生物多样性 ›› 2016, Vol. 24 ›› Issue (1): 3-11.doi: 10.17520/biods.2015157

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莲品种DNA指纹图谱的构建

薛建华1, , A;*(), 姜莉2, #, 马晓林2, 邴艳红1, 赵思晨2, 马克平1   

  1. 1 中国科学院植物研究所植被与环境变化国家重点实验室, 北京 100093
    2 北京市海淀区圆明园管理处, 北京 100084
  • 收稿日期:2015-06-08 接受日期:2015-09-13 出版日期:2016-01-20
  • 通讯作者: 薛建华 E-mail:xuejianhua@ibcas.ac.cn
  • 基金项目:
    基金项目: 国家自然科学基金(31270262)、科技基础性工作专项(2013FY112300)、北京市海淀区科委项目(K2012004S)

Identification of lotus cultivars using DNA fingerprinting

Jianhua Xue1, *(), Li Jiang2, #, Xiaolin Ma2, Yanhong Bing1, Sichen Zhao2, Keping Ma1   

  1. 1 State Key Laboratory of Vegetation and Environmental Change, Institute of Botany, Chinese Academy of Sciences, Beijing 100093
    2 Yuanmingyuan Administration, Beijing 100084
  • Received:2015-06-08 Accepted:2015-09-13 Online:2016-01-20
  • Contact: Xue Jianhua E-mail:xuejianhua@ibcas.ac.cn

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

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

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)"

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
等位基因数
Alleles number
基因型数
Genotype number
特有等位
基因数
Specific allele
number
PIC N. lutea成分的品种数 Number in cultivars of Nelumbo lutea
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"

表2

17对SSR核心引物等位基因编码标准"

引物
Primer
编码 Code
1 2 3 4 5 6 7 8 9 0a 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
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