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

RichHTML

25

PDF

648

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"

[1] 王汉中, 殷艳 . 我国油料产业形势分析与发展对策建议. 中国油料作物学报, 2014,36(3):414-421.
WANG H Z, YIN Y . Analysis and strategy for oil crop industry in China. Chinese Journal of Oil Crop Science, 2014,36(3):414-421. (in Chinese)
[2] PEARSON O H . A Dominant white flower color in Brassica oleracea L. American Naturalist, 1929,63:561-565.
[3] CHEN B, HENEEN W, JONSSON R . Independent inheritance of erucic acid content and flower colour in the C-genome of Brassica napus L.. Plant Breeding, 1988,100:147-149.
[4] HENEEN W, CHEN B, CHENG B, JONSSON A, SIMONSEN V, JORGENSEN R, DAVIK J . Characterization of the A and C genomes of Brassica campestris and B.alboglabra. Hereditas, 1995,123:251-267.
[5] ZHANG B, LU C M, KAKIHARA F, KATO M . Effect of genome composition and cytoplasm on petal color in resynthesized amphidiploids and sesquidiploids derived from crosses between Brassica rapa and Brassica oleracea. Plants Breeding, 2002,121:297-300.
[6] LEE S, LEE S C, BYUN D H, LEE D Y, PARK J Y, LEE J H, LEE H O, SUNG S H, YANG T J . Association of molecular markers derived from the BrCRTISO1 gene with prolycopene-enriched orange-colored leaves in Brassica rapa. Theoretical and Applied Genetics, 2014,127:179-191.
[7] RAHMAN M H . Inheritance of petal colour and its independent segregation from seed colour in Brassica rapa. Plant Breeding, 2001,120:197-200.
[8] JAMBHULKAR S, RAUT R . Inheritance of flower colour and leaf waxiness in Brassica carinata A.Br. Cruciferae Newsletter, 1995,17:66-67.
[9] RAWAT D S, ANAND I J . Inheritance of flower colour in mustard mutant. Indian Journal of Agricultural Science, 1986,56:206-208.
[10] SINGH K H, CHAUHAN J S . Genetics of flower colour in Indian mustard ( Brassica juncea L.Czern $ Coss). Indian Journal of Genetics & Plant Breeding, 2011,71:377-378.
[11] ZHANG B, LIU C, WANG Y, YAO X, WANG F, WU J, KING G J, LIU K . Disruption of a CAROTENOID CLEAVAGE DIOXYGENASE 4 gene convents flower colour from white to yellow in Brassica species. New Phytology, 2015,206:1513-1526.
[12] HUANGE Z, BAN Y Y, BAO R, ZHANG X X, XU A X, DING J . Inheritance and gene mapping of the white flower in Brassica napus L. New Zealand Journal of Crop and Horticultural Science, 2014,42(2):111-117.
[13] LIU X P, TU J X, CHEN B Y, FU T D . Identification of the linkage relationship between the flower colour and the content of erucic acid in the resynthesized Brassica napus L.. Acta Genetica Sinica, 2004,31:357-362.
[14] HAN F Q, YANG C, FANG Z Y, YANG L M, ZHUANG M, LV H H, LIU Y M, LI Z S, LIU B, YU H L, LIU X P, ZHANGH Y Y . Inheritance and InDel markers closely linked to petal color gene (cpc-1) in Brassica oleracea. Molecular Breeding, 2015,35:160.
[15] MITHRA S V A, KAR M K, MOHAPATRA T, ROBIN S, SARLA N, SESHASHAYEE M, SINGH K, SINGH N K, SHARMA R P . DBT propelled national effort in creating mutant resource for functional genomics in rice. Current Science, 2016,110(4):543-548.
[16] HENRY I M, NAGALAKSHMI U, LIEBERMAN M C, NGO K J, KRASILEVA K V, VASQUEZ-GROSS H, AKHUNOVA A, AKUNOV E, DUBCOVSKY J, TAI T H, COMAI L . Efficient genome-wide detection and cataloging of EMS-induced mutations using exome capture and next-generation sequencing. The Plant Cell, 2014,26(4):1382-1397.
[17] WEI F J, DROC G, GUIDERDONI E, HSING Y I C . International consortium of rice mutagenesis: Resources and beyond. Rice, 2013,6(1):39.
[18] TSUDA M, KAGA A, ANAI T, SHIMIZU T, SAYAMAT , TAKAGI K, MACHITA K, WATANABE S, NISHIMURA M, YAMADA N, MORI S, SASAKI H, KANAMORI H, KATAYOSEY , ISHIMOTO M . Construction of a high-density mutant library in soybean and development of a mutant retrieval method using amplicon sequencing. BMC Genomics, 2015,16:1014.
[19] JUST D, GARCIA V, FERNANDEZ L, BRES C, MAUXION J P, PETIT J, JORLY J, ASSALI J, BOURNONVILLE C, FERRAND C, BALDET P, LEMAIRE-CHAMLEY M, MORI K, OKABE Y, ARIIZUMI T, ASAMIZU E, EZURA H, ROTHAN C . Micro-Tom mutants for functional analysis of target genes and discovery of new alleles in tomato. Plant Biotechnology Journal, 2013,30(3):225-231.
[20] LIN T, WANG S H, ZHONG Y, GAO D L, CUI Q Z, CHEN H M, ZHANG Z H, SHEN H L, WENG Y Q, HUANG S W . A truncated F-box protein confers the dwarfism in cucumber. Journal of Genetics Genomics, 2016,43(4):223-226.
[21] LUN Y Y, WANG X, ZHANG C Z, YANG L, GAO D L, CHEN H M, HUANG S W . A CsYcf54 variant conferring light green coloration in cucumber. Euphytica, 2016,208(3):509-517.
[22] ZHOU Q, WANG S H, HU B W, CHEN H M, ZHANG Z H, HUANG S W . An ACCUMULATION AND REPLICATION OF CHLOROPLASTS 5 gene mutation confers light green peel in cucumber. Journal of Integrative Plant Biology, 2015,57(11):936-942.
[23] YAO Y M, LI K X, LIU H D, DUNCAN R W, GUO S M, XIAO L, DU D Z . Whole-genome re-sequencing and fine mapping of an orange petal color gene ( BNPE 1) in spring Brassica napus L.to a 151-kb region. Euphytica, 2017,213:165.
[24] ZHANG X X, LI R H, NIU S L, CHEN L, GAO J, WEN J, YI B, MA C Z, TH J X, FU T D, SHEN J X . Fine-mapping and candidate gene analysis of the Brassica juncea white-flowered mutant Bjpc2 using the whole-genome resequencing. Molecular Genetics & Genomics, 2017,293(2):359-370.
[25] TAKAGI H, ABE A, YOSHIDA K, KOSUGI S, NATSUME S, MITSUOKA C, UEMURA A, UTSUSHI H, TAMIRU M, TAKUMO S, INNAN H, CANO L M, KAMOUN S, TERAUCHI R . QTL-seq: Rapid mapping of quantitative trait loci in rice by whole genome resequencing of DNA from two bulked populations. The Plant Journal, 2013,74(1):174-183.
[26] JONATHON T H, BRADLEY L D, BRENT W B, BUSHRA G, YI C S, H J Y, . MMAPPR: Mutation mapping analysis pipeline for pooled RNA-seq. Genome Research, 2013,23:687-697.
[27] CHALHOUB B, DENOEUD F, LIU S, PARKIN A P, TANG H, WANG X, CHIQUET J . Early allopolyploid evolution in the post- Neolithic Brassica napus oilseed genome. Science, 2014,345:950-953.
[28] SUN F M, FAN G Y, HU Q, ZHOU Y M, GUAN M, TONG C B, LI J N, DU D Z, QI C K, JIANG L C, LIU W Q, HUANG S M, CHEN W B, YU J Y, MEI D S, MEN J L, ZENG P, SHI J Q, LIU K D, WANG X, WANG X F, LONG Y, LIANG X M, HU Z Y, HUANG G D, DONG C H, ZHANG H, LI J, ZHANG Y L, LI L W, SHI C C, WANG J H, MING-YUEN L S, GUAN C, XU X, LIU S Y, LIU X, CHALHOUB B, HUA W, WANG H Z . The high-quality genome of Brassica napus cultivar ‘ZS11’ reveals the introgression history in seni-winter morphotype. The Plant Journal, 2017,92:452-468.
[29] BAYER P E, HURGOBIN B, GOLICZ A A, CHAN C K, YUAN Y X, LEE H T, RENTON M, MENG J L, LI R Y, LONG Y, ZOU J, BANCROFF L, CHALHOUB B, KING G J, BATLEY J, EDWARDS D . Assembly and comparison of two closely related Brassica napus genomes. Plant Biotechnology Journal, 2017,15:1602-1610.
[30] XIAO S, XU J, LI Y, ZHANG L, SHI S, SHI S, WU J, LIU K . Generation and mapping of SCAR and CAPS markers linked to the seed coat color gene in Brassica napus using a genome-walking technique. Genome, 2007,50(7):611-618.
[31] LIU S Y, LIU Y M, YANG X H, TONG C B, EDWARDS D, PARKIN IA, ZHAO M X, MA J X, YU J Y, HUANG S M, WANG X Y, WANG Y J, LU K, FANG Z Y, BANCROFT L, YANG T, HU Q, WANG X F, YUE Z, LI H J, YANG L F, WU Q, WANG W X, KING G J, PIRES J, LU C X, WU Z Y, SAMPATH P, WANG Z, GUO H, PAN S K, YANG L M, MIN J M, ZHANG D, JIN D C, LI W S, BELCRAM H, TU J X, GUAN M, QI C K, DU D Z, LI J N, JIANG L C, BATELY J, SHARPE A G, PARK B, RUPERAO P, CHENG F, WAMINAL N E, HUANG Y, DONG C H, WANG L, LI J P, HU Z Y, LI Z Y, LI X, ZHANG J F, XIAO L, ZHOU Y M, LIU Z S, LIU X Q, QIN R, TANG X, LIU W B, WANG Y P, ZHANG Y Y, LEE J H, KIM H H, DENOEUD F, XU X, LIANG X M, HUA W, WANG X W, WANG J, CHALHOUB B, PATERSON A H . The Brassica oleracea genome reveals the asymmetrical evolution of polyploid genomes. Nature Communications, 2014,5:3930.
[1] ZHU Qi, JIA ZhenPeng, Tahir SHAH, XU ChenSheng, LI ZhiQi, LÜ HuiShuai, ZHU PengChao, WEI XiaoMin, HUANG DongLin, SUN YanNi, CAO WeiDong, GAO YaJun, WANG ZhaoHui, ZHANG DaBin. Green Manure Crops Combined with Enhanced-Efficiency Products Reduced Greenhouse Gas Emissions and Carbon Footprints in Dryland Wheat Fields [J]. Scientia Agricultura Sinica, 2026, 59(7): 1507-1522.
[2] LIU HaiQing, JIN JiaoJiao, SUN WanCang, CHAI Peng, QI WeiLiang, YANG Gang, LI Chan, LUO XueMei, SU YunYun, QIN XueXue. Morphogenesis of the Low-Growth Point and Its Multi-Hormonal Regulatory Mechanism During Overwintering in Winter Rapeseed (Brassica napus L.) [J]. Scientia Agricultura Sinica, 2026, 59(5): 951-966.
[3] YANG Fan, HU XiaoQian, WANG Yu, YUE CaiXia, ZHANG Rui, TIAN Wen, WANG TingTing, LI Yang, JI MeiQuan, ZHANG LiHui, AN KeJing. Optimization of Detection Conditions for Aerobic Spore-Forming Bacillus in Honey and Analysis of Its Contamination Characteristics [J]. Scientia Agricultura Sinica, 2026, 59(4): 887-899.
[4] SHEN LiQiong, HE LinLi, LIU Ni, LU JunXing, ZHU Bo, ZHANG Tao. Effects of Potassium Levels on Waterlogging Resistance and Endogenous Hormone Balance of Rapeseed During Seedling Stage [J]. Scientia Agricultura Sinica, 2026, 59(3): 528-542.
[5] HE ZhiLin, SUN CuiXia, YUE HongLi, TAN YueXia, ZHANG YaoHai, WANG FuSheng, LIU SiTao, JIANG Dong. Genetic Diversity Analysis and GWAS of Alloocimene Based on Resequencing of Citron, Lemon Germplasm Resources [J]. Scientia Agricultura Sinica, 2026, 59(2): 386-401.
[6] TAO ZhiYun, XU WenJuan, LU LiZhi, SONG WeiTao, ZHANG ShuangJie, LIU HongXiang, WANG ZhiCheng, GU HaoTian, ZHU ChunHong, LI HuiFang. Atlas Construction and Regulatory Analysis of Duck Testicular Cell Development [J]. Scientia Agricultura Sinica, 2026, 59(2): 441-458.
[7] WANG ZhongNi, LEI Yue, LI JiaLi, GONG YanLong, ZHU SuSong. Functions of ABC Transporter OsARG1 in Rice Heading Date Regulation [J]. Scientia Agricultura Sinica, 2026, 59(1): 1-16.
[8] WANG JiYing, LI JingXuan, WANG YanPing, GUO JianFeng, LIN HaiChao, ZHAO XueYan. Weighted Gene Co-Expression Network Analysis Reveals Potential Candidate Genes Affecting Fat Deposition in Pigs [J]. Scientia Agricultura Sinica, 2025, 58(9): 1845-1855.
[9] ZOU XiaoWei, XIA Lei, ZHU XiaoMin, SUN Hui, ZHOU Qi, QI Ji, ZHANG YaFeng, ZHENG Yan, JIANG ZhaoYuan. Analysis of Disease Resistance Induced by Ustilago maydis Strain with Overexpressed UM01240 Based on Transcriptome Sequencing [J]. Scientia Agricultura Sinica, 2025, 58(6): 1116-1130.
[10] WANG LiYuan, WANG Hui, WANG MuMu, WANG DongJian, LI RuYu, ZHENG YongSheng, ZHANG Han. Construction and Application of DNA Fingerprint Database for Known Varieties in Upland Cotton DUS Testing [J]. Scientia Agricultura Sinica, 2025, 58(22): 4570-4588.
[11] PAN Yuan, WANG De, LIU Nan, MENG XiangLong, DAI PengBo, LI Bo, HU TongLe, WANG ShuTong, CAO KeQiang, WANG YaNan. Evaluation of the Effectiveness of Two High-Throughput Sequencing Techniques in Identifying Apple Viruses and Identification of Two Novel Viruses [J]. Scientia Agricultura Sinica, 2025, 58(2): 266-280.
[12] LUO JiaRui, WU SanLing, GUO Fu, LIU Zhen, SONG JingHan, TAN YuanYuan, SHU QingYao. Identification and Characterization of Retrotransposon Tos17 in the Genomes of Indica Rice [J]. Scientia Agricultura Sinica, 2025, 58(15): 2933-2947.
[13] DONG Xue, CHEN MengQiu, SHAO Jin, WU XueYou, TANG PeiAn. Construction of a Differential Gene Expression and Quality Regulation Network in Stored Rice Grain Using WGCNA [J]. Scientia Agricultura Sinica, 2025, 58(14): 2885-2903.
[14] DENG LiCheng, LI Cheng, HE Lei, AN HongQiang, WANG CaiLin, ZHANG YaDong, ZHAO ChangJiang, LU Kai. Physiological Characteristics in Response to Salt Stress and Allelic Variation and Expression of Salt-Responsive Genes in Seedling Stage of Nangeng Rice Varieties with Salt-Tolerance Ability [J]. Scientia Agricultura Sinica, 2025, 58(12): 2275-2290.
[15] XU Na, TANG Ying, XU ZhengJin, SUN Jian, XU Quan. Genetic Analysis and Candidate Gene Identification on Fertility and Inheritance of Hybrid Sterility of XI and GJ Cross [J]. Scientia Agricultura Sinica, 2024, 57(8): 1417-1429.
Viewed
Full text


Abstract

Cited
  Shared   
  Discussed   
No Suggested Reading articles found!
京ICP备09089781号-30
Copyright 2018 © Editorial office of Scientia Agricultura Sinica
Tel: 86-010-82106279    E-mail: zgnykx@mail.caas.net.cn
Supported by Beijing Magtech Co.Ltd