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Acta Agronomica Sinica ›› 2021, Vol. 47 ›› Issue (11): 2163-2172.doi: 10.3724/SP.J.1006.2021.04236

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

Location and InDel markers for candidate interval of the orange petal gene in Brassica napus L. by next generation sequencing

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

  1. 1College of Agronomy and Biotechnology, Southwest University, Chongqing 400715, China
    2Chongqing Engineering Research Center for Rapeseed, Chongqing 400715, China
  • Received:2020-10-31 Accepted:2021-01-13 Online:2021-11-12 Published:2021-02-18
  • Contact: WANG Rui E-mail:1833266719@qq.com;1822701415@qq.com;Ruiwang71@163.com
  • About author:First author contact:** Contributed equally to this work
  • Supported by:
    Innovation Project of Southwest University Students(X202010635478);National Key Research and Development Program of China(2016YFD0101300)

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Abstract:

The petal color has been one of the major goals of breeding and genetic research in Brassica napus L. To date, there have been no reports about interval location of dominant orange petal gene trait in B. napus L. In this study, we constructed an F2 mapping population with 458 individuals from the cross between DH Y05 (yellow petal) and DH R08 (orange petal). Whole-genome re-sequencing of DNAs and transcriptome sequencing of RNAs were from two populations each composed of 30 individuals showing extreme opposite trait for a given phenotype in a segregating progeny. Then we performed 30× and 6G of sequencing. Darmor-bzh as the reference genome was aligned to sequence data from the two bulks and parents. QTL-seq and Mutation Mapping Analysis Pipeline for Pooled RNA-seq (MMAPPR) workflow were applied to identify the candidate region of the orange petal gene. 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. The results indicated that the orange petal trait was controlled by a dominant major gene. A major candidate region was identified on chromosome A07 (18-19 Mb) of Darmor-bzh. Three InDel markers linked to the orange petal gene were screened by Polyacrylamide gel electrophoresis (PAGE). This study may provide a novel idea for fine mapping dominant orange petal gene as well as marker assisted selection.

Key words: Brassica napus L., orange petal, gene mapping, sequencing, molecular marker

Fig. 1

Morphology comparision of yellow petal Y05 and orange petal R08 in Brassica napus L. A1 and A2: yellow petal Y05; B1 and B2: orange petal R08."

Table 1

Segregation ratio of F2 population between yellow petal Y05 and orange petal R08"

群体
Population		 总株数
Total plants		 桔红花
Orange petal		 黄花
Yellow petal		 期望比
Expected ratio		 χ2 P
2019 149 108 41 3:1 0.37 > 0.05
2020 458 333 125 3:1 1.16 > 0.05

Fig. 2

Average depth of re-sequencing and coverage of bases on 19 chromosomes in Brassica napus L. Black lines: refer to the average sequencing depth curve using a sliding window."

Fig. 3

Delta (SNP-index) on 19 chromosomes in Brassica napus L. Blue dot: delta (SNP-index); Red line: sliding window average of delta (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. 4

ED4 (Loess fit) on 19 chromosomes and A07 chromosome in Brassica napus L."

Fig. 5

Visualization of InDel variation in candidate intervals by IGV"

Table 2

InDel primer sequences designed in candidate intervals"

引物名称
Primer name		 InDel位点
InDel site		 InDel 正向引物序列
Positive Primer sequence (5′-3′)		 反向引物序列
Reverse primer sequence (5′-3′)
InDel-65 18,968,634-18,968,641 D7 GGGTCGAGATACGGTTACGG CGTCGGTAAGTCTTCTTCACC
InDel-69 18,949,999-18,950,007 D8 TACCGAGCTCCAGGTCTCTC TGCGCAACTGTAACCATTCT
InDel-56 18,789,060-18,789,066 D6 ACAAGCAAGGCCTATATTTTGC TCGGGTGAAGCAAATGTGGA

Fig. 6

Electrophoretic bands of parents and individuals of F2 population 1: parents with yellow petal; 2: parents with orange petal; 3-13: 11 yellow petal plants in F2 population; 14-24: 11 orange petal plants in F2 population."

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