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Acta Agronomica Sinica ›› 2022, Vol. 48 ›› Issue (3): 759-769.doi: 10.3724/SP.J.1006.2022.14025

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Development of linkage InDel markers of the white petal gene based on whole-genome re-sequencing data in Brassica napus L.

WANG Rui1,2(), CHEN Xue1,2, GUO Qing-Qing1,2, ZHOU Rong1,2, CHEN Lei1,2, LI Jia-Na1,2,*()   

  1. 1College of Agronomy and Biotechnology, Southwest University, Chongqing 400715, China
    2Chongqing Engineering Research Center for Rapeseed, Chongqing 400715, China
  • Received:2021-02-08 Accepted:2021-07-12 Online:2022-03-12 Published:2021-08-09
  • Contact: LI Jia-Na E-mail:Ruiwang71@163.com;ljn1950@swu.edu.cn
  • Supported by:
    Project of Intellectual Base for Discipline Innovation in Colleges and Universities (111 Program)(B12006)

Abstract:

InDel is widely distributed across the genome and occurs in a high density and large numbers in a genome. To date, the researches about linkage InDel markers of the white petal gene in B. napus L are very less yet. In this study, we constructed the F2 mapping population from the cross between DH Y05 (yellow petal) and DH W01 (white petal). Two bulks with 30 yellow petal lines and 30 white petal lines of F2 population were constructed by mixing an equal amount of DNA. Then two bulks and parents were performed 30× whole-genome re-sequencing. Darmor-bzh as the reference genome was aligned to sequence data from the two bulks and parents. QTL-seq and PoPoolation2 workflow were applied to identify the candidate region of the white petal gene. A major candidate region was identified on chromosome C03 (52-54 Mb) of Darmor-bzh. The insertion-deletion (InDel) sites can be visualized in candidate interval by Integrative Genomics Viewer (IGV). Based on these Indel variations, we used Vector and Blast to design InDel primers. Eight InDel markers closely linked to the white petal gene were screened by Polyacrylamide gel electrophoresis (PAGE). In summary, these results provide a basis for fine mapping white petal gene and InDel molecular marker located on functional genes as well as molecular marker assisted selection breeding.

Key words: Brassica napus L., re-sequencing, white petal genes, InDel markers

Fig. 1

∆(SNP-index) distribution on each chromosome Blue dot: ∆(SNP-index); Red line: sliding window average of ∆(SNP-index); Green lines: sliding window average of 95%-confidence interval upper/lower side; Orange line: sliding window average of 99%-confidence interval upper/lower side."

Fig. 2

White genes located on chromosome C03 (Chr. 13) using PoPoolation2"

Fig. 3

Visualization of InDel variation in candidate interval by IGV A: InDel-2; B: InDel-3; C: InDel-4; D: InDel-5; E: InDel-6; F: InDel-8; G: InDel-9; H: InDel-10."

Table 1

Primer information for InDels"

引物名称
Primer name
Chr. C03 InDel 正向引物序列
Forward sequence (5°-3°)
反向引物序列
Reverse sequence (5°-3°)
InDel-2 52375493-52375505 D10 GCTAAGCGTGTATCTTATCCT CGGGAATGGCGATCTAAAAA
InDel-3 52396484 I14 GAAAAACCATTGGAAATGCC TGGGAAGTTTGTTAAAGGGT
InDel-4 52348627-52348640 D12 GACAAGTCTTGGACAGCTAA CGAGAGGAATCCAAAGATGT
InDel-5 52377696-52377718 D20 TAGAGAGAGCTTAGACCTCC CGGCTATGAGTGATGTTCTT
InDel-6 52374279 I12 GGTTCTCTCTCTCTTCAGGTT TAATACTAATCGAAATTGTTGTCCC
InDel-8 52358540-52358551 D11 ATGCAACATCCAAAACACCT TTTTGCTCTTTTCGCGTTTG
InDel-9 52358775-52358788 D11 TCTGCGATCACGAATACCT CAAATCGAAAGGAAAGGAACA
InDel-10 52373701-52373721 D19 ACGTTAAAACAGCTTCACTTTC TCCGTGTCTGCCAAGTATC

Fig. 4

Electrophoresis of parents and individuals of F2 population A: InDel-2; B: InDel-3; C: InDel-4; D: InDel-5; E: InDel-6; F: InDel-8; G: InDel-9; H: InDel-10; M: 20 bp ladder. 1: parents with yellow petal; 2: parents with white petal; 3-13: 11 yellow petal plants of F2; 14-24: 11 white petal plants of F2."

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