Molecular Identity Card Establishment of Dendrobium Germplasms by TP-M13-SSR

doi:10.3969/j.issn.1000-2561.2021.10.002

Abstract

Abstract:

A total of 29 Dendrobium germplasms were studied with tailed primer M13 microsatellite markers (TP-M13- SSR). Analysis was made to perform the genetic diversity and establish the germplasms molecular ID. The results showed that by using 14 selected SSR markers, 191 alleles were detected and the detection rate was 79.31%-100% per primer. The polymorphism information content was 0.3877-0.9484, 0.7956 on average. Shannon’s information index was 0.8702-3.1553 and the average value was 2.1606. The genetic similarity coefficient of the germplasms ranged from 0.3037 to 0.9005, and all germplasms could be divided into 6 groups at 0.7000. Based on transferability and Shannon’s information index of the primers, more and more loci were added to distinguish all the germplasms. Finally, DoeSSR5, DoeSSR87 and DoeSSR97 at least were screened. Loci that amplified by the markers were coded to establish the unique and identifiable molecular ID of the germplasms.

Key words: Dendrobium, TP-M13-SSR, molecular identity card

CLC Number:

S682.31

XIAO Wenfang, LI Zuo, CHEN Heming, LYU Fubing. Molecular Identity Card Establishment of Dendrobium Germplasms by TP-M13-SSR[J]. Chinese Journal of Tropical Crops, 2021, 42(10): 2751-2757.

Figures/Tables 6

References 24

[1]	王雁, 周进昌, 郑宝强, 等. 石斛兰[M]. 北京: 中国林业出版社, 2014.
[2]	李会勇, 王天宇, 黎裕, 等. TP-M13自动荧光检测法在高粱SSR基因型鉴定中的应用[J]. 植物遗传资源学报, 2005, 6(1):68-70.
[3]	万映伶, 张嘉, 刘爱青, 等. 基于SSR标记构建中国芍药品种资源分子身份证[J]. 西北农林科技大学学报(自然科学版), 2018, 46(12):90-97.
[4]	陶乃奇, 张斌, 刘信凯, 等. 利用荧光标记SSR鉴别21个茶花新品种[J]. 植物学报, 2019, 54(1):37-45.
[5]	徐雷锋, 葛亮, 袁素霞, 等. 利用荧光标记SSR构建百合种质资源分子身份证[J]. 园艺学报, 2014, 41(10):2055-2064.
[6]	李程, 杨云尧, 张克中, 等. 牡丹花茶品种的筛选及SSR分子身份证的构建[J]. 分子植物育种, 2019, 17(20):6724-6735.
[7]	肖文芳, 李佐, 陈和明, 等. 利用SSR荧光标记毛细管电泳法检测蝴蝶兰组培突变体[J]. 热带作物学报, 2016, 37(10):1938-1944.
[8]	艾叶, 陈璐, 谢泰祥, 等. 基于SSR荧光标记构建建兰品种核心种质[J]. 园艺学报, 2019, 46(10):1999-2008
[9]	韩晓霞, 章静钢, 张鹏博, 等. 基于SSR的铁皮石斛实生群体遗传多样性研究[J]. 江苏农业科学, 2016, 44(6):90-93.
[10]	刘士辉, 李怀志, 沈若刚, 等. 石斛种质资源遗传多样性的SSR分析及指纹图谱构建[J]. 浙江农业科学, 2017, 58(7):1183-1185, 1189.
[11]	Zhao T M, Zheng S G, Hua Y D, et al. Classification of interspecific and intraspecific species by genome-wide SSR markers on Dendrobium[J]. South African Journal of Botany, 2019, 127:136-146. DOI
[12]	Xie J K, Zuo J H, Huang Y H, et al. The origin and germplasm collection for cultivated Dendrobium officinale K. Kimura & Migo individuals revealed by EST-SSR markers[J]. Genetic Resources and Crop Evolution, 2020, 67(5):1209-1219. DOI URL
[13]	宋爽, 周洋帆, 刘正杰, 等. 利用ISSR和AFLP标记分析石斛种质资源的遗传多样性[J]. 云南农业大学学报(自然科学), 2016, 31(4):688-695.
[14]	林榕燕, 叶秀仙, 钟淮钦, 等. 基于SRAP分子标记的石斛兰种质资源遗传多样性分析[J]. 福建农业学报, 2018, 33(5):469-473.
[15]	Li Q G, Mo J M, Wu W R, et al. Genetic diversity, population structure and identification of Dendrobium cultivars with high polysaccharide contents using SCoT, SCAR and nested PCR markers[J]. Genetic Resources and Crop Evolution, 2019, 66(1):71-88. DOI URL
[16]	Lu J J, Suo N N, Hu X, et al. Development and characterization of 110 novel EST-SSR markers for Dendrobium officinale (Orchidaceae)[J]. American Journal of Botany, 2012, 99(10):415-420.
[17]	Cai X Y, Feng Z Y, Hou B W, et al. Development of microsatellite markers for genetic diversity analysis of Dendrobium loddigesii Rolfe, an endangered orchid in China[J]. Biochemical Systematics & Ecology, 2012, 43(1):42-47.
[18]	Yuan Y H, Hou B W, Xu H J, et al. Identification of the geographic origin of Dendrobium thyrsiflorum on Chinese herbal medicine market using trinucleotide microsatellite markers[J]. Biological & Pharmaceutical Bulletin, 2011, 34(12):1794-1800.
[19]	Fan W J, Luo Y M, Li X X, et al. Development of microsatellite markers in Dendrobium fimbriatum Hook, an endangered Chinese endemic herb[J]. Molecular Ecology Resources, 2009, 9(1):373-375. DOI PMID
[20]	胡利娟, 贾元, 田忠静, 等. 基于ISSR标记的29种石斛遗传多样性分析[J]. 江苏农业科学, 2020, 48(17):76-80.
[21]	李怀志, 刘士辉, 沈若刚, 等. 铁皮石斛种质资源遗传多样性的SRAP分析及指纹图谱构建[J]. 安徽农业科学, 2017, 45(14):126-128.
[22]	付涛, 胡仲义, 何月秋, 等. 铁皮石斛EST-SSR分子标记的开发[J]. 核农学报, 2017, 31(4):663-670.
[23]	李永清, 江金兰, 叶炜, 等. 37份药用石斛种质资源亲缘关系的ISSR分析[J]. 福建农业学报, 2015, 30(2):131-135.
[24]	Lu J J, Liu Y Y, Xu J, et al. High-density genetic map construction and stem total polysaccharide content-related QTL exploration for Chinese endemic Dendrobium (Orchidaceae)[J]. Frontiers in Plant Science, 2018, 9:398. DOI URL

编号Number	中文名 Chinese name	拉丁名 Latin name	编号 Number	中文名 Chinese name	拉丁名 Latin name
Den01	茉香石斛	Den. hymenanthum	Den16	线叶石斛	Den. chryseum
Den02	雪山石斛	Den. Hibiki	Den17	晶帽石斛	Den. crystallinum
Den03	密花石斛	Den. densiflorum	Den18	木石斛	Den. crumenatum
Den04	尖刀唇石斛	Den. heterocarpum	Den19	纯白紫瓣石斛	Den. parishii album
Den05	曲径石斛	Den. flexicaule	Den20	棒叶石斛	Den. wassellii
Den06	齿瓣石斛	Den. devonianum	Den21	美花石斛	Den. loddigesii
Den07	喇叭唇石斛	Den. lituiflorum	Den22	绒毛石斛	Den. senile
Den08	勐腊石斛	Den. chrysocrepis	Den23	兜唇石斛	Den. aphyllum
Den09	霍山石斛	Den. huoshanense	Den24	报春石斛	Den. primulinum
Den10	玫瑰石斛	Den. crepidatum	Den25	黄喉石斛	Den. signatum
Den11	距唇石斛	Den. linawianum	Den26	铁皮石斛	Den. catenatum
Den12	金耳石斛	Den. hookerianum	Den27	河口石斛	Den. hekouense
Den13	黄贝壳石斛	Den. polyanthum	Den28	聚石斛	Den. lindleyi
Den14	紫婉石斛	Den. transparens	Den29	铅笔石斛	Den. teretifolium
Den15	重唇石斛	Den. hercoglossum

编号Number	中文名 Chinese name	拉丁名 Latin name	编号 Number	中文名 Chinese name	拉丁名 Latin name
Den01	茉香石斛	Den. hymenanthum	Den16	线叶石斛	Den. chryseum
Den02	雪山石斛	Den. Hibiki	Den17	晶帽石斛	Den. crystallinum
Den03	密花石斛	Den. densiflorum	Den18	木石斛	Den. crumenatum
Den04	尖刀唇石斛	Den. heterocarpum	Den19	纯白紫瓣石斛	Den. parishii album
Den05	曲径石斛	Den. flexicaule	Den20	棒叶石斛	Den. wassellii
Den06	齿瓣石斛	Den. devonianum	Den21	美花石斛	Den. loddigesii
Den07	喇叭唇石斛	Den. lituiflorum	Den22	绒毛石斛	Den. senile
Den08	勐腊石斛	Den. chrysocrepis	Den23	兜唇石斛	Den. aphyllum
Den09	霍山石斛	Den. huoshanense	Den24	报春石斛	Den. primulinum
Den10	玫瑰石斛	Den. crepidatum	Den25	黄喉石斛	Den. signatum
Den11	距唇石斛	Den. linawianum	Den26	铁皮石斛	Den. catenatum
Den12	金耳石斛	Den. hookerianum	Den27	河口石斛	Den. hekouense
Den13	黄贝壳石斛	Den. polyanthum	Den28	聚石斛	Den. lindleyi
Den14	紫婉石斛	Den. transparens	Den29	铅笔石斛	Den. teretifolium
Den15	重唇石斛	Den. hercoglossum

引物名称 Primer name	重复碱基 Repeat base	序列（5′-3′） Sequence (5′-3′)	荧光标记 Fluorescent labelled	检出率 Detection rate/%	峰值 Peak size/bp
DoeSSR4-F	(ATT)9	GATGATTGGTAAAGTGGAGTG	5-FAM	96.55	130~214
DoeSSR4-R	(ATT)9	AACCTACAAATTCAACCCTCT	5-FAM	96.55	130~214
DoeSSR5-F	(TCT)8	CAAAGAGTGGGTAGTTTGTCA	5-FAM	100.00	133~151
DoeSSR5-R	(TCT)8	TCAGAGAGATCTTCGGTTATG	5-FAM	100.00	133~151
DoeSSR15-F	(AT)12	GAATAATGCTTGAGAGGAGGT	5-FAM	96.55	130~207
DoeSSR15-R	(AT)12	CAACATTTACATAGCAAAGCA	5-FAM	96.55	130~207
DoeSSR23-F	(GCC)7	GACATTCTCCGTCAGGTACTA	5-FAM	89.66	103~152
DoeSSR23-R	(GCC)7	CTCACGCCACTAAAATCTTC	5-FAM	89.66	103~152
DoeSSR26-F	(TC)11	TGATGATATAATCCTCGCAAT	5-FAM	89.66	97~183
DoeSSR26-R	(TC)11	TGCAGTTCTATCGTACCAGTT	5-FAM	89.66	97~183
DoeSSR28-F	(GA)11	CGGTGGTCGTACAGAGAG	5-FAM	79.31	131~170
DoeSSR28-R	(GA)11	CTTGTGCTCATGAATCTTCTC	5-FAM	79.31	131~170
DoeSSR41-F	(GA)10	AACAAGCTTTGTTTTCCACTA	5-FAM	93.10	122~164
DoeSSR41-R	(GA)10	GCTCAAACTATAATGGCAGAG	5-FAM	93.10	122~164
DoeSSR51-F	(CT)13	TAACTAGAAGCCTTTGCTCAG	5-FAM	96.55	132~163
DoeSSR51-R	(CT)13	GAAGCTCCATGTTAATCACAG	5-FAM	96.55	132~163
DoeSSR67-F	(TGA)8	GGCCTAGAGGATGAAGAAATA	5-FAM	96.55	131~157
DoeSSR67-R	(TGA)8	CGAGACTTCTCCTTTGTGAC	5-FAM	96.55	131~157
DoeSSR84-F	(GCC)5	ATTCGGAAAATGGTTATCAT	5-FAM	79.31	114~161
DoeSSR84-R	(GCC)5	TGGGAGGGAGACATTCACA	5-FAM	79.31	114~161
DoeSSR87-F	(TTTC)5	ATGACGCCATGTACCACTCC	5-HEX	100.00	272~292
DoeSSR87-R	(TTTC)5	ACAACGATTCGCACCAGTTC	5-HEX	100.00	272~292
DoeSSR93-F	(TGT)7	TGGTTCTATCTTACCTCGGA	5-HEX	79.31	175~206
DoeSSR93-R	(TGT)7	GATTGTTTTATTCTGATTTGCA	5-HEX	79.31	175~206
DoeSSR97-F	(CT)7	CTTTCTACCACCAGGTTGTCA	5-HEX	96.55	239~310
DoeSSR97-R	(CT)7	CCATAAAGTCGGTCCATAGC	5-HEX	96.55	239~310
DoeSSR107-F	(AGA)6	TCAGGGAAGACTTTAGCAGA	5-HEX	79.31	228~246
DoeSSR107-R	(AGA)6	CTCAGGCATCAACAAACACC	5-HEX	79.31	228~246

引物名称 Primer name	重复碱基 Repeat base	序列（5′-3′） Sequence (5′-3′)	荧光标记 Fluorescent labelled	检出率 Detection rate/%	峰值 Peak size/bp
DoeSSR4-F	(ATT)9	GATGATTGGTAAAGTGGAGTG	5-FAM	96.55	130~214
DoeSSR4-R	(ATT)9	AACCTACAAATTCAACCCTCT	5-FAM	96.55	130~214
DoeSSR5-F	(TCT)8	CAAAGAGTGGGTAGTTTGTCA	5-FAM	100.00	133~151
DoeSSR5-R	(TCT)8	TCAGAGAGATCTTCGGTTATG	5-FAM	100.00	133~151
DoeSSR15-F	(AT)12	GAATAATGCTTGAGAGGAGGT	5-FAM	96.55	130~207
DoeSSR15-R	(AT)12	CAACATTTACATAGCAAAGCA	5-FAM	96.55	130~207
DoeSSR23-F	(GCC)7	GACATTCTCCGTCAGGTACTA	5-FAM	89.66	103~152
DoeSSR23-R	(GCC)7	CTCACGCCACTAAAATCTTC	5-FAM	89.66	103~152
DoeSSR26-F	(TC)11	TGATGATATAATCCTCGCAAT	5-FAM	89.66	97~183
DoeSSR26-R	(TC)11	TGCAGTTCTATCGTACCAGTT	5-FAM	89.66	97~183
DoeSSR28-F	(GA)11	CGGTGGTCGTACAGAGAG	5-FAM	79.31	131~170
DoeSSR28-R	(GA)11	CTTGTGCTCATGAATCTTCTC	5-FAM	79.31	131~170
DoeSSR41-F	(GA)10	AACAAGCTTTGTTTTCCACTA	5-FAM	93.10	122~164
DoeSSR41-R	(GA)10	GCTCAAACTATAATGGCAGAG	5-FAM	93.10	122~164
DoeSSR51-F	(CT)13	TAACTAGAAGCCTTTGCTCAG	5-FAM	96.55	132~163
DoeSSR51-R	(CT)13	GAAGCTCCATGTTAATCACAG	5-FAM	96.55	132~163
DoeSSR67-F	(TGA)8	GGCCTAGAGGATGAAGAAATA	5-FAM	96.55	131~157
DoeSSR67-R	(TGA)8	CGAGACTTCTCCTTTGTGAC	5-FAM	96.55	131~157
DoeSSR84-F	(GCC)5	ATTCGGAAAATGGTTATCAT	5-FAM	79.31	114~161
DoeSSR84-R	(GCC)5	TGGGAGGGAGACATTCACA	5-FAM	79.31	114~161
DoeSSR87-F	(TTTC)5	ATGACGCCATGTACCACTCC	5-HEX	100.00	272~292
DoeSSR87-R	(TTTC)5	ACAACGATTCGCACCAGTTC	5-HEX	100.00	272~292
DoeSSR93-F	(TGT)7	TGGTTCTATCTTACCTCGGA	5-HEX	79.31	175~206
DoeSSR93-R	(TGT)7	GATTGTTTTATTCTGATTTGCA	5-HEX	79.31	175~206
DoeSSR97-F	(CT)7	CTTTCTACCACCAGGTTGTCA	5-HEX	96.55	239~310
DoeSSR97-R	(CT)7	CCATAAAGTCGGTCCATAGC	5-HEX	96.55	239~310
DoeSSR107-F	(AGA)6	TCAGGGAAGACTTTAGCAGA	5-HEX	79.31	228~246
DoeSSR107-R	(AGA)6	CTCAGGCATCAACAAACACC	5-HEX	79.31	228~246

引物名称 Primer name	观测等位基因数 Na	有效等位基因数 Ne	观测杂合度 Ho	期望杂合度 He	多态性信息含量 PIC	Shannon信息指数 I
DoeSSR4	20	15.4286	0.2414	0.9352	0.9315	2.8688
DoeSSR5	7	3.1456	0.2414	0.6821	0.6369	1.4135
DoeSSR15	23	14.9492	0.4483	0.9331	0.9295	2.9344
DoeSSR23	10	6.8109	0.3448	0.8532	0.8366	2.0737
DoeSSR26	27	20.2530	0.4483	0.9506	0.9484	3.1553
DoeSSR28	15	7.8808	0.4444	0.8731	0.8637	2.3986
DoeSSR41	13	7.8208	0.6897	0.8721	0.8597	2.2641
DoeSSR51	12	8.1345	0.4828	0.8771	0.8649	2.2433
DoeSSR67	9	5.5556	0.4138	0.8200	0.7977	1.9061
DoeSSR84	11	3.9032	0.3448	0.7438	0.7220	1.8138
DoeSSR87	9	3.5193	0.2759	0.7159	0.6918	1.6574
DoeSSR93	8	4.1667	0.2414	0.7600	0.7311	1.7047
DoeSSR97	22	16.7937	0.5517	0.9405	0.9373	2.9452
DoeSSR107	5	1.6846	0.0870	0.4064	0.3877	0.8702
平均值	13.6429	8.5747	0.3754	0.8116	0.7956	2.1606