杜仲性别相关EST-SSR标记的开发

doi:10.11707/j.1001-7488.20161018

摘要/Abstract

摘要： [目的] 杜仲为雌雄异株多年生木本植物，具有重要的药用价值与经济价值，其利用价值因植株性别而异，尤其是杜仲胶含量及其他次生代谢产物在雌雄植株中都存在差别，但杜仲在开花前采用形态学和细胞学方法很难分辨其性别，且需要7年以上才能开花。因此，本研究应用EST-SSR标记开发杜仲性别相关的分子标记，以期能在早期鉴定出雌雄植株，提高杜仲资源利用率。[方法] 根据杜仲雌雄植株转录组文库中EST-SSR的两翼序列设计引物，利用来源于7个产地的杜仲雌雄植株各6份DNA样品筛选引物，PCR扩增获得具性别差异的扩增产物，将扩增产物进行克隆及序列分析，应用雌雄植株各12份DNA样品检验性别标记的可靠性。[结果] 从140对EST-SSR引物中筛选出1对引物EST-Eu059，在雌、雄植株中产生差异条带，雌、雄植株均有1条约160 bp的序列（分别命名为Eu-f160和Eu-m160），而雄株多1条约120 bp的序列（EuMSM）。序列分析发现，Eu-f160与Eu-m160均为156 bp，具有重复单元（GT）₆，且仅有1个碱基不相同，即雌株重复单元内第3个G碱基变成了A，可能发生了突变；EuMSM为112 bp，缺失了（GT）₆重复单元。经Blastn比对，EuMSM序列与GenBank数据库中的现有序列没有同源性。用杜仲雌、雄植株基因组DNA各12份进行验证表明，在所有雄株中都有1条雄性特异序列。[结论] 本研究筛选出了1对可以鉴别杜仲雌雄株性别的引物EST-Eu059，该标记在雄株中具有1条特异序列片段EuMSM。该标记可在杜仲生长早期快速、可靠地鉴定其性别。

关键词: 杜仲, EST-SSR, 雄性特异序列, 性别鉴定

Abstract: [Objective] Eucommia ulmoides is a dioecious perennial woody plant with high medicinal and economic values. The utilization value was different between male and female of E. ulmoides, especially of the gutta-percha and other secondary metabolites. Also, it was difficult to identify sex by morphological or cytological methods before flowering, and the trees need more than 7 years to bloom. EST-SSR markers were used to find sex-associated ones, and expected to identify sex at early stage for improving the utilization of E. ulmoides resources.[Method] We screened primers by using DNA samples of 6 male and 6 female plants from 7 different regions, and the primers ware designed based on the SSRs locus flanking sequences of dioecious transcriptome library of E. ulmoides. We obtained gender different products by PCR which has been cloned and analyzed, then another 12 male and 12 female plants were used to test reliability of the gender markers.[Result] The result showed that only one pair of primers(EST-Eu059) out of 140 pairs of EST-SSR primers displayed different bands between male and female, a common band was about 160 bp(named as Eu-f160 and Eu-m160) for both male and female plants, with an extra band of 120 bp approximately(named as EuMSM) in all males. Sequence analysis indicated that Eu-f160 and Eu-m160 were 156 bp, with the repeat unit (GT)₆, and only 1 base was different, that was, the third G base in the female repeat unit became A; and EuMSM was 112 bp without (GT)₆. By Blastn comparison, EuMSM was not homologous with the sequences in the GenBank database. We confirmed that there was a male-specific band in all male plants.[Conclusion] We selected one pair of primers EST-Eu059 that can be used to identify male and female plants of E. ulmoides, which had a specific band EuMSM in male plants. This marker can be used to identify sex of E. ulmoides quickly and reliably at an early stage.

Key words: Eucommia ulmoides, EST-SSR, male-specific sequence, sex identification

中图分类号:

S718.46

林开勤, 赵德刚, 李岩, 何选泽, 王韶敏. 杜仲性别相关EST-SSR标记的开发[J]. 林业科学, 2016, 52(10): 146-152.

Lin Kaiqin, Zhao Degang, Li Yan, He Xuanze, Wang Shaomin. Development of Gender-Related EST-SSR Markers in Eucommia ulmoides[J]. Scientia Silvae Sinicae, 2016, 52(10): 146-152.

参考文献

陈峰, 张士永, 朱文银, 等. 2010. 分子标记辅助选择改良圣稻13和圣稻14的条纹叶枯病抗性. 中国农业科学, 43(16):3271-3279.
(Chen F, Zhang S Y, Zhu W Y, et al. 2010. Improving resistance of Japonica varieties Shengdao13 and Shengdao14 to rice stripe virus disease by molecular marker-assisted selection. Scientia Agricultura Sinica, 43(16):3271-3279.[in Chinese])
程晓建, 王白坡, 郑炳松, 等. 2002. 银杏雌雄株性别鉴别研究进展. 浙江林学院学报, 19(2):217-221.
(Cheng X J, Wang B P, Zheng B S, et al. 2002. Progress on sex identification of dioecism in Ginkgo biloba. Journal of Zhejiang Forestry College, 19(2):217-221.[in Chinese])
丁一, 徐启江. 2014. 被子植物成花诱导和性别决定机制的研究进展. 植物生理学报, 50(1):19-36.
(Ding Y, Xu Q J. 2014. Progress on floral induction and sex determination in angiosperm. Plant Physiology Journal, 50(1):19-36.[in Chinese])
杜红岩, 杜兰英, 傅建敏, 等. 2006. 杜仲不同器官含胶率的差异及其相关性分析. 中南林学院学报, 26(4):1-4.
(Du H Y, Du L Y, Fu J M, et al. 2006. The difference and its pertinence analysis of the gutta-percha content in organs of Eucommia ulmoides Oliv. Journal of Central South Forestry University, 26(4):1-4.[in Chinese])
高武军, 谢璐, 卢靖雯, 等. 2010. 重复序列及异染色质化在植物性染色体重组抑制中的作用. 遗传, 32(1):25-30.
(Gao W J, Xie L, Lu J W, et al. 2010. Role of repetitive sequence and heterochromatize in recombination suppression of plant sex chromosomes. Hereditas (Beijing), 32(1):25-30.[in Chinese])
康宗利, 王宁, 杨玉红, 等. 2011. 猕猴桃性别鉴定研究进展. 北方园艺, (20):184-187.
(Kang Z L, Wang N, Yang Y H, et al. 2011. Research progress on the sex identification of Actinidia. Northern Horticulture, (20):184-187.[in Chinese])
李进波, 夏明元, 戚华雄, 等. 2006. 水稻抗褐飞虱基因Bph14和Bph15的分子标记辅助选择. 中国农业科学, 39(10):2132-2137.
(Li J B, Xia M Y, Qi H X, et al. 2006. Marker-assisted selection for brown planthopper (Nilaparvata lugens Stål) resistance genes Bph14 and Bph15 in rice. Scientia Agricultura Sinica, 39(10):2132-2137.[in Chinese])
李同华, 姜静, 陈建名, 等. 2004. 种子植物性别的多态性. 东北林业大学学报, 32(5):48-52.
(Li T H, Jiang J, Chen J M, et al. 2004. The sexual polymorphism of seed plant. Journal of Northeast Forestry University, 32(5):48-52.[in Chinese])
刘涛. 2011. 贵州玉米种质资源遗传多样性分析及种子纯度鉴定研究. 贵阳:贵州大学硕士学位论文, 22-25.
(Liu T. 2011. Studies on genetic diversity analysis of corn germplasms and purity identification of hybrid corn in Guizhou. Guiyang:MS thesis of Guizhou University, 22-25.)
马希汉, 张康健. 2011. 中国杜仲近代认识史上的四次飞跃. 中成药, 33(8):1393-1396.
(Ma X H, Zhang K J. 2011. Four leap of knowledge of China Eucommia in modern history. Chinese Traditional Patent Medicine, 33(8):1393-1396.[in Chinese])
莫俊, 赵德刚. 2014. 杜仲胶累积与异戊二烯代谢途径相关基因表达关系研究. 山地农业生物学报, 11(3):28-32.
(Mo J, Zhao D G. 2014. The relationship between the expression of isoprene metabolic pathway genes and gutta-percha rubber contents in Eucommia ulmoides Oliver.Journal of Mountain Agriculture and Biology, 11(3):28-32.[in Chinese])
裴庆利, 王春连, 刘丕庆, 等. 2011. 分子标记辅助选择在水稻抗病虫基因聚合上的应用. 中国水稻科学, 25(2):119-129.
(Pei Q L, Wang C L, Liu P Q, et al. 2011. Marker-assisted selection for pyramiding disease and insect resistance genes in rice. Chinese Journal of Rice Science, 25(2):119-129.[in Chinese])
时秋香, 刘世强, 李征, 等. 2009. 与黄瓜M基因连锁的三个共显性标记. 园艺学报, 36(5):737-742.
(Shi Q X, Liu S Q, Li Z, et al. 2009. Three co-dominant markers linked to M gene in Cucumis sativus. Acta Horticulturae Sinica,36(5):737-742.[in Chinese])
王丙武,王雅清,莫华,等. 1999. 杜仲雌雄株细胞学、顶芽及叶含胶量的比较. 植物学报, 41(1):11-15.
(Wang B W, Wang Y Q, Mo H, et al. 1999. Comparison of cytology, apical buds and gutta content between staminate and pistillate of Eucommia ulmoides trees. Acta Botanica Sinica, 41(1):11-15.[in Chinese])
王克霞. 2004. 芦笋雌雄株间生理生化特性的差异研究. 杭州:浙江大学硕士学位论文.
(Wang K X. 2004. Comparative studies on the differences of physiological and biochemical characteristics between male and female asparagus. Hangzhou:MS thesis of Zhejiang University.[in Chinese])
王弦云. 2013. ISSRs在杜仲遗传多样性研究及性别鉴定中的应用. 合肥:安徽农业大学硕士学位论文.
(Wang X Y. 2013. Application of ISSRs to analysis of genetic diversity and sex identification for Eucommia ulmoides. Hefei:MS thesis of Anhui Agricultural University.[in Chinese])
吴金红, 蒋江松, 陈惠兰, 等. 2002. 水稻稻瘟病抗性基因Pi-2(t)的精细定位. 作物学报, 28(4):505-509.
(Wu J H, Jiang J S, Chen H L, et al. 2002. Fine mapping of rice blast resistance gene Pi-2 (t). Acta Agronomica Sinica,28(4):505-509.[in Chinese])
许智宏, 刘春明. 1998. 植物发育的分子机理. 北京:科学出版社, 225-238.
(Xu Z H, Liu C M. 1998. Molecular mechanism of plant development. Beijing:Science Press, 225-238.[in Chinese])
赵德刚, 李岩, 赵懿琛, 等. 2015. 杜仲雌雄株转录组测序数据组装及基因功能注释. 山地农业生物学报, 34(1):1-12.
(Zhao D G, Li Y, Zhao Y Z, et al. 2015. Transcriptome data assembly and gene function annotation of female and male plants in Eucommia ulmoides. Journal of Mountain Agriculture and Biology, 34(1):1-12.[in Chinese])
赵云云, 刘捷平. 1991. 雌雄异株植物的生理生化特性及性别鉴定.北京师范学院学报:自然科学版, 12(4):27-33.
(Zhao Y Y, Liu J P. 1991. Physiological and biochemical characteristics and identification of the sexes in dioecious plants. Journal of Capital Normal University:Natural Science Edition, 12 (4):27-33.[in Chinese])
周胜军,张鹏, 朱育强, 等. 2013. 与黄瓜全雌性基因连锁的SSR分子标记. 浙江大学学报:农业与生命科学版, 39(3):291-298.
(Zhou S J, Zhang P, Zhu Y Q, et al. 2013. Identification of SSR marker linked to gynoecious loci in cucumber (Cucumis sativus L.). Journal of Zhejiang University:Agriculture and Life Sciences, 39(3):291-298.[in Chinese])
Charlesworth B. 1991. The evolution of sex chromosomes. Science, 251(4997):1030-1033.
Charlesworth D. 2002. Plant sex determination and sex chromosomes. Heredity, 88(2):94-101.
Costich D E, Meagher T R, Yurkow E J. 1991. A rapid means of sex identification in Silene latifolia by use of flow cytometry. Plant Mol Biol Rep, 9(4):359-370.
Dolezel J, Gohde W. 1995. Sex determination in dioecious plants Melandrium album and M. rubrum using high-resolution flow cytometry. Cytometry, 19(2):103-106.
Di S V, Kesseli R V, Mulcahy D L. 1998. A pseudoautosomal random amplified polymorphic DNA marker for the sex chromosomes of Silene dioica. Genetics, 149(4):2057-2062.
Elmeer L K, Mattat I. 2012. Marker-assisted sex differentiation in date palm using simple sequence repeats. Biotech, 2(2):241-247.
Golenberg E M, West N W. 2013. Hormonal interactions and gene regulation can link monoecy and environmental plasticity to the evolution of dioecy in plants. American Journal of Botany, 100(6):1022-1037.
Li G, Quiros C F. 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. Theoretical & Applied Genetics, 103(2):455-461.
Liao L Q, Liu J, Dai Y X, et al. 2009. Development and application of SCAR markers for sex identification in the dioecious species Ginkgo biloba L. Euphytica, 169(1):49-55.
Nanda S, Kar B, Nayak S, et al. 2013. Development of an ISSR based STS marker for sex identification in pointed gourd (Trichosanthes dioica Roxb.). Scientia Horticulturae, 150:11-15.
Peil A, Flachowsky H, Schumann E, et al. 2003. Sex-linked AFLP markers indicate a pseudoautosomal region in hemp (Cannabis sativa L.). Theoretical & Applied Genetics, 107(1):102-109.
Rode J, Chol I K, Saal B, et al. 2005. Sex-linked SSR markers in hemp. Plant Breeding, 124:167-170.
Sakamoto K, Akiyama Y, Fukui K, et al. 1998. Characterization; genome size and morphology of sex chromosomes in hemp (Cannabis sativa L.). Cytologia, 63(4):459-464.
Shibata F, Hizume M, Kuroki Y. 1999. Chromosome painting of Y chromosome and isolation of a Y chromosome-specific repetitive sequences in the dioecious plant Rumex acetosa. Chromosoma, 108(4):266-270.
Wang D W, Li Y, Li Z Q. 2011. Identification of a male-specific amplified fragment length polymorphism (AFLP) and a sequence characterized amplified region (SCAR) marker in Eucommia ulmoides Oliv. International Journal of Molecular Sciences, 12(1):857-864.
Xu W J, Wang B W, Cui K M. 2004. RAPD and SCAR markers linked to sex determination in Eucommia ulmoides Oliv. Euphytica, 136(3):233-238.
Zhou Y J, Wang X G, Zhang X Q. 2011. Development and application of a SRAP marker for the identification of sex in Buchloe dactyloides. Euphytica, 181(2):261-266.

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