植物学报 ›› 2017, Vol. 52 ›› Issue (6): 723-732.doi: 10.11983/CBB16237

• 研究报告 • 上一篇    下一篇

山葡萄种质资源DNA条形码通用序列的筛选

宋慧芳, 刘海双, 杨义明, 范书田, 李昌禹*(), 艾军*()   

  1. 中国农业科学院特产研究所, 长春 130112
  • 收稿日期:2016-12-02 接受日期:2017-01-24 出版日期:2017-11-01 发布日期:2018-02-22
  • 通讯作者: 李昌禹,艾军 E-mail:lcy_lcy2002@163.com;aijun1005@163.com
  • 基金资助:
    农业部物种资源保护(农作物)项目(No.2016NWB043)和国家现代农业产业技术体系建设专项(No.CARS-30-7)

Screening of Universal DNA Barcodes for Vitis amurensis

Song Huifang, Liu Haishuang, Yang Yiming, Fan Shutian, Li Changyu*(), Ai Jun*()   

  1. Institute of Special Wild Economic Animals and Plants, Chinese Academy of Agricultural Sciences, Changchun 130112, China
  • Received:2016-12-02 Accepted:2017-01-24 Online:2017-11-01 Published:2018-02-22
  • Contact: Li Changyu,Ai Jun E-mail:lcy_lcy2002@163.com;aijun1005@163.com

摘要:

对山葡萄(Vitis amurensis)种质资源样品的ITS、ITS2、psbA-trnHrbcLmatK序列进行PCR扩增及测序, 优化PCR反应的退火温度, 比较各序列的扩增效率、测序成功率、品种间和品种内的差异及barcoding gap图, 使用BLAST和NJ树法比较不同序列的鉴定能力, 最终从5条DNA片段中筛选出可用于山葡萄种质资源鉴定的DNA条形码通用序列。结果表明, 在采集的11份33个山葡萄样品中, psbA-trnH和ITS2序列的扩增与测序成功率较高, 其品种间、品种内差异及barcoding gap较ITS、rbcLmatK序列具有明显的优势, 且ITS2序列能够鉴别psbA-trnH序列无法鉴别的品种。实验证明, ITS2和psbA-trnH序列是较适合鉴别山葡萄资源的DNA条形码序列组合。DNA条形码弥补了形态学鉴定的不足, 可为山葡萄种质资源的准确鉴定提供科学依据。

关键词: 山葡萄, DNA条形码, 鉴定, ITS2, psbA-trnH, 序列

Abstract:

To determine the best candidate sequence that can be used as a universal DNA barcode to identify Vitis amurensis varieties, we used PCR amplification with the ideal annealing temperature and sequenced the DNA fragments of five different regions (ITS, ITS2, psbA-trnH, rbcL and matK) of Amur grape samples. The effective sequence ratios obtained by PCR amplification and sequencing, divergence of intra- and inter-varieties, DNA barcoding gap and identification by using BLAST and NJ tree methods were used to evaluate the discrimination ability of these candidate sequences. ITS2 and psbA-trnH had fairly high amplification efficiency and sequencing efficiency in 33 samples belonging to 11 V. amurensis resources; they had larger divergences of intra- and inter-varieties and better DNA barcoding gaps than the other regions tested (ITS, rbcL and matK). In addition, ITS2 could discriminate some varieties that could not be successfully discriminated by the psbA-trnH region. ITS2 and psbA-trnH may be promising DNA barcode region combinations for identifying V. amurensis resources. DNA barcoding can compensate for morphological identification and help in the exact identification of V. amurensis resources.

Key words: Vitis amurensis, DNA barcode, identification, ITS2, psbA-trnH, sequence

表1

山葡萄品种信息"

Number Varieties name Locality of origin Parents or source Flower type
1 Zuoyouhong Zuojia, Jilin Varieties Bisexual
2 Shuanghong Zuojia, Jilin Varieties Bisexual
3 Zuoshan1 Zuojia, Jilin Wild resource Male
4 Zuoshan2 Zuojia, Jilin Wild resource Male
5 4N1 Zuojia, Jilin Genetic material Tetraploid
6 4N2 Zuojia, Jilin Genetic material Tetraploid
7 Shuangqing Zuojia, Jilin Varieties Bisexual
8 Shuangfeng Zuojia, Jilin Varieties Bisexual
9 Shuangyou Ji’an, Jilin Varieties Bisexual
10 75047 Shangzhi, Heilongjiang Wild resource Female
11 73061 Dunhua, Jilin Wild resource Female

表2

山葡萄DNA条形码标记引物序列及退火温度"

Fragment Eight annealing temperature gradient (°C) Annealing temperature (°C)
ITS2 54.8-55.4-56.0-56.6-57.2-57.8-58.4-59.0 56.0
psbA-trnH 52.2-52.8-53.4-54.0-54.6-55.2-55.8-56.4 55.2
matK 48.9-49.5-50.1-50.7-51.3-51.9-52.5-53.1 50.1
rbcL 52.1-52.7-53.3-53.9-54.5-55.1-55.7-56.3 54.5
ITS 49.9-50.5-51.1-51.7-52.3-52.9-53.5-54.1 52.9

图1

5个候选序列PCR反应的梯度退火温度(A) ITS2; (B) matK; (C) rbcL; (D) psbA-trnH; (E) ITS。M: DNA marker DL2000; 1-8: 45-60°C之间的温度梯度"

表3

不同DNA条形码序列扩增获得的有效序列比例"

Marker Amplification
efficiency
(%)
Sequencing success rate
(%)
Effective sequence
ratio (%)
ITS2 100.0 96.9 96.9
matK 100.0 96.9 96.9
psbA-trnH 100.0 100.0 100.0
rbcL 96.9 90.9 88.1
ITS 45.5 30.3 13.8

表4

4个候选序列/序列组合的长度、GC含量及品种间和品种内差异"

Potential barcode Aligned length
(bp)
Number of variable sites Mean intra-
distance
Mean inter-
distance
Average of GC content
(%)
ITS2 483 397 0.0015 0.1162 64.50
matK 896 44 0.0032 0.0110 35.40
psbA-trnH 422 238 0.0089 0.0921 27.50
rbcL 697 82 0.0068 0.0180 44.20
ITS2+matK 1379 441 0.0024 0.0652 49.95
ITS2+psbA-trnH 905 635 0.0058 0.0985 46.00
ITS2+rbcL 1180 479 0.0045 0.0655 54.35
matK+psbA-trnH 1318 282 0.0064 0.0550 31.45
matK+rbcL 1543 126 0.0051 0.0220 41.35
psbA-trnH+rbcL 1119 320 0.0076 0.0560 39.80

表5

候选序列品种间差异的Wilcoxon检验"

w+ w- Inter relative ranks n P value Result
ITS2 rbcL w+=33035.00, w-=13630.00 319 0.000 P<0.01, ITS2>rbcL
ITS2 matK w+=1444453.00, w-=0.00 577 0.000 P<0.01, ITS2>matK
ITS2 psbA-trnH w+=11562.00, w-=8660.00 500 0.000 P<0.01, ITS2>psbA-trnH
rbcL matK w+=8673.00, w-=6903.00 319 0.174 P>0.05, rbcL=matK
rbcL psbA-trnH w+=4350.00, w-=8720.00 620 0.000 P<0.01, rbcL<psbA-trnH
matK psbA-trnH w+=12556.00, w-=25330.00 422 0.000 P<0.01, matK<psbA-trnH

表6

候选序列品种内差异的Wilcoxon检验"

w+ w- Inter relative ranks n P value Result
ITS2 rbcL w+=39345.00, w-=18966.00 384 0.000 P<0.01, ITS2>rbcL
ITS2 matK w+=142845.00, w-=0.00 577 0.000 P<0.01, ITS2>matK
ITS2 psbA-trnH w+=17550.00, w-=18326.00 366 0.056 P>0.05, ITS2=psbA-trnH
rbcL matK w+=11952.00, w-=5253.00 384 0.000 P<0.01, rbcL>matK
rbcL psbA-trnH w+=5968.00, w-=13589.00 469 0.000 P<0.01, rbc<psbA-trnH
matK psbA-trnH w+=3985.00, w-=12578.00 580 0.000 P<0.01, matK<psbA-trnH

图2

山葡萄品种间和品种内变异barcoding gap图(A) ITS2序列; (B) psbA-trnH序列; (C) rbcL序列; (D) matK序列"

图3

利用不同序列构建的11份山葡萄资源品种系统进化树(A) ITS2序列; (B) psbA-trnH序列; (C) rbcL序列; (D) matK序列"

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