能源作物芒属双药芒组SSR引物的筛选及其评价

doi:10.3724/SP.J.1003.2011.08109

生物多样性 ›› 2011, Vol. 19 ›› Issue (5): 535-542. DOI: 10.3724/SP.J.1003.2011.08109

能源作物芒属双药芒组SSR引物的筛选及其评价

马洪峥¹^,², 李珊珊², 葛颂², 戴思兰¹^,^*(), 陈文俐²^,^*()

¹北京林业大学园林学院, 北京 100083
²中国科学院植物研究所系统与进化植物学国家重点实验室, 北京 100093

收稿日期:2011-07-11 接受日期:2011-08-24 出版日期:2011-09-20 发布日期:2011-10-08
通讯作者: 戴思兰,陈文俐
基金资助:
中国科学院知识创新工程重要方向项目(KZCX2-YW-034);中国科学院知识创新工程重要方向项目(KSCX2-YW-Z-0943)

Isolation of SSR markers for two related second-generation energy crop species, Miscanthus nepalensis and M. nudipes (Poaceae)

Hongzheng Ma¹^,², Shanshan Li², Song Ge², Silan Dai¹^,^*(), Wenli Chen²^,^*()

¹College of Landscape Architecture, Beijing Forestry University, Beijing 100083
²State Key Laboratory of Systematic and Evolutionary Botany, Institute of Botany, Chinese Academy of Sciences, Beijing 100093

Received:2011-07-11 Accepted:2011-08-24 Online:2011-09-20 Published:2011-10-08
Contact: Silan Dai, Wenli Chen

1. 附录I 本研究中所用的21个国内牡丹品种和68个引进品种及来源.pdf(249KB)

摘要/Abstract

摘要：

禾本科芒属植物具有在边际性土地(marginal land)上驯化成为高产能源作物的巨大潜力, 其中尼泊尔芒(Miscanthus nepalensis)和双药芒(M. nudipes)两个双药芒组物种分布于喜马拉雅-横断山地区, 是具有耐寒及抗旱特性的优良种质资源。为了了解其遗传多样性和群体遗传结构,需要筛选出有效的分子标记。目前在适用于核心芒属植物群体遗传学分析的42对SSR引物中, 有23对来自于核心芒属物种,19对来自玉米。我们利用来自双药芒组2个种的6个代表性个体, 筛选得到14对可在双药芒组中稳定扩增的引物, 其中有12对来自核心芒属,显示同属引物的扩增效率为52.2%(12/23)。成功扩增的14个位点中11个为多态。利用所得引物对双药芒组4个自然居群进行群体遗传学初步分析发现, 所获得的SSR引物多态性高、表现稳定, 适于双药芒组物种的群体遗传多样性分析, 可为芒属植物资源的研究和利用提供重要的分子标记。

关键词: Miscanthus nepalensis, M. nudipes, SSR引物, 能源作物, 多态性

Abstract

Miscanthusspecies have shown great potential as second-generation energy crops, especially when planted on marginal land. Within the genus, M. nepalensisand M. nudipes are much different from other species owing to the presence of only two anthers, and are thus treated as sect. Diandra Keng taxonomically. Distributed in the Himalaya and Hengduan Mountains, the two species exhibit a very high level of tolerance to cold and drought stresses. In order to scientifically develop these crop species, the first step would be to carry out a population-genetic investigation to elucidate the genetic basis of phenotypes such as tolerance-related and morphological characteristics. For this purpose, we need to select practical and high-resolution molecular markers. We sequenced six individuals of M. nepalensisandM. nudipes to validate the efficacious use of 42 previously-used SSR primer pairs. A total of 14 primer pairs were identified as applicable, of which 12 were from Miscanthusspecies, indicating that the amplification ratio within genus Miscanthus is 52.2% (12/23). Of the 14 primer pairs, 11 were polymorphic. Screening four natural populations of M. nepalensisand M. nudipesusing the 14 SSR markers, we found that these primers had high power in detecting genetic diversity and population genetic structure in M. nepalensisand M. nudipes, and thus provide feasible genetic markers for population genetics studies and molecular breeding.

Key words: Miscanthus nepalensis, Miscanthus nudipes, SSR primers, energy crop, polymorphism

马洪峥, 李珊珊, 葛颂, 戴思兰, 陈文俐 (2011) 能源作物芒属双药芒组SSR引物的筛选及其评价. 生物多样性, 19, 535-542. DOI: 10.3724/SP.J.1003.2011.08109.

Hongzheng Ma, Shanshan Li, Song Ge, Silan Dai, Wenli Chen (2011) Isolation of SSR markers for two related second-generation energy crop species, Miscanthus nepalensis and M. nudipes (Poaceae). Biodiversity Science, 19, 535-542. DOI: 10.3724/SP.J.1003.2011.08109.

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链接本文: https://www.biodiversity-science.net/CN/10.3724/SP.J.1003.2011.08109

https://www.biodiversity-science.net/CN/Y2011/V19/I5/535

图/表 6

参考文献 26

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样品代号 Sample code	地理位置 Locality	样品数 Sample size	海拔 Altitude (m)	经度 Longitude (E)	纬度 Latitude (N)
引物筛选 Primer selection
双药芒 M. nudipes
Nu06-18	中国西藏 Tibet, China	1	2,887	88º57′00.16″	27º24′37.20″
Nu08-08	中国云南 Yunnan, China	1	2,751	100º15′40.61″	27º01′43.77″
Nu18-20	中国四川 Sichuan, China	1	2,466	102º55′43.46″	32º07′18.89″
尼泊尔芒 M. nepalensis
GS01-20	印度北安恰尔邦 Uttaranchal, India	1	2,200	79º27′14.58″	29º23′34.70″
Ne11-09	中国云南 Yunnan, China	1	2,270	100º07′40.00″	25º42′23.08″
Ne20-05	中国四川 Sichuan, China	1	878	102º46′44.96″	30º19′09.16″
适用性检测 Applicability test
双药芒 M. nudipes
Nu11	中国四川 Sichuan, China	20	2,235	102º23′29.66″	27º52′43.25″
Nu06	中国西藏 Tibet, China	20	2,887	88º57′00.16″	27º24′37.20″
尼泊尔芒 M. nepalensis
Ne09	中国云南 Yunnan, China	20	2,759	98º42′01.42″	25º58′40.00″
Ne18	中国四川 Sichuan, China	20	2,047	102º02′32.08″	28º25′26.24″

样品代号 Sample code	地理位置 Locality	样品数 Sample size	海拔 Altitude (m)	经度 Longitude (E)	纬度 Latitude (N)
引物筛选 Primer selection
双药芒 M. nudipes
Nu06-18	中国西藏 Tibet, China	1	2,887	88º57′00.16″	27º24′37.20″
Nu08-08	中国云南 Yunnan, China	1	2,751	100º15′40.61″	27º01′43.77″
Nu18-20	中国四川 Sichuan, China	1	2,466	102º55′43.46″	32º07′18.89″
尼泊尔芒 M. nepalensis
GS01-20	印度北安恰尔邦 Uttaranchal, India	1	2,200	79º27′14.58″	29º23′34.70″
Ne11-09	中国云南 Yunnan, China	1	2,270	100º07′40.00″	25º42′23.08″
Ne20-05	中国四川 Sichuan, China	1	878	102º46′44.96″	30º19′09.16″
适用性检测 Applicability test
双药芒 M. nudipes
Nu11	中国四川 Sichuan, China	20	2,235	102º23′29.66″	27º52′43.25″
Nu06	中国西藏 Tibet, China	20	2,887	88º57′00.16″	27º24′37.20″
尼泊尔芒 M. nepalensis
Ne09	中国云南 Yunnan, China	20	2,759	98º42′01.42″	25º58′40.00″
Ne18	中国四川 Sichuan, China	20	2,047	102º02′32.08″	28º25′26.24″

位点名称 Locus	引物序列 Primer sequences (5′-3′)	参考重复式样 Reference motif	参考片段长度 Reference length	退火温度 Annealing temperature (℃)
Bgln176	F: AGTTCACGTCCAGCTGAATGACAG R: CGCGCATCGCATGCTTATCCTA	-	140	54
Bgln434	F: GTGCAAAGGGGAGAGAGG R: TCGCCGTTCTTCGCCTTAG	-	85	54
DSSR09	F: CGCACCACTCCCTGACAAT R: GCCAGTAACATCCCCAACG	(AC)₁₇	294	58
DSSR24	F: AAGGGCGAGGCAAAGCAAG R: AGATTTCGTGACAAAGGGAGC	(GAT)₇(GA)₂	100	58
DSSR32	F: GATTAGACTGTTTGGTAGGGATTC R: CTGGTTTTGCGAGATTTCAT	(GA)₂₄	305	54
MSSR10	F: CCTGGGATTCTTTGATTTGAG R: GGATTTGGCTTTCGCTGTC	(AG)₁₉	159	54
MSSR14	F: AACTAAAGGCGAAAGCTAGGAGG R: CAGATGCTGGCTGTTGGTGATGT	(AC)₁₈A₉	155	58
MSSR21	F: TATGGGTGAATGTTGGTTT R: GCCCGTTTGTGCGAGTGC	(AC)₈A₈	187	58
MSSR36	F: TAAGCCCAAACAAAGGAAAT R: CAAATGGCAATAGTGAGCAA	(AG)₁₃	228	54
MSSR37	F: CAGATGCCATTACTGTAGCGA R: TACCACAACGAAACCAAAAC	(AC)₅(AG)₂(AC)₁₁	243	54
MSSR38	F: GAGTGAGCAGTGGCAACG R: ATCTGGCTGGACAACCTTTT	(GAT)₇	250	54
MSSR42	F: TGCCACGCCTTCTTCACCTATC R: GCATCCAGCCATCCACCCTC	(TG)₈ (AG)₁₄	179	58
M&M02	F: AAGACGGTGGTAAGCCACTT R: AGACAACACCAAAGTATTATC	(CAC)₉	110	54
M&M39	F: AGAAATGAAAGTGCAGTGAC R: AAGGAGTGCTTCTCCCTCTC	(AGGC)₆ (CGCA)₃	180	54

位点名称 Locus	引物序列 Primer sequences (5′-3′)	参考重复式样 Reference motif	参考片段长度 Reference length	退火温度 Annealing temperature (℃)
Bgln176	F: AGTTCACGTCCAGCTGAATGACAG R: CGCGCATCGCATGCTTATCCTA	-	140	54
Bgln434	F: GTGCAAAGGGGAGAGAGG R: TCGCCGTTCTTCGCCTTAG	-	85	54
DSSR09	F: CGCACCACTCCCTGACAAT R: GCCAGTAACATCCCCAACG	(AC)₁₇	294	58
DSSR24	F: AAGGGCGAGGCAAAGCAAG R: AGATTTCGTGACAAAGGGAGC	(GAT)₇(GA)₂	100	58
DSSR32	F: GATTAGACTGTTTGGTAGGGATTC R: CTGGTTTTGCGAGATTTCAT	(GA)₂₄	305	54
MSSR10	F: CCTGGGATTCTTTGATTTGAG R: GGATTTGGCTTTCGCTGTC	(AG)₁₉	159	54
MSSR14	F: AACTAAAGGCGAAAGCTAGGAGG R: CAGATGCTGGCTGTTGGTGATGT	(AC)₁₈A₉	155	58
MSSR21	F: TATGGGTGAATGTTGGTTT R: GCCCGTTTGTGCGAGTGC	(AC)₈A₈	187	58
MSSR36	F: TAAGCCCAAACAAAGGAAAT R: CAAATGGCAATAGTGAGCAA	(AG)₁₃	228	54
MSSR37	F: CAGATGCCATTACTGTAGCGA R: TACCACAACGAAACCAAAAC	(AC)₅(AG)₂(AC)₁₁	243	54
MSSR38	F: GAGTGAGCAGTGGCAACG R: ATCTGGCTGGACAACCTTTT	(GAT)₇	250	54
MSSR42	F: TGCCACGCCTTCTTCACCTATC R: GCATCCAGCCATCCACCCTC	(TG)₈ (AG)₁₄	179	58
M&M02	F: AAGACGGTGGTAAGCCACTT R: AGACAACACCAAAGTATTATC	(CAC)₉	110	54
M&M39	F: AGAAATGAAAGTGCAGTGAC R: AAGGAGTGCTTCTCCCTCTC	(AGGC)₆ (CGCA)₃	180	54

位点名称 Locus	重复式样 Repeat motif	等位基因数 N	长度变异范围 Sequence range (bp)	多态信息含量 PIC	期望杂合度 H_E	观察杂合度 Ho
Bgln176	(CTAG)₃(CT)₆(CGGC)₄	4	146-158	0.6260	0.5189	1.0000
Bgln434	(CT)₅C₄(CT)₃	1	84	0.0000	0.0000	0.0000
DSSR09	(AC)₇	1	272	0.0000	0.0000	0.0000
DSSR24	(GAT)₇(GA)₂	1	94	0.0000	0.0000	0.0000
DSSR32	(GA)₁₀	21	296-345	0.5198	0.3843	0.2625
MSSR10	(GA)₂GC(GA)₄T(GA)₂	6	117-127	0.5970	0.4808	0.8500
MSSR14	(AC)₄A₁₁	7	118-124	0.7189	0.4859	0.2375
MSSR21	T₇(CA)₆(CA)₆(AC)₄	10	167-208	0.7556	0.6055	0.9375
MSSR36	(AG)₉	8	218-226	0.6904	0.7210	0.3000
MSSR37	(CA)₄GAA(CA)₅	11	240-261	0.6691	0.3529	0.1625
MSSR38	(GAT)₄	2	236-239	0.3047	0.2564	0.5000
MSSR42	(AG)₁₀	8	177-194	0.6329	0.5881	0.8625
M&M02	(CAC)₅	5	90-106	0.6622	0.7096	0.3750
M&M39	(GAA)₃(CGCA)₂	2	141-154	0.3158	0.3984	0.5375
平均值 Average	-	6	-	0.4637	0.3930	0.4304

能源作物芒属双药芒组SSR引物的筛选及其评价

Isolation of SSR markers for two related second-generation energy crop species, Miscanthus nepalensis and M. nudipes (Poaceae)

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参考文献 26

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居群 Population	等位基因总数 N_a	近交系数 F_IS	居群分化指数 F_ST	个体分化指数 F_IT
M. nepalensis(Ne09)	22	-0.0412	0.3854	-
M. nepalensis(Ne18)	28	-0.2940	0.4793	-
M. nudipes(Nu06)	42	-0.1798	0.1775	-
M. nudipes(Nu11)	70	0.0358	0.0464	-
平均值 Average	40.5	-0.1198	0.2722	0.2447

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