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作物学报

作物学报 ›› 2018, Vol. 44 ›› Issue (7): 956-965.doi: 10.3724/SP.J.1006.2018.00956

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

利用SNP芯片构建我国冬油菜参试品种DNA指纹图谱

赵仁欣1,李森业1,郭瑞星2,曾新华2,文静1,马朝芝1,沈金雄1,涂金星1,傅廷栋1,易斌1,*()   

  1. 1 华中农业大学植物科学技术学院 / 作物遗传改良国家重点实验室, 湖北武汉 430000
    2 中国农业科学院油料作物研究所, 湖北武汉 430062
  • 收稿日期:2017-11-29 接受日期:2018-03-26 出版日期:2018-07-10 网络出版日期:2018-04-16
  • 通讯作者: 易斌
  • 基金资助:
    本研究由国家重点研发计划项目(2017YFD0102000)和中央高校基本科研业务费专项(2662016PY063)资助

Construction of DNA Fingerprinting for Brassica napus Varieties Based on SNP Chip

Ren-Xin ZHAO1,Sen-Ye LI1,Rui-Xing GUO2,Xin-Hua ZENG2,Jing WEN1,Chao-Zhi MA1,Jin-Xiong SHEN1,Jin-Xing TU1,Ting-Dong FU1,Bin YI1,*()   

  1. 1 National Key Laboratory of Crop Genetic Improvement, College of Plant Science and Technology, Huazhong Agricultural University, Wuhan 430000, Hubei, China
    2 Oil Crops Research Institute, Chinese Academy of Agricultural Sciences, Wuhan 430062, Hubei, China
  • Received:2017-11-29 Accepted:2018-03-26 Published:2018-07-10 Published online:2018-04-16
  • Contact: Bin YI
  • Supported by:
    This study was supported by the National Key Research and Development Program of China (2017YFD0102000) and the Fundamental Research Funds for the Central Universities (2662016PY063).

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摘要：

以2016?2017年度187份国家油菜参试品种及2015?2016年度33份续试品种为材料, 利用油菜60K Illumina Infinium SNP (single nucleotide polymorphism)芯片进行基因分型, 得到52 157个SNP标记。按照分型为AA和BB频率不为0、最小基因型频率(minor freq)大于0.05、SNP得率(call freq)大于0.80、样品缺失率小于5%及分型明晰的要求筛选出5374个SNP标记。这些标记的PIC (polymorphism information content)均值为0.27, PIC值大于0.25的SNP标记占55.94%。其中有5143个SNP标记能对应到A1~A10、C1~C9连锁群上, 且比较全面地覆盖了油菜的基因组。用PowerMarker、MEGA等软件对224份样品的5374个标记分析, 结果显示: (1)设置8个品种重复2次点样对照, 得出Infinium芯片的技术误差为0.36%, 与Illumina公司公布的0.1%的技术误差十分接近。(2) NJ (Neighbor-joining)聚类分析中, 97.86%的区试品种的相似系数小于95%、87.88%的续试品种相似系数大于95%, 相似系数95%可作为品种特异性及一致性鉴定的阈值。这5374个SNP标记在油菜全基因组上分布均匀、多态性高、区分度好, 可用于甘蓝型油菜品种特异性和一致性的鉴定分析及甘蓝型油菜品种DNA指纹图谱构建的研究。

关键词: 甘蓝型油菜, DNA指纹图谱, SNP, 基因芯片

Abstract:

Using the Brassica 60K Infinium SNP (single nucleotide polymorphism) array, a total of 52 157 SNP markers were obtained by genotyping 33 annual test varieties in 2015-2016 and 187 varieties in 2016-2017. A total of 5374 high quality SNPs were retained after removing SNPs with AA or BB frequency = 0, main gene frequency < 0.80, secondary gene frequency < 0.05, sample loss rate > 0.50, or SNPs that did not show clearly defined clusters. The mean PIC (polymorphism information content) value of selected 5374 markers was 0.27, SNPs with the PIC value >0.25 accounted for 55.94% of total number. Among 5374 SNPs, 5143 corresponded to the A1-A10 and C1-C9 linkage groups, and covered the rapeseed genome. We analyzed 5374 SNPs from 224 samples with softwares like PowerMarker and MEGA, etc. The technical error of the SNP array was 0.36%, when comparing two replicates of eight samples. It was close to the 0.1% technical error announced by Illumina. The NJ (Neighbor- joining) cluster analysis revealed that 97.86% of the tested varieties had a similarity coefficient of less than 93%, 87.88% renewed varieties had a similarity coefficient of greater than 95%, which could be further used as criteria for the varietal specificity and the annual consistency assessment. The 5374 SNP markers screened in this study were highly polymorphic and evenly distributed in the rapeseed genome, and could be used for future variety identification and the construction of DNA fingerprinting.

Key words: Brassica napus, DNA fingerprinting, SNP, gene chip

表1

2016-2017我国冬油菜试验品种名称和编号"

No. 品种 Variety No. 品种 Variety No. 品种 Variety
1 阳光151 Yangguang 151 41 油研26J5# Youyan 26J5 # 81 南油846 Nanyou 846
2 川油53 Chuanyou 53 42 华油杂18# Huayouza 18 # 82 华油杂12 (CK) Huayouza 12 (CK)
3 内油835# Neiyou 835 # 43 陕油1508 Shaanyou 1508 83 希望402 Xiwang 402
4 希望152# Xiwang 152 # 44 川油杂1701 Chuanyouza 1701 84 大地199 Dadi 199
5 渝油35 Yuyou 35 45 蓉油29 Rongyou 29 85 华169 Hua 169
6 黔油37 Qianyou 37 46 庆油1号 Qingyou 1 86 中油613 Zhongyou 613
7 德5油66# De 5 you 66 # 47 绵油30 Mianyou 30 87 陕油1309 Shaanyou 1309
8 云油杂29 Yunyouza 29 48 中双3370 Zhongshuang 3370 88 华油杂28 Huayouza 28
9 杂优16 Zayou 16 49 大地193 Dadi 193 89 湘杂油373 Xiangzayou 373
10 蓉油18 (CK) Rongyou 18 (CK) 50 阳光165 Yangguang 165 90 亚华油207 Yahuayou 207
11 川农油5号 Chuannongyou 5 51 汉油10号 Hanyou 10 91 德5油319 De 5 you 319
12 宜油29 Yiyou 29 52 希望122# Xiwang 122 # 92 秦荣5号# Qinrong 5 #
13 中油611 Zhongyou 611 53 2616 93 景油517 Jingyou 517
14 南油546 Nanyou 546 54 沣油643 Fengyou 643 94 大地192 Dadi 192
15 蓉油22# Rongyou 22 # 55 川油56 Chuanyou 56 95 宁杂601 Ningza 601
16 卓油17# Zhuoyou 17 # 56 渝油36 Yuyou 36 96 苏6006 Su 6006
17 大地78# Dadi 78 # 57 创油16 Chuangyou 16 97 希望131 Xiwang 131
18 汉油11 Hanyou 11 58 华油杂12 (CK) Huayouza 12 (CK) 98 宁杂108# Ningza 108 #
19 蓉油26 Rongyou 26 59 希望152 Xiwang 152 99 荣华油50 Ronghuayou 50
20 阳光161 Yangguang 161 60 中油619 Zhongyou 619 100 中油600 Zhongyou 600
21 内油543 Neiyou 543 61 阳光736 Yangguang 736 101 德徽油88# Dehuiyou 88 #
22 蓉油18 (CK) Rongyou 18 (CK) 62 华油杂703 Huayouza 703 102 川油57 Chuanyou 57
23 陕油2016 Shaanyou 2016 63 华协82 Huaxie 82 103 核杂22 Heza 22
24 豪油18 Haoyou 18 64 沣油306# Fengyou 306 # 104 浙油杂319# Zheyouza 319 #
25 希望209 Xiwang 209 65 西油6号 Xiyou 6 105 科乐油2号 Keleyou 2
26 川油55 Chuanyou 55 66 蓉油30 Rongyou 30 106 宁杂158 Ningza 158
27 南油646# Nanyou 646 # 67 豪油29 Haoyou 29 107 浙油杂108# Zheyouza 108 #
28 黔油32# Qianyou 32 # 68 景油599 Jingyou 599 108 宁杂118# Ningza 118 #
29 庆油8号# Qingyou 8 # 69 湘杂油352 Xiangzayou 352 109 杂优15# Zayou 15 #
30 D27x54 D27x54 70 华油杂12 (CK) Huayouza 12 (CK) 110 核杂19# Heza 19 #
31 黔油35 Qianyou 35 71 渝华3号 Yuhua 3 111 甲14F06 Jia 14F06
32 希望291 Xiwang 291 72 圣光128 Shengguang 128 112 华油杂704 Huayouza 704
33 绵油35 Mianyou 35 73 湘杂油512# Xiangzayou 512 # 113 创油17 Chuangyou 17
34 蓉油18 (CK) Rongyou 18 (CK) 74 华油杂653 Huayouza 653 114 合油杂4501 Heyouza 4501
35 SZ15128 SZ15128 75 金油杂158 Jinyouza 158 115 嘉油1427 Jiayou 1427
36 大地195 Dadi 195 76 中油159 Zhongyou 159 116 秦优29 Qinyou 29
37 兴油540 Xingyou 540 77 中油153 Zhongyou 153 117 金地油559# Jindiyou 559 #
38 黔油38 Qianyou 38 78 希望202 Xiwang 202 118 浙核杂1401# Zheheza 1401 #
39 华油杂28 Huayouza 28 79 兴油杂19 Xingyouza 19 119 华油杂29 Huayouza 29
40 孝油800# Xiaoyou 800 # 80 秦优1619 Qinyou 1619 120 湘杂油456 Xiangzayou 456

表1

续"

No. 品种 Variety No. 品种 Variety No. 品种 Variety
121 浙杂1314 Zheza 1314 145 陕油21 Shaanyou 21 169 中油735# Zhongyou 735 #
122 陕油98 Shaanyou 98 146 荣华油505 Ronghuayou 505 170 大地69# Dadi 69 #
123 秦荣6号 Qinrong 6 147 双油1503 Shuangyou 1503 171 华油杂701# Huayouza 701 #
124 金油杂6号 Jinyouza 6 148 陕油1603 Shaanyou 1603 172 沣油568# Fengyou 568 #
125 镇13S629 Zhen 13S629 149 荣密1号 Rongmi 1 173 中油607 Zhongyou 607
126 沪油杂19 Huyouza 19 150 陕油1609 Shaanyou 1609 174 华油杂644 Huayouza 644
127 豪油38 Haoyou 38 151 陕油1924 Shaanyou 1924 175 Z14-1
128 沣油640 Fengyou 640 152 荣华油18 Ronghuayou 18 176 希望759 Xiwang 759
129 合油杂555# Heyouza 555 # 153 兴油576 Xingyou 576 177 沣油112 Fengyou 112
130 浙油杂315# Zheyouza 315 # 154 陕油1617 Shaanyou 1617 178 湘油422 Xiangyou 422
131 庆油1号 Qingyou 1 155 运杂1302 Yunza 1302 179 大地190 Dadi 190
132 浙油杂413 Zheyouza 413 156 陕油1506 Shaanyou 1506 180 华油杂29 Huayouza 29
133 秦优99 Qinyou 99 157 秦优7号(CK) Qinyou 7 (CK) 181 圣光158# Shengguang 158 #
134 陕油1203 Shaanyou 1203 158 黔油早6号 Qianyouzao 6 182 秦优1618 Qinyou 1618
135 合油杂2501 Heyouza 2501 159 黔油早5号 Qianyouzao 5 183 陕油1609 Shaanyou 1609
136 天禾油17 Tianheyou 17 160 玉油1号 Yuyou 1 184 阳光161 Yangguang 161
137 圣光158# Shengguang 158 # 161 云油杂28 Yunyouza 28 185 中油153 Zhongyou 153
138 南油758# Nanyou 758 # 162 川早油2号 Chuanzaoyou 2 186 秦优10号(CK) Qinyou 10 (CK)
139 秦优30 Qinyou 30 163 Z14-1 187 青杂10号(CK) Qingza 10 (CK)
140 陕油1607 Shaanyou 1607 164 蓉早2号 Rongzao 2 188 蓉油29 Rongyou 29
141 陕油33 Shaanyou 33 165 华油杂651 Huayouza 651 189 杂优15# Zayou 15 #
142 秦优1699 Qinyou 1699 166 阳光131 Yangguang 131 190 秦优10号(CK) Qinyou 10 (CK)
143 秦优1618 Qinyou 1618 167 川早油1号 Chuanzaoyou 1 191 青杂10号(CK) Qingza 10 (CK)
144 陕油1606 Shaanyou 1606 168 大地112 Dadi 112

图1

基于Genome Studio分析的不同类型的AA、AB、BB基因型的分型图A: 3种基因型分型边界不明显; B: 杂合群与纯合群相互重叠; C: 3种基因型向一侧偏移; D: 3种基因型分型界限明显。"

图2

5143个SNP标记在油菜基因组的位置分布图"

表2

8组点样对照品种的差异系数"

品种
Variety		 样品数
Number of samples		 差异系数
Variation coefficient (%)
阳光161 Yangguang 161 2 0. 06
蓉油29 Rongyou 29 2 0.12
中油153 Zhongyou 153 2 0.07
杂优15# Zayou 15# 2 0.07
华油杂29 Huayouza 29 2 1.45
秦优1618 Qinyou 1618 2 0.02
圣光158# Shengguang 158# 2 0.26
陕油1609 Shaanyou 1609 2 0.80
平均值 Mean 0.36

图3

2016-2017年度国家冬油菜参试品种相似系数分布"

图4

2016-2017年度国家冬油菜区试品种聚类分析结果编号对应的品种名称见表1。"

图5

2016-2017年度国家冬油菜续试品种聚类分析结果带*号的是2016-2017年度的种子, 不带的是2015-2016年度的种子。红色方框中圈出的是年度间相似系数小于95%的品种。"

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