Scientia Agricultura Sinica ›› 2020, Vol. 53 ›› Issue (6): 1108-1117.doi: 10.3864/j.issn.0578-1752.2020.06.003

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

Location and Linkage Markers for Candidate Interval of the White Petal Gene in Brassica napus L. by Next Generation Sequencing

Xue CHEN,Rui WANG(),FuYu JING,ShengSen ZHANG,LeDong JIA,MouZheng DUAN,Yu WU   

  1. College of Agronomy and Biotechnology, Southwest University, Chongqing 400715
  • Received:2019-08-21 Accepted:2019-10-30 Online:2020-03-16 Published:2020-04-09
  • Contact: Rui WANG E-mail:ruiwang71@163.com

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Abstract:

【Objective】 Since the petal colour can be used for ornamental and landscaping purposes, the petal color has been one of the major goals of breeding and genetic research in Brassica napus L.. In this paper, Genetic analysis, candidate interval identification, linkage markers and synteny analysis were applied to elucidate the genetic control of the white petal in Brassica napus L.. 【Method】 To Map the white petal locus, an inbred line Y05, which has yellow flowers, was crossed with an inbred line W01, which has white flowers. The F1 plants were self-crossed to develop F2 mapping population. For BSA, parental and two pools with 30 yellow petal lines and 30 white petal lines of F2 were constructed by mixing an equal amount of DNA or RNA respectively. 30× or 5× depth of genome-sequencing was conducted. Darmor-bzh, Zhongshuang11(ZS11), Darmor and Tapidor as the reference genome were aligned to sequence data from the 2 bulks and parents using QTL-seq workflow. The sliding window method with a window size of 2Mb and a step size of 50kb was used to present the SNP indexes of the whole genome. The difference between the SNP indexes of the two pools was calculated as the delta (SNP- index). Candidate regions for petal color were identified from the chromosomes with 95% confidence intervals. Mutation Mapping Analysis Pipeline for Pooled RNA-seq (MMAPPR) without parental strain information and requiring Darmor-bzh reference genome calculated allelic frequency by Euclidean distance followed by Loess regression analysis, and identified the region where the mutation lies, and generated a list of putative coding region mutations in the linked genomic segment. The SSR primers were designed by using MISA and Prime 3 for repeated sequence identification, and the SSR primers were screened by polyacrylamide gel electrophoresis in the F2 population. 【Result】 The segregation of white petal and yellow petal among F2 population fitted the Mendelian segregation ratio of 3:1. This indicates that the white petal trait was controlled by a major gene and that white petal was dominant over yellow petal. The results of the candidate interval using whole-genome re-sequencing showed that a candidate interval (52-55 Mb) exceeding the threshold value was identified for the petal color on chromosome C03 when Darmor-bzh was used as reference genome. While ZS11, Darmor and Tapidor were aligned to sequence data, candidate intervals for white petal were all identified on chromosome C03. Linked region peaks (54-55 Mb) identified by MMAPPR for the petal color was on chromosome C03 of Darmor-bzh. Six SSR markers that were located in the interval (760 kb) were closely linked to the white flower gene. Synteny analysis showed that the interval 760 kb (52.81-53.57 Mb) was corresponding to chromosome A02 (56.76-57.40 Mb) of Brassica rapa and chromosome C03 (10.99-11.28 Mb) of Brassica oleracea. 【Conclusion】 The white petal was controlled by a major gene which was dominant over yellow petal. Six SSR markers closely linked to the white petal gene were selected. A candidate interval for white petal gene was identified on chromosome C03 (52-55 Mb). The present study may facilitate cloning of the white petal gene as well as marker assisted selection.

Key words: Brassica napus L., white petal, sequencing, candidate interval, SSR

Fig. 1

Comparison of yellow petal and white petal in rapeseed A: Yellow petal Y05; B: Yellow petal of F2; C: White petal W01; D: White petal of F2"

Table 1

Segregation of F2 between yellow petal Y05 and white petal W01"

年份
Year		 总株数
Total plants		 白花
White petal		 黄花
Yellow petal		 期望比
Expected ratio		 χ2 P
2018 213 154 59 3﹕1 0.8279 >0.05
2019 176 135 41 3﹕1 0.2877 >0.05

Fig. 2

Candidate interval for white petal in C03 chromosomes of four reference genomes in Brassica napus L. A: Darmor-bzh reference; B: ZS11 reference; C: Tapidor reference; D: Darmor reference. 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. 3

ED4（Loess fit）of genome-wide Genome-wide (A) and chromosome 13 (B) Loess fit curves for SNP allele frequency Euclidean distance"

Table 2

SSR Primer sequence"

引物名
Primer name		 Chr.C03 SSR 正向引物序列
Forward primer sequence (5′-3′)		 反向引物序列
Reverse primer sequence (5′-3′)
SSR149 52811241—52811250 (A)10 CCTTAAAGAATACGCTGTGTCT TCGATCACAGAAGCCCCTAAGT
SSR154 52859725—52859754 (CTT)10 AGCAAAACCCACATCACTCATT TGGTTGGTTGGTGGAGGAGATA
SSR157 52891130—52891141 (CT)6 TGTGGTTCTGGCTGCTGTATTC CCCCACCAACGAGTCCAAAAC
SSR161 52941029—52941061 (A)10 GCGGATCAAGCTCTAGTATCT CCAAAGGGGTTAGGTGTTTAGT
SSR180 53152240—53152251 (TA)6 TGTGAAGCCCGGTTTGCAACTT ACTCCCACTCTCCCACGATTGT
SSR222 53582130—53582142 (A)13 AGAGTCCTTAGCGTTTTGTATT ACGGATGCAGTGGAAGTGAAT

Fig. 4

SSR markers of F2 individuals with yellow and white petals A: SSR149; B: SSR154; C: SSR157; D: SSR161; E: SSR180; F: SSR222; M: 20 bp ladder; 1: Y05; 2: W01; 3-13: 11 yellow petal plants of F2; 14-24: 11 white petal plants of F2"

Fig. 5

Synteny analysis of white petal candidate interval between Brassica napus L. and Brassica oleracea or Brassica rapa"

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