莲品种DNA指纹图谱的构建

doi:10.17520/biods.2015157

摘要/Abstract

摘要：

为了克服单纯依据形态特性鉴定品种的局限性, 我们开展了莲品种DNA指纹图谱构建研究, 旨在对其品种的快速准确鉴定及专利权保护等起一定作用。本研究以圆明园保存的72个莲品种为实验材料, 用来自不同地点的1,409份野生莲(Nelumbo nucifera)和58份美洲黄莲(N. lutea)群体样本作遗传背景参照。从104对核微卫星引物(nSSR)中筛选出15对, 从17对叶绿体微卫星(cpSSR)引物中筛选出2对, 共17对引物作为72个莲品种DNA指纹鉴定的条码。15对nSSR引物共检测到94个等位基因(平均6.27个), 其中11个属于美洲黄莲, 65个属于野生莲, 18个不能区分; 多态信息含量(PIC)介于0.3899-0.8023之间 (平均0.5748)。2对cpSSR引物共检测到13个单倍型, 其中9个属于野生莲, 4个属于美洲黄莲。全部17对引物标记结果显示, 共有19个品种含有美洲黄莲遗传组分, 其中8个母系来源于美洲黄莲; 有36个品种(涉及12对引物)具有至少1个特有基因型; 最少8对引物组合可完全区分开68个品种。有2组共4个品种组内全部17对引物均不能区分。本研究通过核心引物组合法使68个莲品种获得特异性DNA指纹。推荐13对nSSR和2对cpSSR共15对引物作为莲品种鉴定的核心条码, 并建议将形态特征与DNA指纹相结合作为莲品种的鉴定标准。

关键词: DNA指纹, 微卫星, Nelumbo, 品种鉴定, 分子身份证

Abstract

DNA fingerprinting is a fast and accurate method for cultivar identification, which can overcome the limitation of morphological traits. We used DNA fingerprinting to identify 72 lotus (Nelumbo) cultivars collected from the Resources Garden of the Yuanmingyuan Park in Beijing. We used 1,409 samples of N. nucifera and 58 samples of N. lutea as a genetic background reference. Fifteen out of 104 pairs of nucleus microsatellite primers (nSSR) and 2 out of 17 pairs of chloroplast microsatellite primers (cpSSR), for a total of 17 pairs of fluorescent primers, were selected as the barcode for fingerprint identification of the 72 lotus cultivars. For the 15 nSSR primers, 94 alleles were examined (with an average of 6.27). Out of the 94 alleles, 11 belong to N. lutea, 65 belong to N. nucifera, and the remaining 18 alleles could not be identified. The polymorphism information content (PIC) range between 0.3899 and 0.8023, with an average value of 0.5748. For the two pairs of cpSSR primers, 13 haplotypes were examined. Among them, 9 haplotypes belong to N. nucifera and 4 haplotypes belong to N. lutea. Results of identification of all 17 pairs of primer markers showed that 19 cultivars included genes from N. lutea, and 8 cultivars had female parents from N. lutea. There were 36 cultivars (using 12 pairs of primers) which had at least one unique genotype. With a minimum of 8 pairs of primers, 68 cultivars could be distinguished. Among the 72 cultivars, four cultivars in two groups could not be distinguished based on the whole set of 17 primers. Using the core primer combination method, we developed specific DNA fingerprints for each of the 68 lotus cultivars. Based on the above results, we recommended 15 pairs of primers including 13 pairs of nSSR and 2 pairs of cpSSR as the core barcode for lotus cultivar identification.

Key words: DNA fingerprint, microsatellites, Nelumbo, cultivar identification, molecular ID

薛建华, 姜莉, 马晓林, 邴艳红, 赵思晨, 马克平 (2016) 莲品种DNA指纹图谱的构建. 生物多样性, 24, 3-11. DOI: 10.17520/biods.2015157.

Jianhua Xue, Li Jiang, Xiaolin Ma, Yanhong Bing, Sichen Zhao, Keping Ma (2016) Identification of lotus cultivars using DNA fingerprinting. Biodiversity Science, 24, 3-11. DOI: 10.17520/biods.2015157.

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链接本文: https://www.biodiversity-science.net/CN/10.17520/biods.2015157

https://www.biodiversity-science.net/CN/Y2016/V24/I1/3

图/表 3

表1 17对SSR核心引物扩增的72个莲品种的等位基因数、基因型数及多态信息含量(PIC)

Table 1 Number of alleles, number of genotype, and polymorphic information content (PIC ) of 17 core SSR primers

引物 Primer	72个品种莲 72 lotus cultivars					1,409份野生莲等位基因数 Alleles number in 1,409 samples of N. nucifera	58份美洲黄莲等位基因数 Alleles number in 58 samples of N. lutea	种间共享等位基因数 Shared alleles between species
引物 Primer	等位基因数 Alleles number	基因型数 Genotype number	特有等位基因数 Specific allele number	PIC	有N. lutea成分的品种数 Number in cultivars of Nelumbo lutea		58份美洲黄莲等位基因数 Alleles number in 58 samples of N. lutea	种间共享等位基因数 Shared alleles between species
SSR030^[1]**	10	26	0	0.8023	0	15	2	0
SSR040^[1]	12	24	2	0.7426	0	10	5	2
SSR064^[1]	8	18	0	0.7199	4	15	1	0
SSR016^[1]	9	18	1	0.6576	6	16	6	3
SSR088^[1]	5	10	0	0.6284	0	4	2	0
Nelumbo14^[2]	6	12	0	0.6051	11	5	5	0
NS034^[3]	8	14	1	0.6022	0	9	5	4
SSR067^[1]	5	9	0	0.5846	0	6	2	0
SSR285^[1]	7	12	3	0.5795	0	6	4	0
SSR074^[1]	7	9	0	0.5379	0	10	3	1
Nelumbo13^[2]	4	6	1	0.5010	19	5	2	0
SSR472^[1]	3	5	0	0.4575	12	3	1	0
SSR049^[1]	3	4	0	0.4134	0	7	1	0
SSR482^[1]	3	5	0	0.4003	0	4	5	0
SSR045^[1]	4	7	0	0.3899	0	3	1	0
Lotus12^[4]	10	10	0	-	8	16	3	0
Lotus17^[4]	3	3	0	-	8	1	2	0

图1 本研究中72个莲品种花的形态图

Fig. 1 The floral morphologies of 72 lotus cultivars used in this study 1. 建选17号‘Jian Xuan Shiqihao’; 2. 沂蒙颂‘Yimeng Song’; 3. 绿如意‘Lvruyi’; 4. 蟹爪红‘Xiezhua Hong’; 5. 卓越‘Zhuoyue’; 6. 红唇‘Hong Chun’; 7. 千瓣莲‘Qianban Lian’; 8. 红樱桃‘Hongyingtao’; 9. 白芍药莲‘Bai Shaoyao Lian’; 10. 案头春‘Antou Chun’; 11. 一丈青‘Yizhangqing’; 12. 白湘莲‘Baixianglian’; 13. 黄舞妃‘Huang Wu Fei’; 14. 冰心‘Bing Xin’; 15. 珠峰翠影 ‘Zhufeng Cuiying’; 16. 新红‘Xin Hong’; 17. 大洒锦‘Dasajin’; 18. 中日友谊莲‘Zhong-ri Youyi Lian’; 19. 心洁‘Xin Jie’; 20. 晨光‘Chenguang’; 21. 星空牡丹‘Xingkong Mudan’; 22. 栀子碗莲‘Zhizi Wanlian’; 23. 粉千叶‘Fen Qianye’; 24. 日出‘Richu’; 25. 素质冰姿‘Suzhi Bing Zi’; 26. 红牡丹‘Hong Mudan’; 27. 古代莲‘Gudai Lian’; 28. 白雪公主‘Baixue Gongzhu’; 29. 紫重阳‘Zi Chongyang’; 30. 伯里夫人‘Boli Furen’; 31. 玫园秀色‘Meiyuan Xiu Se’; 32. 丹鹤‘Dan He’; 33. 金凤展翅‘Jinfeng Zhanchi’; 34. 英华‘Yinghua’; 35. 杏花春雨‘Xinghua Chunyu’; 36. 喜笑颜开‘Xixiaoyankai’; 37. 普者黑白荷‘Puzhehei Baihe’; 38. 粉楼春‘Fenlou Chun’; 39. 建乡壮士‘Jianxiang Zhuangshi’; 40. 希望‘Xiwang’; 41. 红领巾‘Honglingjin’; 42. 丹凤朝阳‘Dangfeng Chaoyang’; 43. 小洒锦‘Xiaosajin’; 44. 普者黑红荷‘Puzhehei Honghe’; 45. 蝶恋花‘Die Lian Hua’; 46. 小精灵‘Xiao Jingling’; 47. 红边玉蝶‘Hongbian Yudie’; 48. 佛见笑‘Fojianxiao’; 49. 莺莺‘Yingying’; 50. 奔月‘Benyue’; 51. 粉娃莲‘Fenwalian’; 52. 睡美人‘Shui Meiren’; 53. 东湖夕照‘Donghu Xi Zhao’; 54. 粉青莲‘Fen Qing Lian’; 55. 红湘莲‘Hongxianglian’; 56. 姬妃莲‘Jifei Lian’; 57. 点额妆‘Dian E Zhuang’; 58. 曙光‘Shuguang’; 59. 露半唇‘Lu Ban Chun’; 60. 桃红宿雨‘Tao Hong Su Yu’; 61. 春桃 ‘Chun Tao’; 62. 彩蝶‘Cai Die’; 63. 小佛手‘Xiao Foshou’; 64. 飞虹‘Fei Hong’; 65. 红楼‘Hong Lou’; 66. 粉霞‘Fen Xia’; 67. 丹绣球‘Dan Xiuqiu’; 68. 钗头凤‘Chai Tou Feng’; 69. 红盏托珠‘Hong Zhan Tuo Zhu’; 70. 平山芙蓉‘Pingshan Furong’; 71. 中山红台‘Zhongshan Hongtai’; 72. 至尊千瓣‘Zhizun Qianban’

图1 本研究中72个莲品种花的形态图

表2 17对SSR核心引物等位基因编码标准

Table 2 Alleles encoded standard of 17 core SSR primers

引物 Primer	编码 Code
引物 Primer	1	2	3	4	5	6	7	8	9	0^a	1	2
Lotus12	374^*	375	379^*	380	385	390	395	400	410	415
SSR040	205⁺	211	213^*	215	217	223	225	229	231	233	235	237⁺
SSR030	130^*	142	145	148	151	154	160	163	166	169
SSR016	190^*	215	218	221	224	233	236	242	254⁺
SSR285	262	272	274	276	292⁺	300⁺	316⁺
NS034	98	102	104	112	114	116	118	124⁺
Nelumbo14	147	149	151	153	171^*	173 ^*
SSR074	154	160	163	169	172	178	181
SSR049	233	245	248
SSR064	178^*	187	190	193	199	202	205	208
SSR067	238	244	247	253	262^*
SSR088	285	297	301	305^*	309^*
SSR482	172	200	203
Lotus17	231^*	242	246^*
Nelumbo13	142^*	146	156⁺	160
SSR045	187^*	211	214	217
SSR472	196^*	223	229

参考文献 25

1	Archak S, Lakshminarayanareddy V, Nagaraju J (2007) High-throughput multiplex microsatellite marker assay for detection and quantification of adulteration in Basmati rice (Oryza sativa). Electrophoresis, 28, 2396-2405.
2	Botstein D, White RL, Skolnick M, Davis RW (1980) Construction of a genetic linkage map in man using restriction fragment length polymorphisms. American Journal of Human Genetics, 32, 314-331.
3	Chen CW, Cao K, Wang LR, Zhu GR, Fang WC (2011) Molecular ID establishment of main China peach varieties and peach related species. Scientia Agricultura Sinica, 44, 2081-2093. (in Chinese with English abstract)
	[陈昌文, 曹珂, 王力荣, 朱更瑞, 方伟超 (2011) 中国桃主要品种资源及其野生近缘种的分子身份证构建. 中国农业科学, 44, 2081-2093.]
4	Faria MA, Nunes E, Oliveira MBPP (2008) Relative quantification of Vitis vinifera L. varieties in musts by microsatellite DNA analysis. European Food Research and Technology, 227, 845-850.
5	Jeffreys AJ, Wilson V, Thein SL (1985a) Individual-specific ‘fingerprints’ of human DNA. Nature, 316, 76-79.
6	Jeffreys AJ, Wilson V, Thein SL (1985b) Hypervariable ‘Minisatellite’ regions in human DNA. Nature, 314, 67-73.
7	Kubo N, Hirai M, Kaneko A, Tanaka D, Kasumi K (2009) Development and characterization of simple sequence repeat (SSR) markers in the water lotus (Nelumbo nucifera). Aquatic Botany, 90, 191-194.
8	Lai YP, Wang LR, He QL, Zhang ZF, Zhang XM, Du YY, Yu Y (2014) Screening of SSR core primers and fingerprinting construction of Brassica napus L. Southwest China Journal of Agricultural Sciences, 27, 2290-2299. (in Chinese with English abstract)
	[赖运平, 王丽容, 何巧林, 张浙峰, 张新明, 堵苑苑, 余毅 (2014) 甘蓝型油菜核心SSR引物筛选及指纹图谱构建. 西南农业学报, 27, 2290-2299.]
9	Li Y, Svetlana P, Yao JX, Li CS (2014) A review on the taxonomic, evolutionary and phytogeographic studies of the lotus plant (Nelumbonaceae: Nelumbo). Acta Geologica Sinica, 88, 1252-1261.
10	Liu K, Muse SV (2005) PowerMarker: an integrated analysis environment for genetic marker analysis. Bioinformatics, 21, 2128-2129.
11	Madhou M, Normand F, Bahorun T, Hormaza JI (2013) Fingerprinting and analysis of genetic diversity of litchi (Litchi chinensis Sonn.) accessions from different germplasm collections using microsatellite markers. Tree Genetics & Genomes, 9, 387-396.
12	Miura K, Ikegami S (2012) Photographic Reference Book of World Lotus Flowers. Bensei Publish Company, Tokyo. (in Japan)
13	Ming R, VanBuren R, Liu YL, Yang M, Han YP, Li LT, Zhang Q, Kim MJ, Schatz MC, Campbell M, Li JP, Bowers JE, Tang HB, Lyons E, Ferguson AA, Narzisi G, Nelson DR, Blaby-Haas CE, Gschwend AR, Jiao YN, Der JP, Zeng FC, Han J, Min XJ, Hudson KA, Singh R, Grennan AK, Karpowicz SJ, Watling JR, Ito K, Robinson SA, Hudson ME, Yu QY, Mockler TC, Carroll A, Zheng Y, Sunkar R, Jia RZ, Chen NC, Arro J, Wai CM, Wafula E, Spence A, Han YN, Xu LM, Zhang JS, Peery R, Haus MJ, Xiong WW, Walsh JA, Wu J, Wang ML, Zhu YJ, Paull RE, Britt AB, Du CG, Downie SR, Schuler MA, Michael TP, Long SP, Ort DR, Schopf JW, Gang DR, Jiang N, Yandell M, dePamphilis CW, Merchant SS, Paterson AH, Buchanan BB, Li SH, Shen-Miller J (2013) Genome of the long-living sacred lotus (Nelumbo nucifera Gaertn.). Genome Biology, 14, R41.
14	Nie XH, You CY, Li XF, Qin JH, Huang C, Guo HL, Wang XQ, Zhao WX, Lin ZX (2014) Construction of DNA fingerprinting and analysis of genetic diversity for Xinluzao cotton varieties. Acta Agronomica Sinica, 40, 2104-2117. (in Chinese with English abstract)
	[聂新辉, 尤春源, 李晓方, 秦江鸿, 黄聪, 郭欢乐, 王夏青, 赵文霞, 林忠旭(2014) 新陆早棉花DNA指纹图谱的构建及遗传多样性分析. 作物学报, 40, 2104-2117.]
15	Song HB, Cui XB, Ma HY, Zhu ZC, Luan FS (2012) Construction of DNA fingerprint database based on SSR marker for varieties (Lines) of Cucumis melo L. Scientia Agricultura Sinica, 45, 2676-2689. (in Chinese with English abstract)
	[宋海斌, 崔喜波, 马鸿艳, 朱子成, 栾非时 (2012) 基于SSR标记的甜瓜品种(系)DNA指纹图谱库的构建. 中国农业科学, 45, 2676-2689.]
16	Tian HL, Chen XQ, Wang JX, Xue JH, Wen J, Mitchell G, Zhou SL (2008) Development and characterization of microsatellite loci for lotus (Nelumbo nucifera). Conservation Genetics, 9, 1385-1388.
17	Wang QC, Zhang XY (2005) Lotus Flower Cultivars in China. China Forestry Publishing House, Beijing. (in Chinese)
	[王其超, 张行言 (2005) 中国荷花品种图志. 中国林业出版社, 北京.]
18	Wu ZY, Lu AM, Tang YC, Chen ZD, Li DZ (2003) The Families and Genera of Angiosperms in China. Science Press, Beijing. (in Chinese)
	[吴征镒, 路安民, 汤彦承, 陈之端, 李德铢 (2003) 中国被子植物科属综论. 科学出版社, 北京.]
19	Xue JH, Dong WP, Zhou SL (2012) Nelumbonaceae: Systematic position and species diversification revealed by the complete chloroplast genome. Journal of Systematics and Evolution, 50, 477-487.
20	Xue JH, Wang S, Zhou SL (2012) Polymorphic chloroplast microsatellite loci in Nelumbo (Nelumbonaceae). American Journal of Botany, 99, E240-E244.
21	Yang M, Han YN, VanBuren R, Ming R, Xu LM, Han YP, Liu YL (2012) Genetic linkage maps for Asian and American lotus constructed using novel SSR markers derived from the genome of sequenced cultivar. BMC Genomics, 13, 653.
22	Zhang XY, Chen LQ, Wang QC (2011) New Lotus Flower Cultivars in China I. China Forestry Publishing House, Beijing. (in Chinese)
	[张行言, 陈龙清, 王其超 (2011)中国荷花新品种图志I. 中国林业出版社, 北京.]
23	Zhao JR, Wang FG (2009) Research and Application of Maize Cultivars’ DNA Fingerprint Identification Technology. China Agricultural Science and Technology Press, Beijing. (in Chinese)
	[赵久然, 王凤格 (2009) 玉米品种DNA指纹鉴定技术研究与应用. 中国农业科学技术出版社, 北京.]
24	Zhao JR, Wang FG, Yi HM, Tian HL, Yang Y (2015) Progress of construction of Chinese maize varieties standard DNA fingerprint database. Crops, 165, 1-6. (in Chinese with English abstract).
	[赵久然, 王凤格, 易红梅, 田红丽, 杨扬 (2015) 我国玉米品种标准DNA指纹库构建研究及应用进展. 作物杂志, (165), 1-6.]
25	Zhao SJ, Zhu HJ, Lu XQ, He N, Liu WG (2014) Studies on DNA fingerprinting and genetic diversity of seedless watermelon (Citrullus lanatus) varieties using core simple sequence repeat (SSR) markers. Journal of Agricultural Biotechnology, 22, 188-194.
	(in Chinese with English abstract) [赵胜杰, 朱红菊, 路绪强, 何楠, 刘文革 (2014) 利用核心简单重复序列(SSR)标记分析无籽西瓜品种的DNA指纹及遗传多样性. 农业生物技术学报, 22, 188-194.]

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