發(fā)布時(shí)間：2018-04-09 12:23

  本文選題：藥材山藥　切入點(diǎn)：薯蕷　出處：《南京農(nóng)業(yè)大學(xué)》2016年博士論文

【摘要】：山藥(Dioscorea.opposita Thunb.)是中國(guó)的傳統(tǒng)藥材,其基源植物為薯厀科(Dioscoreaceae)薯蕷屬(Dioscorea Linn.)植物薯厀(D.opposita Thunb.在《Flora of China》中,已將其正名為D.polystachyaTurcz.),在其所有品種中又以鐵棍山藥(D.polystachya 'Tiegun')為最佳,目前公認(rèn)的道地性產(chǎn)區(qū)為河南溫縣。國(guó)內(nèi)市場(chǎng)上山藥的基源植物存在著三個(gè)亂象:薯蕷近緣種的冒充造成物種混亂、鐵棍山藥的冒充造成品種混亂以及非道地產(chǎn)區(qū)冒充河南溫縣鐵棍山藥所造成的產(chǎn)區(qū)混亂。傳統(tǒng)的形態(tài)學(xué)鑒別對(duì)于山藥藥材及其基源植物難以鑒定,顯微鑒別需要較高的專業(yè)知識(shí)及復(fù)雜的操作技術(shù),分子標(biāo)記的指紋圖譜等傳統(tǒng)分子鑒定方法操作繁瑣、花費(fèi)的成本較大,因此基于基源植物鑒定的山藥藥材需要一種簡(jiǎn)單、快速和準(zhǔn)確的方法。本研究搜集了山藥基源植物薯蕷5個(gè)易混近緣種、14個(gè)薯蕷主要品種以及8個(gè)鐵棍山藥主產(chǎn)區(qū)的實(shí)驗(yàn)材料,分別在物種、品種及區(qū)域特異性三個(gè)層面建立分子鑒別體系。珠芽是薯蕷重要的營(yíng)養(yǎng)器官和無性繁殖器官,但目前未見對(duì)其發(fā)生的分子機(jī)理的相關(guān)研究。本研究通過對(duì)產(chǎn)生珠芽的鐵棍山藥同株著生和不著生珠茅的葉腋、不產(chǎn)生珠芽的花籽山藥的葉腋等3個(gè)組織,進(jìn)行轉(zhuǎn)錄組n,序分析,旨在發(fā)掘參與珠芽發(fā)生的基因。具體研究的內(nèi)容及結(jié)果如下:(1)通過 SRAP (Sequence-Related Amplified Polymorphism)分子標(biāo)記對(duì)薯蕷及 5 個(gè)近緣種的遺傳多樣性研究發(fā)現(xiàn),物種之間遺傳多樣性十分豐富:所篩選出的21對(duì)能擴(kuò)增出清晰并呈現(xiàn)多態(tài)性條帶的引物組合總共擴(kuò)增產(chǎn)生了 355條重復(fù)性好、清晰的譜帶,其中338條帶為多態(tài)性帶,占總擴(kuò)增條帶的95.27%;平均每對(duì)引物擴(kuò)增16.90條帶譜和16.10條多態(tài)性帶。對(duì)遺傳多樣性分析發(fā)現(xiàn)遺傳多樣性總位點(diǎn)系數(shù)為Na為1.8963,有效位點(diǎn)系數(shù)Ne為1.7506。遺傳多樣性指數(shù)H為0.4840,香農(nóng)指數(shù)I為0.6662。6個(gè)物種的總的雜合度Ht為0.4541,物種內(nèi)的平均雜合度Hs為0.1661。物種間的Gst為0.5711,表明所有遺傳變異中的57.11%都出現(xiàn)在物種之間。物種之間存在較為豐富的基因交流(Nm=0.3754)的主要原因是野生薯蕷屬物種之間存在有性繁殖的方式。對(duì)物種之間的遺傳結(jié)構(gòu)研究發(fā)現(xiàn),日本薯蕷(D.japonica Thunb.)的Fis值最低,表明其種內(nèi)分化程度最高,褐苞薯蕷(D.persisimisPrainBurkill)的種內(nèi)分化程度最低,山薯(D.fordii PrainBurkill)與其他5個(gè)物種的遺傳差異最大(Fst=0.155),薯蕷與其它5個(gè)物種之間的遺傳差異最小(Fst=0.038)。對(duì)遺傳關(guān)系的研究發(fā)現(xiàn),薯蕷與日本薯蕷之間遺傳關(guān)系最近。本研究結(jié)合SCAR (Sequence Characterized Amplified Region)標(biāo)記開發(fā)的物種特異性鑒定SRAP-SCAR分子標(biāo)記,能有效的將薯蕷從其近緣種中鑒定出來。(2)利用 ISAP (Intron Sequence Amplified Polymorphism)分子標(biāo)記對(duì)薯蕷14 個(gè)品種進(jìn)行了遺傳關(guān)系的分析,結(jié)果表明:11對(duì)ISAP引物共擴(kuò)增出95條帶,其中91條(95.79%)為多態(tài)性帶。遺傳多樣性分析結(jié)果顯示薯蕷品種之間存在著豐富的遺傳多樣性:總位點(diǎn)系數(shù)Na變化范圍為1.8999到2.000,平均值為1.9651。有效位點(diǎn)Ne變化范圍為1.1529到1.4271,平均值為1.3018。,遺傳多樣性指數(shù)H變化范圍為0.1327到0.2673,平均值為0.2002。香農(nóng)指數(shù)I變化范圍為0.2011到0.5832,平均值為0.3102。遺傳相似系數(shù)與遺傳距離分析結(jié)果表明,與鐵棍山藥遺傳關(guān)系由近到遠(yuǎn)的品種分別為:太谷山藥、糙山藥、麻山藥、安順山藥、無架雙胞山藥、細(xì)長(zhǎng)毛山藥、米山藥、華鎣山藥、花籽山藥、小白嘴山藥、粗牛腿山藥、九斤黃及白玉山藥。薯蕷品種遺傳關(guān)系分析表明,薯蕷品種之間可以明顯的分成兩大支,并且與形態(tài)學(xué)特征相關(guān)。從11對(duì)引物中篩選出1對(duì)鐵棍山藥的特異性引物,擴(kuò)增得到1條大為435bp的特異性片段。對(duì)14個(gè)品種140個(gè)單株植物進(jìn)行驗(yàn)證的結(jié)果顯示鐵棍山藥10個(gè)單株全部在435bp處出現(xiàn)SCAR特異帶,其他13個(gè)品種沒有出現(xiàn)此帶。因此本研究開發(fā)的ISAP-SCAR標(biāo)記可以作為鐵棍山藥的特異分子標(biāo)記,用來鑒別鐵棍山藥。(3)利用ISSR (Inter-Simple Sequence Repeat)分子標(biāo)記對(duì)鐵棍山藥8個(gè)產(chǎn)區(qū)的產(chǎn)品的遺傳多樣性特征進(jìn)行分析,結(jié)果表明:遺傳多樣性總位點(diǎn)系數(shù)Na變化范圍從1.7789到2.0000,有效位點(diǎn)Ne變化范圍為1.0417到1.5503,遺傳多樣性指數(shù)H為0.0794到0.3751以及香農(nóng)指數(shù)I變化范圍為0.1864到0.5388,表明了區(qū)域之間存在較為豐富的遺傳多樣性水平。對(duì)區(qū)域之間的遺傳關(guān)系研究發(fā)現(xiàn),與河南溫縣產(chǎn)區(qū)遺傳關(guān)系由近到遠(yuǎn)的地區(qū)分別是江蘇徐州、山西太原、山東濟(jì)寧、湖南瀏陽、河北滄州、四川南充和廣東東莞,產(chǎn)區(qū)間遺傳關(guān)系的遠(yuǎn)近與引種年限有關(guān)。利用SCAR (Sequence Characterized Amplified Region)技術(shù)進(jìn)行鐵棍山藥的區(qū)域特異性相關(guān)的分子鑒別研究,通過ISSR擴(kuò)增,在引物ISSR4 (ACACACACACACACACC)的擴(kuò)增產(chǎn)物中獲得河南溫縣鐵棍山藥的特異性譜帶,再將這個(gè)條帶轉(zhuǎn)化為SCAR標(biāo)記并進(jìn)行單株檢測(cè),結(jié)果表明與河南溫縣鐵棍山藥的ISSR-SCAR標(biāo)記在廣東東莞、山東濟(jì)寧、四川南充、湖南瀏陽、山西太原產(chǎn)區(qū)中未出現(xiàn),證明與這個(gè)標(biāo)記能準(zhǔn)確鑒別引種時(shí)間較長(zhǎng)的非道地產(chǎn)區(qū)的鐵棍山藥。利用三種分子標(biāo)記的數(shù)據(jù)對(duì)薯蕷不同區(qū)域、不同品種及不同物種進(jìn)行聯(lián)合分析發(fā)現(xiàn),薯蕷及其近緣種在系統(tǒng)樹上的位置與單獨(dú)使用SRAP標(biāo)記的分析結(jié)果相似:與薯蕷遺傳關(guān)系由近到遠(yuǎn)的物種依次是日本薯蕷、參薯、無翅參薯、褐苞薯蕷和山薯。(4)本研究通過對(duì)鐵棍山藥同株生成珠芽、不生成珠芽及花籽山藥不生成珠芽等3個(gè)組織進(jìn)行轉(zhuǎn)錄組n,序,結(jié)果為:共得到Unigene70480個(gè),對(duì)轉(zhuǎn)錄組的差異基因分析表明,鐵棍山藥不生成珠芽的組織相對(duì)于生成珠芽的組織,共有6263個(gè)Unigene表達(dá)上調(diào),有8929個(gè)Unigene表達(dá)下調(diào);鐵棍山藥生成珠芽的組織相對(duì)于花籽山藥,共有13026個(gè)Unigene表達(dá)上調(diào),有11352個(gè)Unigene表達(dá)下調(diào)。鐵棍山藥生成珠芽的組織同時(shí)相對(duì)于其不生成珠芽的部分及花籽山藥共有2698個(gè)Unigene上調(diào),4949個(gè)Unigene下調(diào)。將差異基因在多個(gè)數(shù)據(jù)庫(kù)中進(jìn)行BLAST比對(duì),最終得到這些Unigenes的注釋信息。依據(jù)已報(bào)道的調(diào)控龍舌蘭(Agave tequilana Linn.)、臺(tái)閩苣苔(Titanotrichum oldhamii (Hemsley) Solereder)珠芽發(fā)生的關(guān)鍵基因,并結(jié)合調(diào)控植物側(cè)枝形成的關(guān)鍵基因等,最終選擇23個(gè)可能參與薯厀珠芽發(fā)生的候選基因。此外,本研究基于轉(zhuǎn)錄組數(shù)據(jù)的SSR (simple sequence repeats)位點(diǎn)分布特征開發(fā)得到11102對(duì)SSR引物,為進(jìn)一步的薯蕷育種和遺傳圖譜構(gòu)建等工作提供更加豐富的標(biāo)記選擇。
[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.

【學(xué)位授予單位】：南京農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：S632.1

【相似文獻(xiàn)】

相關(guān)期刊論文 前10條

1 陜西王耘;;解讀植物的珠芽[J];中國(guó)花卉盆景;2004年01期

2 金翁;;鐵樹珠芽繁殖法[J];中國(guó)花卉盆景;2010年08期

3 徐文果;陳志雄;張宏芳;余選禮;唐李軍;;紅魔芋珠芽繁種技術(shù)[J];中國(guó)熱帶農(nóng)業(yè);2007年05期

4 H.Sudasrip;董婉秋;;椰子珠芽無性繁殖[J];熱帶作物譯叢;1979年04期

5 趙祥云,程廉,邢尤美,謝麗萍,賈學(xué)文;百合珠芽組培及脫毒研究[J];園藝學(xué)報(bào);1993年03期

6 李艾蓮;梯度磁場(chǎng)對(duì)山藥珠芽的苗期生長(zhǎng)效應(yīng)[J];中國(guó)中藥雜志;2000年06期

7 陳俊義,潘祥初,謝舜松,張紅衛(wèi);珠芽繁育商品百合種技術(shù)初探[J];上海農(nóng)業(yè)科技;2004年05期

8 潘炳文;半夏珠芽生態(tài)觀察[J];中國(guó)中藥雜志;1998年09期

9 常莉;徐有明;薛建平;;離體培養(yǎng)條件下半夏葉柄形成珠芽過程中內(nèi)源激素的變化[J];華中農(nóng)業(yè)大學(xué)學(xué)報(bào);2007年05期

10 王道寬;半夏珠芽生態(tài)觀察[J];中藥材;1986年01期

相關(guān)會(huì)議論文 前1條

1 楊薇;楊柏云;羅麗萍;;腳板薯(Dioscorea batatas Decne．)脫毒試管苗培育及其試管珠芽誘導(dǎo)[A];中國(guó)園藝學(xué)會(huì)十屆二次理事會(huì)暨學(xué)術(shù)研討會(huì)論文摘要集[C];2007年

相關(guān)博士學(xué)位論文 前1條

1 彭斌;山藥基源植物分子鑒別體系建立與珠芽相關(guān)基因的轉(zhuǎn)錄組分析[D];南京農(nóng)業(yè)大學(xué);2016年

相關(guān)碩士學(xué)位論文 前5條

1 張偉;卷丹與布魯拉諾百合種間雜交后代形態(tài)性狀遺傳分析及珠芽性狀A(yù)FLP標(biāo)記[D];沈陽農(nóng)業(yè)大學(xué);2016年

2 葉德;基于高通量測(cè)序半夏珠芽轉(zhuǎn)錄組研究[D];浙江理工大學(xué);2017年

3 張恪;卷丹百合珠芽發(fā)育與花青素的積累研究[D];青海大學(xué);2017年

4 楊薇;腳板薯（Dioscorea batatas Decne.）脫毒試管苗培育及其試管珠芽誘導(dǎo)[D];南昌大學(xué);2006年

5 常莉;半夏珠芽的位置效應(yīng)及其影響因素[D];華中農(nóng)業(yè)大學(xué);2007年

，

本文編號(hào)：1726432

boshi