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作物学报 ›› 2023, Vol. 49 ›› Issue (6): 1455-1465.doi: 10.3724/SP.J.1006.2023.21048

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

顶芒山羊草特异寡核苷酸探针开发和oligo-FISH核型构建

史培瑶(), 陈丽娟, 孙昊杰, 程梦豪, 肖进, 袁春霞, 王秀娥, 王海燕*()   

  1. 南京农业大学作物遗传与种质创新国家重点实验室/细胞遗传研究所/现代作物生产省部共建协同创新中心，江苏南京 210095
  • 收稿日期:2022-07-06 接受日期:2022-10-10 出版日期:2023-06-12 网络出版日期:2022-10-21
  • 通讯作者: *王海燕, E-mail: hywang@njau.edu.cn
  • 作者简介:E-mail: 2019101115@njau.edu.cn
  • 基金资助:
    国家重点研发计划项目(2020YFE0202900);中央高校基本科研业务费(KYZZ2022003);江苏省现代农业产业技术体系(JATS[2021]463);江苏省种业振兴项目(JBGS[2021]006);江苏省种业振兴项目(013);江苏省种业振兴项目(047)

Development of specific oligonucleotide probe library of Aegilops comosa and construction of oligo-FISH karyotype

SHI Pei-Yao(), CHEN Li-Juan, SUN Hao-Jie, CHENG Meng-Hao, XIAO Jin, YUAN Chun-Xia, WANG Xiu-E, WANG Hai-Yan*()   

  1. National Key Laboratory of Crop Genetics & Germplasm Enhancement and Utilization/Cytogenetics Institute/Collaborative Innovation Center for Modern Crop Production Co-sponsored by Province and Ministry (CIC-MCP), Nanjing Agricultural University, Nanjing 210095, Jiangsu, China
  • Received:2022-07-06 Accepted:2022-10-10 Published:2023-06-12 Published online:2022-10-21
  • Contact: *E-mail: hywang@njau.edu.cn
  • Supported by:
    National Key Research and Development Program of China(2020YFE0202900);Fundamental Research Funds for the Central University(KYZZ2022003);Jiangsu Province Modern Agricultural Industry Technology System(JATS[2021]463);Seed Industry Revitalization Project of Jiangsu Province(JBGS[2021]006);Seed Industry Revitalization Project of Jiangsu Province(013);Seed Industry Revitalization Project of Jiangsu Province(047)

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摘要：

栽培小麦近缘物种顶芒山羊草(Aegilops comosa, 2n=2x=14, MM)是小麦改良的三级基因库。为准确鉴定顶芒山羊草M基因组染色体或染色体区段, 本研究利用二代测序获得顶芒山羊草M基因组序列信息, 从中鉴定出16条可能的特异卫星重复序列。根据这些序列设计12个寡核苷酸(oligo)探针进行oligo-FISH, 结果表明, 其中10个探针可在顶芒山羊草染色体上产生明显的杂交信号。对探针特异性分析发现, 5个探针仅在顶芒山羊草染色体上产生杂交信号, 在小麦染色体上未观察明显杂交信号, 可作为顶芒山羊草特异探针鉴定小麦背景中的顶芒山羊草染色体。选择在顶芒山羊草染色体上信号分布丰富的3个探针(oligo-pAc89、oligo-pAc148、oligo-pAc225)组成探针套ONPS#AC1, 结合利用本实验室根据小麦D亚基因组开发的寡核苷酸探针库, 构建了顶芒山羊草的oligo-FISH核型。本研究构建的FISH核型可以准确识别顶芒山羊草各条染色体, 为挖掘、转移和利用顶芒山羊草优异基因提供了快速准确的鉴定手段。

关键词: 顶芒山羊草, 二代测序, 卫星重复序列, 寡聚核苷酸探针, 荧光原位杂交

Abstract:

Ae. comosa (Aegilops comosa, 2n=2x=14, MM) is a tertiary gene bank for wheat improvement. In order to accurately identify the chromosomes of Ae. comosa M genome or the chromosome segments transferred into wheat, the next-generation sequencing information of Ae. comosa M genome were obtained. Based on the next-generation sequencing information of Ae. comosa M genome, 12 oligonucleotide probes were designed for oligo-FISH analysis according to the 16 possible specific satellite repeats identified. The oligo-FISH results showed that ten of the probes could produce obvious hybridization signals on the chromosomes of Ae. comosa. The probe specificity analysis revealed that the five probes generated hybridization signals on the chromosomes of Ae. comosa, but there was no obvious hybridization signal on the chromosomes in wheat, which used as the specific probes to identify the chromosomes or chromosome segments of M genome in wheat background. Three probes (oligo-pAc89, oligo-pAc148, and oligo-pAc225) with abundant signal distribution on the chromosomes of Ae. comosa were selected to form a probe set named ONPS#AC1. Combined with the oligonucleotide probe library developed according to wheat D sub genome, the oligo-FISH karyotype of Ae. comosa was constructed, which can accurately identify each chromosome of the M genome, providing an important molecular cytogenetic basis for mining, transferring, and utilizing the excellent genes of Ae. comosa.

Key words: Aegilops comosa, second-generation sequencing, satellite repeats, Oligo nucleotide probe, fluorescence in situ hybridization

表1

开发的12个oligo探针的序列信息"

序列名称
Sequence name		 在M基因组
中占比
Percentage in M genome		 基序长度
Consensus
length (bp)		 探针名称
Probe name		 探针长度
Probe length
(bp)		 探针序列
Probe sequence (5′-3′)
CL89 0.35 73 oligo-pAc89 59 AATCCTACCGAGTGGTGAGCAATCCTCCCACTCGGGGGCTTAGCTGCAGTCCAGTGCTC
CL105 0.28 637 oligo-pAc105 59 TAGGAGTCACATAATATTTAGTAGTTTTTGTCTCTTATTGCCGGGGAGACGCCTACGCG
CL146 0.1 553 oligo-pAc146 59 CTGGATAATAAAGTACATAACACATCATCCTCATTATGTTCATGCACACGAAAAATTAT
CL148 0.1 118 oligo-pAc148 59 ACGGGCCATCGAAATCGTGCCCTGACCCCAAA
ACAGTGTGCTATAGCCCACGAAAATGG
CL149 0.1 663 oligo-pAc149 59 ATGGCAAACAATGTTGCCTAAGGAAGTTTTCATTTTCTTTGGACGAAAAAACCATTTTC
CL198 0.042 509 oligo-pAc198 59 ACACACCCCTCACAAACCGGAGCAACTCACTAGAAGGCTCGTGGTTCCGAGAGGGAACA
CL217 0.03 49 oligo-pAc217 49 CCACCTAAGCCTAACCATTAGGGTTTACGGTGCCTCGGGGTCGACGGAG
CL225 0.028 46 oligo-pAc225 46 GAGTGGTGATGAGGTGACCAACCGAGTGGCGACGCGCGGGGCGGCC
CL238 0.025 369 oligo-pAc238 59 ATATGAGATCCAATTACTTGTAGGGAAATGCAAGAGACCTCAGTTATGATGATGCACTA
CL259 0.02 108 oligo-pAc259 59 AGTACCGAAATTAGTGATATACAACTAAGTCTGGTGATCATGGTGCTGTTTTTCAGTAC
CL263 0.02 119 oligo-pAc263 59 AGCTAGCTAAGCATATTGAGTCATTCTGGAGGAAAAAATGCCAAGTATAGGTCATTTTA
CL301 0.0015 90 oligo-pAc301 59 AGGCGGACGTCGTCGCGGTAGCGACGACGGACGCCGAGACGAGCACGTGACACCACTGC

图1

5个oligo探针在顶芒山羊草(PI542176)和中国春有丝分裂中期染色体的oligo-FISH A1, A2: 探针oligo-pAc105; B1, B2: 探针oligo-pAc259; C1, C2: 探针oligo-pAc149; D1, D2: 探针oligo-pAc217; E1, E2: 探针oligo- pAc148。染色体用DAPI套染(蓝色); 5个寡聚核苷酸探针用5′TAMRA修饰(红色)。标尺为10 μm。"

图2

5个oligo探针在顶芒山羊草(PI542176)和中国春有丝分裂中期染色体的oligo-FISH A1, A2: 探针oligo-pAc263; B1, B2: 探针oligo-pAc225; C1, C2: 探针oligo-pAc238; D1, D2: 探针oligo-pAc301; E1, E2: 探针oligo- pAc89。染色体用DAPI套染(蓝色); 探针oligo-pAc225、oligo-pAc238和oligo-pAc263用5′TAMRA修饰(红色); 探针oligo-pAc89和oligo-pAc301用5′FAM修饰(绿色)。标尺为10 μm。"

图3

5个oligo探针在7个二倍体的小麦近缘物种中的oligo-FISH分析 A1-A7: 探针oligo-pAc105; B1-B7: 探针oligo-pAc148; C1-C7: 探针oligo-pAc149; D1-D7: 探针oligo-pAc217; E1-E7: 探针oligo- pAc259。染色体用DAPI套染(蓝色); 5个寡聚核苷酸探针用5′TAMRA修饰(红色)。标尺为10 μm。"

图4

Ae. comosa的oligo-FISH核型 A: 1M~7M信号组合图, 红色为中国春D亚基因组染色体特异寡核苷酸探针涂染信号, 绿色为ONPS#AC1信号, 白色箭头示寡核苷酸探针涂染的染色体; B: 图A中剪切的1M~7M每条染色体的组合图; C: 从图B中分离出的1M~7M各染色体的中国春D亚基因组染色体特异寡核苷酸探针涂染信号图; D: 从图B中分离出的1M~7M每条染色体的ONPS#AC1信号图, 标尺为10 μm; E: 顶芒山羊草的核型模式图。"

图5

5个oligo探针分别与绿色荧光探针套相结合分别在顶芒山羊草(PI542176)有丝分裂中期染色体的oligo-FISH A: 探针oligo-pAc105; B: 探针oligo-pAc148; C: 探针oligo-pAc149; D: 探针oligo-pAc217; E: 探针oligo-pAc259; 染色体用DAPI套染(蓝色)。5个寡聚核苷酸探针用5’TAMRA修饰(红色); 绿色为ONPS#AC1信号。标尺为10 μm。"

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