山药基源植物分子鉴别体系建立与珠芽相关基因的转录组分析

发布时间：2018-04-09 12:23

本文选题：药材山药　切入点：薯蓣　出处：《南京农业大学》2016年博士论文

【摘要】：山药(Dioscorea.opposita Thunb.)是中国的传统药材,其基源植物为薯厀科(Dioscoreaceae)薯蓣属(Dioscorea Linn.)植物薯厀(D.opposita Thunb.在《Flora of China》中,已将其正名为D.polystachyaTurcz.),在其所有品种中又以铁棍山药(D.polystachya 'Tiegun')为最佳,目前公认的道地性产区为河南温县。国内市场上山药的基源植物存在着三个乱象:薯蓣近缘种的冒充造成物种混乱、铁棍山药的冒充造成品种混乱以及非道地产区冒充河南温县铁棍山药所造成的产区混乱。传统的形态学鉴别对于山药药材及其基源植物难以鉴定,显微鉴别需要较高的专业知识及复杂的操作技术,分子标记的指纹图谱等传统分子鉴定方法操作繁琐、花费的成本较大,因此基于基源植物鉴定的山药药材需要一种简单、快速和准确的方法。本研究搜集了山药基源植物薯蓣5个易混近缘种、14个薯蓣主要品种以及8个铁棍山药主产区的实验材料,分别在物种、品种及区域特异性三个层面建立分子鉴别体系。珠芽是薯蓣重要的营养器官和无性繁殖器官,但目前未见对其发生的分子机理的相关研究。本研究通过对产生珠芽的铁棍山药同株着生和不着生珠茅的叶腋、不产生珠芽的花籽山药的叶腋等3个组织,进行转录组n,序分析,旨在发掘参与珠芽发生的基因。具体研究的内容及结果如下:(1)通过 SRAP (Sequence-Related Amplified Polymorphism)分子标记对薯蓣及 5 个近缘种的遗传多样性研究发现,物种之间遗传多样性十分丰富:所筛选出的21对能扩增出清晰并呈现多态性条带的引物组合总共扩增产生了 355条重复性好、清晰的谱带,其中338条带为多态性带,占总扩增条带的95.27%;平均每对引物扩增16.90条带谱和16.10条多态性带。对遗传多样性分析发现遗传多样性总位点系数为Na为1.8963,有效位点系数Ne为1.7506。遗传多样性指数H为0.4840,香农指数I为0.6662。6个物种的总的杂合度Ht为0.4541,物种内的平均杂合度Hs为0.1661。物种间的Gst为0.5711,表明所有遗传变异中的57.11%都出现在物种之间。物种之间存在较为丰富的基因交流(Nm=0.3754)的主要原因是野生薯蓣属物种之间存在有性繁殖的方式。对物种之间的遗传结构研究发现,日本薯蓣(D.japonica Thunb.)的Fis值最低,表明其种内分化程度最高,褐苞薯蓣(D.persisimisPrainBurkill)的种内分化程度最低,山薯(D.fordii PrainBurkill)与其他5个物种的遗传差异最大(Fst=0.155),薯蓣与其它5个物种之间的遗传差异最小(Fst=0.038)。对遗传关系的研究发现,薯蓣与日本薯蓣之间遗传关系最近。本研究结合SCAR (Sequence Characterized Amplified Region)标记开发的物种特异性鉴定SRAP-SCAR分子标记,能有效的将薯蓣从其近缘种中鉴定出来。(2)利用 ISAP (Intron Sequence Amplified Polymorphism)分子标记对薯蓣14 个品种进行了遗传关系的分析,结果表明:11对ISAP引物共扩增出95条带,其中91条(95.79%)为多态性带。遗传多样性分析结果显示薯蓣品种之间存在着丰富的遗传多样性:总位点系数Na变化范围为1.8999到2.000,平均值为1.9651。有效位点Ne变化范围为1.1529到1.4271,平均值为1.3018。,遗传多样性指数H变化范围为0.1327到0.2673,平均值为0.2002。香农指数I变化范围为0.2011到0.5832,平均值为0.3102。遗传相似系数与遗传距离分析结果表明,与铁棍山药遗传关系由近到远的品种分别为:太谷山药、糙山药、麻山药、安顺山药、无架双胞山药、细长毛山药、米山药、华蓥山药、花籽山药、小白嘴山药、粗牛腿山药、九斤黄及白玉山药。薯蓣品种遗传关系分析表明,薯蓣品种之间可以明显的分成两大支,并且与形态学特征相关。从11对引物中筛选出1对铁棍山药的特异性引物,扩增得到1条大为435bp的特异性片段。对14个品种140个单株植物进行验证的结果显示铁棍山药10个单株全部在435bp处出现SCAR特异带,其他13个品种没有出现此带。因此本研究开发的ISAP-SCAR标记可以作为铁棍山药的特异分子标记,用来鉴别铁棍山药。(3)利用ISSR (Inter-Simple Sequence Repeat)分子标记对铁棍山药8个产区的产品的遗传多样性特征进行分析,结果表明:遗传多样性总位点系数Na变化范围从1.7789到2.0000,有效位点Ne变化范围为1.0417到1.5503,遗传多样性指数H为0.0794到0.3751以及香农指数I变化范围为0.1864到0.5388,表明了区域之间存在较为丰富的遗传多样性水平。对区域之间的遗传关系研究发现,与河南温县产区遗传关系由近到远的地区分别是江苏徐州、山西太原、山东济宁、湖南浏阳、河北沧州、四川南充和广东东莞,产区间遗传关系的远近与引种年限有关。利用SCAR (Sequence Characterized Amplified Region)技术进行铁棍山药的区域特异性相关的分子鉴别研究,通过ISSR扩增,在引物ISSR4 (ACACACACACACACACC)的扩增产物中获得河南温县铁棍山药的特异性谱带,再将这个条带转化为SCAR标记并进行单株检测,结果表明与河南温县铁棍山药的ISSR-SCAR标记在广东东莞、山东济宁、四川南充、湖南浏阳、山西太原产区中未出现,证明与这个标记能准确鉴别引种时间较长的非道地产区的铁棍山药。利用三种分子标记的数据对薯蓣不同区域、不同品种及不同物种进行联合分析发现,薯蓣及其近缘种在系统树上的位置与单独使用SRAP标记的分析结果相似:与薯蓣遗传关系由近到远的物种依次是日本薯蓣、参薯、无翅参薯、褐苞薯蓣和山薯。(4)本研究通过对铁棍山药同株生成珠芽、不生成珠芽及花籽山药不生成珠芽等3个组织进行转录组n,序,结果为:共得到Unigene70480个,对转录组的差异基因分析表明,铁棍山药不生成珠芽的组织相对于生成珠芽的组织,共有6263个Unigene表达上调,有8929个Unigene表达下调;铁棍山药生成珠芽的组织相对于花籽山药,共有13026个Unigene表达上调,有11352个Unigene表达下调。铁棍山药生成珠芽的组织同时相对于其不生成珠芽的部分及花籽山药共有2698个Unigene上调,4949个Unigene下调。将差异基因在多个数据库中进行BLAST比对,最终得到这些Unigenes的注释信息。依据已报道的调控龙舌兰(Agave tequilana Linn.)、台闽苣苔(Titanotrichum oldhamii (Hemsley) Solereder)珠芽发生的关键基因,并结合调控植物侧枝形成的关键基因等,最终选择23个可能参与薯厀珠芽发生的候选基因。此外,本研究基于转录组数据的SSR (simple sequence repeats)位点分布特征开发得到11102对SSR引物,为进一步的薯蓣育种和遗传图谱构建等工作提供更加丰富的标记选择。
[Abstract]:Yam (Dioscorea.opposita Thunb.) is Chinese traditional medicine, the original plant for potato Xi, (Dioscoreaceae) (Dioscorea Linn.) plants of Dioscorea tuber Xi (D.opposita Thunb. in , has its name for D.polystachyaTurcz.), in all its varieties in tiegun (D.polystachya'Tiegun') is the best at present, recognized as the genuine producing areas of Henan Wenxian County. The domestic market of yam species there are three chaos: Dioscorea Species as species caused confusion, tiegun posing breed confusion and posing non real estate areas of Henan Wenxian County tiegun caused by producing chaos. The traditional morphological classification for Chinese yam herbs and plants to source identification, microscopic identification requires higher professional knowledge and complex operation technology, fingerprint and other traditional method for molecular identification of molecular markers. As the cost of large, cumbersome, and so on, yam medicine based source plant identification requires a simple, rapid and accurate method. This study has collected 5 species of Dioscorea yam mixed species, the experimental materials of 14 Dioscorea main varieties and 8 tiegun respectively in the main producing areas, species three aspects, varieties and regional specificity to establish a molecular identification system. The bulbils are Dioscorea important vegetative organs and asexual reproduction organs, but no molecular mechanism of the occurrence of the related research. Through the research of producing bulbils tiegun with plant life and not a pearl Mao axil, not have the seed yam bulbils axil of 3 organizations, transcription group n, sequence analysis, aims to explore the participation of bulbil occurrence gene. The specific research contents and results are as follows: (1) by SRAP (Sequence-Related Amplified Polymorphism) branch 5 genetic diversity species of Dioscorea and found on molecular markers, genetic diversity between species is very rich: 21 of the screened amplified clear and polymorphic primer combinations amplified a total generated 355 reproducible and clear bands, of which 338 bands were polymorphic bands, accounted for 95.27%; the average of 16.90 bands per primer spectrum and 16.10 polymorphic bands. The genetic diversity analysis showed that the genetic diversity of the total site coefficient of Na was 1.8963, the effective site coefficient Ne 1.7506. genetic diversity index H is 0.4840, the Shannon index I 0.6662.6 species total heterozygosity of Ht was 0.4541, the average heterozygosity of Hs species in the 0.1661. species of Gst was 0.5711, showed that all the genetic variation in 57.11% species occur in between. There is abundant genetic exchange between species (Nm=0.3754) of the Lord Why is there sexual reproduction between wild Dioscorea Species. Study of genetic structure between species, Japanese yam (D.japonica Thunb.) the lowest Fis value showed that the intraspecific differentiation of the highest level of Dioscorea persimilis (D.persisimisPrainBurkill) species in the lowest level, potato (D.fordii PrainBurkill) and the maximum genetic differences among the other 5 species (Fst=0.155), the genetic differences between the other 5 species of Dioscorea and the minimum (Fst=0.038). The research on genetic relationship, genetic relationship with Japanese yam Dioscorea recently. The study combines SCAR (Sequence Characterized Amplified Region) species specific identification of SRAP-SCAR molecular marker development, can will be effective from the Dioscorea Species identified. (2) using ISAP (Intron Sequence Amplified Polymorphism) markers of 14 cultivars of Dioscorea Analysis of genetic relationship, the results show that: 11 pairs of ISAP primers amplified 95 bands, of which 91 bands (95.79%) were polymorphic. The genetic diversity analysis results show that there exist abundant genetic diversity of Dioscorea Species: the range of the total site coefficient Na is 1.8999 to 2, the average 1.9651. effective site Ne changes in the range of 1.1529 to 1.4271, the average value is 1.3018., the variation range of genetic diversity index H is 0.1327 to 0.2673, with an average of 0.2002. Shannon I index ranges from 0.2011 to 0.5832, with an average of 0.3102. genetic similarity coefficient and genetic distance analysis showed that genetic relationship and tiegun from near to far varieties are: Taigu yam, yam yam, brown, Anshun yam, frameless twin yam, fine hair yam, rice seed yam, Huaying yam, yam, white yam mouth, thick corbel yam, nine pounds of yellow and white yam yam. Analysis of genetic relationship between varieties indicated that diosgenin can be divided into two distinct branches, and correlated with morphological characteristics. From 11 to 1 of tiegun specific primers screened primers, amplified 1 large 435bp specific fragment of 14 varieties of 140 individual plants were conducted to validate the results show tiegun 10 individuals in all 435bp SCAR specific bands, the other 13 varieties do not have this belt. So the ISAP-SCAR markers developed in this study can be used as a specific molecular marker for the identification of tiegun, tiegun. (3) using ISSR (Inter-Simple Sequence Repeat) genetic marker products tiegun yam 8 from the characteristics of diversity analysis showed that the change range of the total genetic diversity of Na locus coefficient from 1.7789 to 2, the effective sites of Ne ranged from 1.0417 to 1.5503, genetic diversity index The number of H is 0.0794 to 0.3751 and the Shannon index I ranged from 0.1864 to 0.5388, indicates the level of genetic diversity between the rich areas. Study on genetic relationship between regions of Henan and Wenxian County found that the genetic relationship is far from near to the area are Jiangsu Xuzhou, Shanxi Taiyuan, Shandong Jining, Hunan Liuyang Hebei, Cangzhou, Sichuan, Nanchong and Guangdong in Dongguan, the genetic relationship between the distance and area introduction years. Using SCAR (Sequence Characterized Amplified Region) molecular identification of region specific related to tiegun technology, was amplified by ISSR with primer ISSR4 (ACACACACACACACACC) Henan Wenxian County tiegun specific bands obtained the PCR products, then the strip into a SCAR marker and single detection, and the result showed that Henan Wenxian County tiegun ISSR-SCAR markers in Guangzhou East Dongguan, Shandong Jining, Sichuan Nanchong, Hunan Liuyang, Shanxi did not appear in Taiyuan, and that this marker to identify the introduction of long non real estate areas of tiegun. By using three kinds of molecular marker data of Dioscorea in different regions, different varieties and different species of Dioscorea and combined analysis found that nearly species in the phylogenetic tree based on the location and analysis of the use of SRAP markers alone were similar with yam genetic relationship from the near to the distant species are Japanese yam, Dioscorea alata, unwinged alata, Dioscorea persimilis and potatoes. (4) this study by generating bulbils of tiegun monoecious, not generation of bulbils and seed yam bulbils do not generate 3 tissue transcriptome sequence, N, results were obtained: Unigene70480, analysis of genetic differences on the transcriptome showed that tiegun does not produce bulbils tissue with respect to the generation of bulbils, Co Expression of 6263 Unigene, 8929 Unigene expression; tiegun generation bead bud tissues relative to seed yam, co expression of 13026 Unigene, 11352 Unigene downregulated. Tiegun generated bulbils with respect to its organization also does not produce bulbils and seed yam has a total of 2698 Unigene rise, 4949 Unigene down. The differentiated genes BLAST comparison in multiple databases, eventually get these Unigenes annotation information. According to the reported regulation of agave (Agave tequilana Linn.), Titanotrichum oldhamii (Titanotrichum oldhamii (Hemsley) Solereder) key genes of bulbil occurrence, and combined with the regulation of plant branches the formation of the key genes, the final choice of the 23 candidate genes may be involved in the occurrence of potato Xi bulbils. In addition, this research is based on the transcriptome data of SSR (simple sequence repeats) site The distribution characteristics of the development of 11102 pairs of SSR primers for further breeding of Dioscorea and genetic map construction work markers provide a richer choice.

【学位授予单位】：南京农业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S632.1

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