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作物学报

作物学报 ›› 2024, Vol. 50 ›› Issue (7): 1867-1876.doi: 10.3724/SP.J.1006.2024.33063

• 研究简报 • 上一篇    下一篇

叶绿体标记在玉米种质资源快速分组中的应用分析

王蕊1,**(), 孙擘1,2,**(), 张云龙1, 张茗起1, 范亚明1, 田红丽1, 赵怡锟1, 易红梅1, 匡猛2,*(), 王凤格1,*()   

  1. 1北京市农林科学院玉米研究所 / 农业农村部农作物DNA指纹创新利用重点实验室(部省共建) / 玉米DNA指纹及分子育种北京市重点实验室, 北京 100097
    2中国农业科学院棉花研究所 / 棉花生物学国家重点实验室, 河南安阳 455000
  • 收稿日期:2023-10-26 接受日期:2024-01-30 出版日期:2024-07-12 网络出版日期:2024-02-23
  • 通讯作者: *王凤格, E-mail: gege0106@163.com; 匡猛, E-mail: kuangmeng007@163.com
  • 作者简介:王蕊, E-mail: skywangr@126.com;
    孙擘, E-mail: sunbo990818@163.com

    **同等贡献

  • 基金资助:
    北京市农林科学院科技创新能力建设专项(KJCX20230301);北京市农林科学院科技创新能力建设专项(KJCX20230303);北京市农林科学院科研创新平台建设专项(PT2023-34)

Application analysis of chloroplast markers on rapid classification in maize germplasm

WANG Rui1,**(), SUN Bo1,2,**(), ZHANG Yun-Long1, ZHANG Ming-Qi1, FAN Ya-Ming1, TIAN Hong-Li1, ZHAO Yi-Kun1, YI Hong-Mei1, KUANG Meng2,*(), WANG Feng-Ge1,*()   

  1. 1Maize Research Institute, Beijing Academy of Agricultural and Forestry Sciences / Key Laboratory of Crop DNA Fingerprinting Innovation and Utilization (Co-construction by Ministry and Province), Ministry of Agriculture and Rural Affairs / Beijing Key Laboratory of Maize DNA Fingerprinting and Molecular Breeding, Beijing 100097, China
    2Institute of Cotton Research, Chinese Academy of Agricultural Sciences / State Key Laboratory of Cotton Biology, Anyang 455000, Henan, China
  • Received:2023-10-26 Accepted:2024-01-30 Published:2024-07-12 Published online:2024-02-23
  • Contact: *E-mail: gege0106@163.com; E-mail: kuangmeng007@163.com
  • About author:

    **Contributed equally to this work

  • Supported by:
    Science and Technology Innovation Capacity Building Project of BAAFS(KJCX20230301);Science and Technology Innovation Capacity Building Project of BAAFS(KJCX20230303);Construction of Scientific Research and Innovation Platforms Special Project of BAAFS(PT2023-34)

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摘要：

叶绿体标记具有遗传保守性高且不受核基因重组干扰等特点, 适于对玉米种质资源进行分类管理。本研究基于Maize6H-60K芯片对玉米叶绿体标记筛选获得叶绿体分组候选位点, 使用上述位点对3549份玉米种质资源进行聚类分析, 将玉米种质资源划分为B、D、H、C、T共5个类群。通过基因型信息对比筛选出29个叶绿体分组特异标记(Varietal Chloroplast Panel, VCP)并设计KASP引物。开发兼容芯片、KASP平台的快速分组分析算法, 算法结果与聚类分析一致。挑选5个类群特异位点利用序贯法对种质资源进行快速分组检测, 构建玉米种质资源快速分组方法, 减少了95%的检测量, 为玉米种质资源提供一种新型高效的分组管理方法。

关键词: 玉米, 种质资源, 叶绿体标记, KASP, 分组方案

Abstract:

The chloroplast marker has high genetic conservation and is not affected by nuclear gene recombination, making it suitable for the classification and management of maize germplasm resources. Based on the Maize6H-60K chip, candidate markers for chloroplast grouping were obtained and 3549 maize germplasm resources were classified into five groups: B, D, H, C, and T. Twenty-nine specific chloroplast group panels (VCP) were selected and KASP primers were designed by the comparison of genotype information. A rapid group analysis algorithm compatible with chip and KASP platform was developed, which was consistent with the cluster analysis. Five group-specific markers were selected using sequential analysis to rapidly detect the germplasm resources, and a new efficient method for rapid grouping of maize germplasm resources was established, reducing the detection volume by 95%. This method provides a new efficient way to classify and manage maize germplasm resources.

Key words: maize, germplasm resources, chloroplast marker, KASP, classification method

附表1

Maize6H-60K芯片上68个叶绿体位点信息"

序号Number 位点名称
Loci number		 探针编号
Probe number		 等位基因
Allele		 在玉米品种B73叶绿体基因组上的位置
Location on the chloroplast genome of maize variety B73
1 CPMIDP02 AX-178078514 - // TCTTT 51,084
2 CPMIDP04 AX-178078490 - // ATGAACTTCTAATG 108,724
3 CPMIDP07 AX-178087616 - // G 33,742
4 CPMSNP01 AX-178078491 T // G 1337
5 CPMSNP02 AX-178078519 A // G 3358
6 CPMSNP05 AX-172772979 A // C 7431
7 CPMSNP07 AX-247287238 T // G 7533
8 CPMSNP09 AX-178078489 T // C 9640
9 CPMSNP10 AX-157921701 A // C 12,456
10 CPMSNP12 AX-157921697 T // C 12,988
11 CPMSNP15 AX-172772981 A // G 13,011
12 CPMSNP16 AX-178078506 A // G 14,852
13 CPMSNP17 AX-157921679 T // C 16,064
14 CPMSNP18 AX-178078503 T // G 16,124
15 CPMSNP19 AX-157921742 A // C 16,309
16 CPMSNP21 AX-247279512 A // C 18,618
17 CPMSNP22 AX-172773004 A // G 18,648
18 CPMSNP23 AX-157921748 T // G 19,299
19 CPMSNP24 AX-172773005 A // G 19,342
20 CPMSNP25 AX-178078507 T // C 19,363
21 CPMSNP26 AX-247233844 A // G 20,323
22 CPMSNP28 AX-157921674 T // G 20,665
23 CPMSNP29 AX-178078513 T // G 21,159
24 CPMSNP30 AX-157921751 A // G 29,999
25 CPMSNP31 AX-157921587 T // G 31,594
26 CPMSNP32 AX-178078509 T // G 31,631
27 CPMSNP33 AX-157921568 T // G 31,660
28 CPMSNP37 AX-157921692 T // C 32,830
29 CPMSNP38 AX-172773021 A // G 33,799
30 CPMSNP39 AX-172772993 A // C 34,124
31 CPMSNP41 AX-157921677 A // T 36,010
32 CPMSNP42 AX-172772994 A // T 36,558
33 CPMSNP44 AX-172773028 A // C 44,905
34 CPMSNP45 AX-157921694 T // G 45,987
35 CPMSNP47 AX-247285852 T // G 48,601
36 CPMSNP49 AX-157921576 T // G 49,550
37 CPMSNP50 AX-172773017 T // G 49,664
38 CPMSNP51 AX-172773018 T // G 49,715
39 CPMSNP52 AX-157921703 T // C 50,718
40 CPMSNP53 AX-172773019 A // G 50,781
41 CPMSNP54 AX-157921584 T // C 52,325
42 CPMSNP55 AX-172773020 A // G 52,460
43 CPMSNP57 AX-172772992 A // G 53,063
44 CPMSNP59 AX-172773002 A // G 56,142
45 CPMSNP62 AX-172773012 T // G 58,831
46 CPMSNP63 AX-172773013 T // G 59,519
47 CPMSNP64 AX-178078502 A // G 61,309
48 CPMSNP65 AX-157921725 A // G 61,560
49 CPMSNP67 AX-172772996 A // C 62,820
50 CPMSNP68 AX-172772997 T // C 64,621
51 CPMSNP70 AX-172772998 A // C 65,052
52 CPMSNP71 AX-172772999 A // C 66,406
53 CPMSNP72 AX-172773000 T // C 66,694
54 CPMSNP73 AX-172772986 T // C 66,832
55 CPMSNP78 AX-172773010 T // G 67,465
56 CPMSNP79 AX-157921686 T // G 69,515
57 CPMSNP80 AX-172773032 A // C 74,978
58 CPMSNP81 AX-172773014 A // G 77,910
59 CPMSNP84 AX-172773015 A // C 79,146
60 CPMSNP85 AX-172772976 T // G 80,107
61 CPMSNP86 AX-178078522 A // C 80,476
62 CPMSNP87 AX-172772977 A // T 105,654
63 CPMSNP88 AX-172772978 A // G 106,410
64 CPMSNP90 AX-178078518 T // G 107,436
65 CPMSNP91 AX-178078493 A // C 107,999
66 CPMSNP92 AX-178078504 T // C 108,597
67 CPMSNP94 AX-172773024 A // G 113,567
68 CPMSNP96 AX-157921671 A // G 115,907

图1

叶绿体分组候选位点筛选情况 a: 位点多态性且分型结果正确(候选); b: 分型结果正确但位点单态(剔除); c: 分型结果缺失(剔除); d: 分型结果散乱(剔除)。"

表1

叶绿体候选位点信息"

序号
No.		 位点名称
Loci name		 特异组别
VCP group		 等位基因
Allele		 特异等位基因
Specific allele		 是否入选
Whether selected
1 CPMSNP02 B A / G A 是Yes
2 CPMSNP17 B T / C C 是Yes
3 CPMSNP30 B A / G A 是Yes
4 CPMSNP92 B T / C C 是Yes
5 CPMSNP07 D T / G T 是Yes
6 CPMSNP19 D A / C C 是Yes
7 CPMSNP09 H T / C T 是Yes
8 CPMSNP10 H A / C A 是Yes
9 CPMSNP31 H T / G G 是Yes
10 CPMSNP33 H T / G G 是Yes
11 CPMSNP37 H T / C T 是Yes
12 CPMSNP45 H T / G G 是Yes
13 CPMSNP49 H T / G G 是Yes
14 CPMSNP52 H T / C T 是Yes
15 CPMSNP65 H A / G A 是Yes
16 CPMSNP79 H T / G T 是Yes
17 CPMSNP96 H A / G G 是Yes
18 CPMSNP18 C T / G G 是Yes
19 CPMSNP38 C A / G A 是Yes
20 CPMSNP39 C A / C C 是Yes
21 CPMSNP44 C A / C C 是Yes
22 CPMSNP57 C A / G A 是Yes
23 CPMSNP72 C T / C C 是Yes
24 CPMSNP81 C A / G A 是Yes
25 CPMSNP29 T T / G T 是Yes
26 CPMSNP47 T T / G G 是Yes
27 CPMSNP67 T A / C C 是Yes
28 CPMSNP73 T T / C C 是Yes
29 CPMSNP86 T A / C C 是Yes
30 CPMSNP12 B, H T / C C 否No
31 CPMSNP54 D, T T / C C 否No
32 CPMSNP64 D, T A / G G 否No
33 CPMSNP91 D, T A / C C 否No
34 CPMSNP22 B-Zi 330 group A / G G 否No
35 CPMSNP41 B-1145 group A / T T 否No
36 CPMSNP62 B-Feng 062 group T / G T 否No
37 CPMSNP85 B- Feng 062 group T / G G 否No
38 CPMSNP80 Evenly distributed A / C / 否No

图2

玉米种质资源自交系的叶绿体分组候选标记聚类分析结果"

图3

玉米种质资源叶绿体分组特异位点筛选"

表2

玉米叶绿体分组特异位点及KASP引物信息"

序号
No.		 特异位点编号
VCP number		 位点名称
Loci name		 上游引物1
Forward primer 1 (5'-3')		 上游引物2
Forward primer 2 (5'-3')		 下游通用引物
Reverse primer (5'-3')
1 VCPMB01 CPMSNP92 CTTGCAATAGGACTTACAACCTCC CTTGCAATAGGACTTACAACCTCT CCCATTTATATGGGAATTTTGGATAAGATT
2 VCPMB02 CPMSNP17 AAATTCATTCATTTCTTTTTTGAAAATGTCC CTAAATTCATTCATTTCTTTTTTGAAAATGTCT GGCATCTCGCACTAAACTAAGTCATAAA
3 VCPMB03 CPMSNP30 TAGGAAATCGCGAATTAGATCATTTGTTT GGAAATCGCGAATTAGATCATTTGTTC GCTCGTGCTTCTCTTGTTGAGGTAA
4 VCPMB04 CPMSNP02 AACAAACATAAACTAATTAGATAGAAAAGGAGT CAAACATAAACTAATTAGATAGAAAAGGAGC GAAAGAAAGGGAGTCTAATCCATAGAACTT
5 VCPMD01 CPMSNP19 AGTTGATGGTTAGGTTAATTCACGGAT GTTGATGGTTAGGTTAATTCACGGAG TAACCTTAAAAAGCTTAAAAAGTAGGGGAT
6 VCPMD02 CPMSNP07 GCAGGGGGTAGAAAGGCTGATA CAGGGGGTAGAAAGGCTGATC CTACATTGAATGTATAGCTGCAGCAATAAA
7 VCPMH01 CPMSNP45 AAGCGCGGGTTTCCTTTACTAATTTT AGCGCGGGTTTCCTTTACTAATTTG AGAGAGAGGGTTCGCATAGAGAGAA
8 VCPMH02 CPMSNP10 ATATTTTATAGGGTATATCCACCTGG CCTATATTTTATAGGGTATATCCACCTGT ACATAGACGGTCGACCCAGACATA
9 VCPMH03 CPMSNP49 AGTGAATCTTAAACCCATTGATAAAAGA AGTGAATCTTAAACCCATTGATAAAAGC TTTATTCCCTAACCATAGTTGTTATCCTTT
10 VCPMH04 CPMSNP96 AAAAGATCCTATTTTAACGAATCACACGTA AGATCCTATTTTAACGAATCACACGTG TACCATTAACTTTTTGTGTACTAGCAATAT
11 VCPMH05 CPMSNP79 GTATTTCTATTTTCTATAGCATAAAACCCG AAGTATTTCTATTTTCTATAGCATAAAACCCT GGATTTCTTGTAAATTTATCTCAAACCTAA
12 VCPMH06 CPMSNP33 ACTGACTTCTTTTACTTATTAAAATACAATTTA ACTGACTTCTTTTACTTATTAAAATACAATTTC CTAACAGGTCTGATTTTCGATTTTGTACTT
13 VCPMH07 CPMSNP65 CGATTTCTGTATCGATCATGATATACG ATCGATTTCTGTATCGATCATGATATACA GATATGCGTTTGAAATAGATGTGCGAGTT
14 VCPMH08 CPMSNP52 CCAAAAGGATAATCCTAGAATCCCG CCCAAAAGGATAATCCTAGAATCCCA ATCGGCACTTCTCCAAACCCAGAAA
15 VCPMH09 CPMSNP37 CAATTTTTATCAGAGGACAATATGAATATTAC CAATTTTTATCAGAGGACAATATGAATATTAT TATAACCCCTTGAGTGTTTTAATGGAACAT
16 VCPMH10 CPMSNP09 TCAACGTCCAATTATGAAATCCTTGG GTTCAACGTCCAATTATGAAATCCTTGA GTAGCAGCTATATTTCGGTTCATCCTTT
17 VCPMH11 CPMSNP31 TTAAGTATACATAAAGCAATTTTTTTTACTTT TAAGTATACATAAAGCAATTTTTTTTACTTG GTTAGCATTCTAAGGTCAAAAGTATAGTTT
18 VCPMC01 CPMSNP81 AGGCGTGGGCGAATTAGAGTC CAGGCGTGGGCGAATTAGAGTT GTCTTTGTTTATGCTTCGGATTGGAACAA
19 VCPMC02 CPMSNP18 GTGCTCGTTTAGTGTTCAGACCA GTGCTCGTTTAGTGTTCAGACCC CTTAGTTTAGTGCGAGATGCCCACAT
20 VCPMC03 CPMSNP57 TATTTAGTACTTGTTTATAGACTCGAC CCTTATTTAGTACTTGTTTATAGACTCGAT AATGCTTTTATCTCTATTCTATGGCGCAAT
21 VCPMC04 CPMSNP44 GCCTATACTACTATTCTATGGATAAAGCT CCTATACTACTATTCTATGGATAAAGCG TCGCTCACTAATTGATCTTTACGGTGTTT
22 VCPMC05 CPMSNP38 ATTCTAAAATCATTCTTTAGAAAGCCACAC CTAAAATCATTCTTTAGAAAGCCACAT GGCCAAGTCAGGTTAGATCTATATCTTTA
23 VCPMC06 CPMSNP72 TCATATACTAAAAAAGAATTCAAAAAGGGGA CATATACTAAAAAAGAATTCAAAAAGGGGG GAGATAGAATTCTTCGTGACATGACGAAA
24 VCPMC07 CPMSNP39 ATGGGAACTCAAAGATATCGAAGAGTA GGGAACTCAAAGATATCGAAGAGTC CAACCAATCACTCTTTTATTCCATCCTTTT
25 VCPMT01 CPMSNP29 ATATTCTAAAAAGATTGGATAGCAAAGATTTC GATATTCTAAAAAGATTGGATAGCAAAGATTTA GCTTTATCCCGTTTCATAGAAAGGAGATA
26 VCPMT02 CPMSNP73 ATTTCAAAAATTTTGTATTCTATTGGATTGGAT TCAAAAATTTTGTATTCTATTGGATTGGAC TTTGTTGTAATTCTTCGAATTCTCGAACAA
27 VCPMT03 CPMSNP67 AGTTGAACTTAATTCAAAAAGTAAAGCAATTCT GTTGAACTTAATTCAAAAAGTAAAGCAATTCG CGGGGACACATTTCTTGTGAGCAAA
28 VCPMT04 CPMSNP47 GAACTATTTATCCTTAAATTATTAACAAATAA GAACTATTTATCCTTAAATTATTAACAAATAC GCCAAGAGATTGGCATTTTCATTTGATCAT
29 VCPMT05 CPMSNP86 TTGAATCCTGCAATGGAGCTTCCA GAATCCTGCAATGGAGCTTCCC GCAGCCGGGTTAATAAAACTGAGAAAATT

图4

叶绿体分组特异标记KASP分型图 蓝色点表示FAM荧光标记等位基因, 红色点表示HEX荧光标记等位基因, 黑色点表示空白对照。"

表3

VCP 分组算法分析示意表"

特异位点编号
VCP number		 Sample 1 Sample 2 Sample 3 Sample 4 Sample 5 Sample 6 Sample 7 Sample 8
VCPMB01 T T T T F F F F
VCPMB02 T T F T F F F F
VCPMB03 T T T T F F F F
VCPMB04 T N T T F F F F
VCPMD01 F F F F T F F F
VCPMD02 F F F F T F F F
VCPMH01 F F F F F T F F
VCPMH02 F F F F F T F F
VCPMH03 F F F F F T F F
VCPMH04 F F F F F T F F
VCPMH05 F F F F F T F F
VCPMH06 F F F F F T F F
VCPMH07 F F F F F T F F
VCPMH08 F F F F F T F F
VCPMH09 F F F F F T F F
VCPMH10 F F F F F T F F
VCPMH11 F F F T F T F F
VCPMC01 F F F F F F T F
VCPMC02 F F F F F F T F
VCPMC03 F F F F F F T F
VCPMC04 F F F F F F T F
VCPMC05 F F F F F F T F
VCPMC06 F F F F F F T F
VCPMC07 F F F F F F T F
VCPMT01 F F F F F F F T
VCPMT02 F F F F F F F T
VCPMT03 F F F F F F F T
VCPMT04 F F F F F F F T
VCPMT05 F F F F F F F T
分组结果判定
Group results		 B group
 B group
 B group
 B group
 D group
 H group
 C group
 T group

表4

玉米叶绿体分组特异位点分组情况统计"

特异引物
编号
VCP number		 特异组别VCP group 特异组别
基因型
Specific group
genotype		 特异组别分型情况
Specific group classification (%)		 非特异组别分型情况
Non-specific group classification (%)
特异组别
基因型比例
Specific group genotype
proportions		 非特异组别
基因型比例
Non-specific group genotype
proportions		 缺失率
Missing rate		 特异组别基
因型比例
Specific group genotype
proportions		 非特异组别
基因型比例
Non-specific group genotype proportions		 缺失率
Missing rate
VCPMB01 B BB 99.97 0 0.03 99.66 0 0
VCPMB02 B BB 99.80 0 0.20 99.66 0 0.34
VCPMB03 B AA 99.70 0.07 0.24 100.00 0 0
VCPMB04 B AA 98.08 0.24 1.69 99.32 0.17 0.51
VCPMD01 D AA 99.73 0 0.27 99.97 0 0.03
VCPMD02 D BB 98.37 0.81 0.81 99.97 0 0.03
VCPMH01 H AA 100.00 0 0 100.00 0 0
VCPMH02 H BB 100.00 0 0 99.91 0 0.09
VCPMH03 H AA 100.00 0 0 99.79 0 0.21
VCPMH04 H AA 100.00 0 0 99.46 0.18 0.36
VCPMH05 H BB 99.49 0 0.51 99.88 0 0.12
VCPMH06 H AA 99.49 0 0.51 99.70 0.12 0.18
VCPMH07 H BB 98.48 0.51 1.02 99.76 0 0.24
VCPMH08 H BB 97.97 0 2.03 99.91 0.09 0
VCPMH09 H BB 96.95 0 3.05 99.88 0 0.12
VCPMH10 H BB 95.43 1.02 3.55 99.76 0 0.24
VCPMH11 H AA 95.43 1.02 3.55 97.76 0.78 1.46
VCPMC01 C BB 100.00 0 0 99.94 0 0.06
VCPMC02 C AA 100.00 0 0 99.92 0 0.08
VCPMC03 C BB 93.75 0 6.25 99.92 0 0.08
VCPMC04 C AA 93.75 6.25 0 99.89 0 0.11
VCPMC05 C BB 93.75 0 6.25 99.86 0 0.14
VCPMC06 C AA 93.75 0 6.25 99.83 0 0.17
VCPMC07 C AA 75.00 0 25.00 99.75 0 0.25
VCPMT01 T BB 100.00 0 0 99.89 0 0.11
VCPMT02 T AA 100.00 0 0 99.83 0 0.17
VCPMT03 T AA 100.00 0 0 99.72 0 0.28
VCPMT04 T AA 100.00 0 0 99.72 0 0.28
VCPMT05 T AA 100.00 0 0 99.72 0.11 0.17

图5

VCP 分组方案流程图"

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