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Acta Agronomica Sinica ›› 2023, Vol. 49 ›› Issue (6): 1455-1465.doi: 10.3724/SP.J.1006.2023.21048

• CROP GENETICS & BREEDING·GERMPLASM RESOURCES·MOLECULAR GENETICS • Previous Articles     Next Articles

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 Online:2023-06-12 Published: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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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

Table 1

Sequence information of 12 oligo nucleotide probes developed in this study"

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

Fig. 1

oligo-FISH of five oligo probes on root tip metaphase chromosomes of Ae. comosa (PI542176) and T. aestivum cv Chinese Spring A1, A2: probe oligo-pAc105; B1, B2: probe oligo-pAc259; C1, C2: probe oligo-pAc149; D1, D2: probe oligo-pAc217; E1, E2: probe oligo-pAc148. Chromosomes were counterstained with DAPI (blue); five oligo probes were modified with 5′TAMRA (red). Bar: 10 μm."

Fig. 2

oligo-FISH of five oligo probes on root tip metaphase chromosomes of Ae. comosa (PI542176) and Chinese Spring A1, A2: probe oligo-pAc263; B1, B2: probe oligo-pAc225; C1, C2: probe oligo-pAc238; D1, D2: probe oligo-pAc301; E1, E2: probe oligo-pAc89. Chromosomes were counterstained with DAPI (blue); oligo probes oligo-pAc225, oligo-pAc238, and oligo-pAc263 were modified with 5′TAMRA (red), and oligo-pAc89 and oligo-pAc301 were modified with 5′FAM (green). Bar: 10 μm."

Fig. 3

oligo-FISH of five oligo probes in seven diploid relative species of wheat A1-A7: probe oligo-pAc105; B1-B7: probe oligo-pAc148; C1-C7: probe oligo-pAc149; D1-D7: probe oligo-pAc217; E1-E7: probe oligo-pAc259. Chromosomes were counterstained with DAPI (blue); five oligo probes were modified with 5′TAMRA (red). Bar: 10 μm."

Fig. 4

oligo-FISH karyotype of Ae. comosa A: the merged signals of chromosome 1M-7M using oligo-painting of sub-genome D chromosome specific oligo-painting probes (red) and oligo-FISH using ONPS#AC1 (green), white arrow points to the painted chromosome with oligonucleotide probe; B: the merged figures of 1 M-7M cutting from (A); C: the oligo-painting FISH signals digitally separated from (B); D: the oligo-FISH using ONPS#AC1 signals digitally separated from (B); E: the oligo-FISH karyotype of Ae. comosa. Bars: 10 μm."

Fig. 5

oligo-FISH on metaphase chromosomes of Ae. comosa (PI542176) using combining probes including five oligo probes and ONPS#AC1, respectively. A: probe oligo-pAc105; B: probe oligo-pAc148; C: probe oligo-pAc149; D: probe oligo-pAc217; E: probe oligo-pAc259. Chromosomes were counterstained with DAPI (blue); five oligo probes were modified with 5’TAMRA (red). ONPS#AC1 probe was modified with 5’FAM (green). Bars: 10 μm."

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