植物学报 ›› 2017, Vol. 52 ›› Issue (6): 723-732.DOI: 10.11983/CBB16237
宋慧芳, 刘海双, 杨义明, 范书田, 李昌禹*(), 艾军*()
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
基金资助:
Song Huifang, Liu Haishuang, Yang Yiming, Fan Shutian, Li Changyu*(), Ai Jun*()
Received:
2016-12-02
Accepted:
2017-01-24
Online:
2017-11-01
Published:
2018-02-22
Contact:
Li Changyu,Ai Jun
摘要: 对山葡萄(Vitis amurensis)种质资源样品的ITS、ITS2、psbA-trnH、rbcL和matK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、rbcL和matK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。
宋慧芳, 刘海双, 杨义明, 范书田, 李昌禹, 艾军. 山葡萄种质资源DNA条形码通用序列的筛选. 植物学报, 2017, 52(6): 723-732.
Song Huifang, Liu Haishuang, Yang Yiming, Fan Shutian, Li Changyu, Ai Jun. Screening of Universal DNA Barcodes for Vitis amurensis. Chinese Bulletin of Botany, 2017, 52(6): 723-732.
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1 山葡萄品种信息
Table 1 Information of Vitis amurensis varieties
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 |
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 |
表2 山葡萄DNA条形码标记引物序列及退火温度
Table 2 The primer information and annealing temperature of PCR from Vitis amurensis
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
Figure 1 Gradient annealing temperature of PCR reaction of 5 candidate sequences(A) ITS2; (B) matK; (C) rbcL; (D) psbA-trnH; (E) ITS. M: DNA marker DL2000; 1-8: Gradient annealing temperature among 45-60°C
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3 不同DNA条形码序列扩增获得的有效序列比例
Table 3 The effective sequence ratio obtained by PCR am- plification of five DNA barcode sequences
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 |
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
Table 4 Measures of inter-varieties and intra-varieties divergence locus length and average of GC content for 4 candidate barcodes/combination sequences
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 |
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5 候选序列品种间差异的Wilcoxon检验
Table 5 Wilcoxon signes tests for inter-varieties divergences of candidate sequences
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 |
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 |
表6 候选序列品种内差异的Wilcoxon检验
Table 6 Wilcoxon signes tests for intra-varieties divergences of candidate sequences
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
Figure 2 Distribution for intra- and inter-varieties variation of Vitis amurensis(A) ITS2 sequence; (B) psbA-trnH sequence; (C) rbcL sequence; (D) matK sequence
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
Figure 3 Neighbor-joining (NJ) tree for 11 varieties of Vitis amurensis by using different sequences(A) ITS2 sequence; (B) psbA-trnH sequence; (C) rbcL sequence; (D) matK sequence
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