers BK, Pedersen HA, 2000. Genetic variation in three species of Epipactis (Orchidaeeae): geographic scale and evolutionary inferences［J］. Biological Journal of the Linneae Society, 69: 411—430
Esselman EJ, Li JQ, Crawford DJ et al., 1999. Clonal diversity in the rare Calamagrostis porteri ssp. in sperata (Poaceae): comparative results for allozymes and RAPD and ISSR markers［J］. Molecular Ecology, 8: 443—451
Forrest AD, Hollingsworth ML, Hollingsworth PM et al., 2004. Population genetic structure in European populations of Spiranthes romanzoffiana set in the context of other genetic studies on orchids［J］. Heredity, 92: 218—227
Francis CY, Yang RC, 2000. POPGENE, Version 132. http://wwwualbertaca/_fyeh/indexhtm
Gao L, Yang B, 2006. Genetic diversity of wild Cymbidium goeringii (Orchidaceae) populations from Hubei based on ISSR analysis［J］. Biodiversity Science, 14: 250—257
George S, Sharma J, Yadon VL, 2009. Genetic diversity of three endangered and narrow endemic Piperia yadonni (Orchidaceae) assessed with ISSR polymorphisms［J］. American Journal of Botany, 96: 2020—2022
Gilbert JE, Lewis RB, Wilkinson MJ et al., 1999. Developing an appropriate strategy to access genetic variability in plant germplasm collections［J］. Theoretical and Applied Genetics, 98: 1125—1131
Godt MJW, Caplow F, Hamrick JL, 2005. Allozyme diversity in the federally threatened golden paintbrush, Castilleja levisecta (Scrophulariaceae) ［J］. Conservation Genetics, 6: 87—99
Hamrick JL, 1982. Plant population genetics and evolution［J］. American Journal of Botany, 69:1685—1693
Hamrick JL, Godt MJ, 1990. Allozyme diversity in plant species［A］. In: Brown AHD, Clegg MT, Kahler AL, Weir BS (eds.), Plant Population Genetics, Breeding, and Genetic Resources［M］. Sinauer, Sunderland, MA, USA, 43—63
Hartl DL, Clark AG, 1989. Principle of Population Genetics, 2nd edn［M］. Sinauer, Sunderland, MA, USA
Jian SG, Zhong Y, Liu N et al., 2006. Genetic variation in the endangered endemic species Cycas fairylakea (Cycadaceae) in China and implications for conservation［J］. Biologic Conservation, 15: 1681—1694
Jiang ZL, Yang XM, Wang R et al., 2005. Genetic diversity of Roegneria thoroldiana (Oliv.) Keng populations based on SSR analyses［J］. Journal of Plant Genetic Resources, 6: 315—331
Li A, Wang KQ, Ge S, 2000. Genetic diversity within and among population of Viola tenuicornis with reference to sampling strategies［J］. Acta Botanica Sinica, 42: 1069—1074
Li A, Luo YB, Xiong ZT et al., 2002a. A preliminary study on conservation genetics of three endangered orchid species［J］. Acta Botanica Sinica, 44: 250—252
Li A, Luo YB, Ge S, 2002b. A preliminary study on conservation genetics of an endangered orchid (Paphiopedilum micranthum) from southwestern China［J］. Biochemical Genetics, 40: 195—201
Li G, Quiros CF, 2001. Sequencerelated amplified polymorphism (SRAP), a new marker system based on a simple PCR reaction: its application to mapping and gene tagging in Brassica［J］. Theoretical and Applied Genetics, 103: 455—461
Li HS, Chen GZ, 2004. Genetic diversity of mangrove plant Sonneratia caseolaris in Hainan Island based on ISSR analysis［J］. Acta Ecologica Sinica, 24: 1656—1662
Liao YJ, Tsai YC, Sun YW, 2011. In vitro shoot induction and plant regeneration from flower buds in Paphiopedilum orchids［J］. In vitro Cellular & Developmental Biology, 47: 702—709
Liu H, Feng CL, Luo YB et al., 2010. Potential challenges of climate change to orchid conservation in a wild orchid hotspot in southwestern China［J］. Botany Review, 76: 174—192
Liu ZJ, Zhang JY, Ru ZZ, 2004. Conservation biology of Paphiopedilum purpuratum (Orchidaceae) ［J］. Biodiversity Science, 12: 509—516
Liu ZJ, Liu KW, Chen LJ, 2006. Conservation ecology of endangered species Paphiopedilum armeniacum (Orchidaceae) ［J］. Acta Ecologica Sinica, 26: 2791—2800
Liu ZJ, Chen SC, Chen LJ et al., 2009. The Genus Paphiopedilum in China［M］. Beijing: Science Press, 4—12
Luan SS, Chiang TY, Gong X, 2006. High genetic diversity vs. low genetic differentiation in Nouelia insignis (Asteraceae), a narrowly distributed and endemic species in China, revealed by ISSR fingerprinting［J］. Annals of Botany, 98: 583—589
Luo YB, Jia JS, Wang CL, 2003. Conservation strategy and potential advantages of the Chinese Paphiopedilum［J］. Biodiversity Science, 11 (6): 491—498
Miller MP, 1997. Tools For Population Genetic Analysis (TFPGA) 13: A Windows Program for the Analysis of Allozyme and Molecular Population Genetic Data［M］. Flagstaff: Department of Biological Sciences, Northern Arizona University, AZ, USA
Nei M, 1978. Estimation of average heterozygosity and genetic distance from a small number of individuals［J］. Genetics, 89: 583—590
Nybom H, 2004. Comparison of different nuclear DNA markers for estimating intraspecific genetic diversity in plants［J］. Molecular Ecology, 13: 1143—1155
Peakall ROD, Smouse PE, 2006. GENALEX 6: genetic analysis in Excel. Population genetic software for teaching and research［J］. Molecular Ecology Notes, 6: 288—295
Rosenberg MS, Anderson CD, 2011. PASSAGE: Pattern Analysis, Spatial Statistics and Geographic Exegesis. Version 2［M］. Methods in Ecology & Evolution, 2: 227—232
Shi J, Cheng J, Luo D, 2007. Pollination syndromes predict broodsite deceptive pollination by female hoverflies in Paphiopedilum dianthum (Orchidaceae) ［J］. Acta Phytotaxonomica Sinica (植物分类学报), 45: 551—560
Shannon CE, Weaver W, 1949. The Mathematical Theory of Communication［M］. Urbana: University of Illinois Press, IL, USA
Slatkin M, Barton NH, 1989. A comparison of three indirect methods for estimating average levels of gene flow［J］. Evolution, 43: 1349—1368
Smith JL, Hunter KL, Hunter RB, 2002. Genetic variation in the terrestrial orchid Tipularia discolor［J］. Southeastern Naturalist, 1: 17—26
Swamy KK, Kumar HNK, Ramakrishna TM et al., 2004. Studies on seed morphometry of epiphytic orchids from western ghats of karnataka［J］. Taiwania, 49: 124—140
Swamy KK, Kumar HNK, Ramaswamy SN, 2007. Studies on seed morphometry of Dendrobium species［J］. Phytomorphology, 57: 33—43
Tsi ZH, Luo YB. Cribb PJ et al., 1999. A preliminary report on the population size, ecology, and conservation status of Paphiopedilum species (Orchidaceae) in southwest China［J］. Lindlyana: the Scientific Journal of the American Orchid, 14: 12—23
Wallace LE, 2003, Molecular evidence for allopolyploid speciation and recurrent origins in Platanthera huronensis (Orchidaceae) ［J］. International Journal of Plant Sciences, 164: 907—916
Wang HZ, Wu ZX, Lu JJ et al., 2009. Molecular diversity and relationships among Cymbidium goeringii cultivars based on intersimple sequence repeat (ISSR) markers［J］. Genetica, 136: 391—399
Wong KC, Sun M, 1999. Reproductive biology and conservation genetics of Goodyera procera (Orchidaceae) ［J］. American Journal of Botany, 86: 1406—1413
Wright S, 1931. Evolution in Mendelian population［J］. Genetics, 16: 97—159
Yan TF, Zhou FJ, Yan XF et al., 1999. Genetic diversity and population differentiation of Rhodiola angusta［J］. Bulletin of Botanical Research, 19: 189—194
Zeng SG, Tian RX, Chen ZL, 2010, Research progress on cross breeding of Paphiopedilum［J］. Journal of Tropical and Subtropical Botany, 18: 459—468
Zhang JJ, 2012. Embryo Development and Seed Morphology of Paphiopedilum, Orchidaceae (MS degree) ［D］. Kunming: Kunming Institute of Botany, Chinese Academy of Sciences, China, 22—32
Zietkiewicz E, Fafalski A, Labuda D, 1994. Genome fingerprinting by simple sequence repeat (SSR)anchored polymerase chain reaction amplification［J］. Genomics, 20: 176—183

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