基于二代测序的甘蓝型油菜白花基因候选区间定位及连锁标记验证

doi:10.3864/j.issn.0578-1752.2020.06.003

摘要/Abstract

摘要：

【目的】近几年随着观光农业的兴起,花色的选育和改良已成为甘蓝型油菜种质资源鉴定和材料创制的重要研究方向。以甘蓝型油菜黄白花分离F₂群体为研究对象,通过二代测序技术,对白花性状基因候选区间定位,开发与白花性状连锁的分子标记,为定位白花候选基因和选育白花新材料提供新思路。【方法】以甘蓝型油菜DH纯系黄花Y05和甘蓝型油菜纯系白花W01杂交,观察F₁和F₂群体的花色分离,分析白花性状遗传模式。在F₂群体中选取30株纯白花和30株纯黄花构建DNA叶片子代池和RNA花瓣子代池,对亲本和DNA叶片子代池进行30×重测序,对RNA花瓣子代池进行5×测序。以法国甘蓝型油菜Darmor-bzh、中双11、Darmor、Tapidor为参考序列,重测序QTL-seq分析流程计算2个DNA子代池的SNP-index和delta（SNP-index）。利用R包画出SNP-index和delta（SNP-index）滑窗分析图,鉴定候选区间。转录组MMAPPR分析流程以法国甘蓝型油菜Darmor-bzh为参考序列,计算SNP频率,ED ⁴（Loess fit）检测峰值和鉴定候选区间。利用MISA进行重复序列鉴定,使用Prime3在候选区间进行SSR引物设计,在F₂群体中采用聚丙烯酰胺凝胶电泳方法对SSR引物进行筛选。【结果】甘蓝型油菜黄花与白花杂交F₂群体中,白花和黄花性状分离比符合3﹕1,暗示白花性状受1对显性主效基因控制。全基因组重测序区间定位结果显示,白花性状基因候选区间在Darmor-bzh C03染色体52—55 Mb。同时以甘蓝型油菜中双11、Darmor、Tapidor分别为参考序列,均鉴定出白花基因候选区间在C03染色体上的一致性和稳定性。转录组测序定位白花性状基因位于Darmor-bzh C03染色体54—55 Mb。转录组测序和重测序定位染色体结果高度一致。在此区间内MISA和Primer3结合设计SSR引物,聚丙烯酰胺凝胶电泳筛选到6个与白花性状紧密连锁共分离的SSR标记。6个SSR标记区间范围在760 kb（52.81—53.57 Mb）。此候选区间与甘蓝、白菜共线性分析,对应白菜A02染色体56.76—57.40 Mb区间,对应甘蓝C03染色体10.99—11.28 Mb区间。【结论】甘蓝型油菜白花性状由1对显性主效基因控制。白花性状基因候选区间在法国甘蓝型油菜Darmor-bzh C03染色体52—55 Mb区间内。此区间760 kb范围内筛选出6个与白花性状基因紧密连锁共分离的SSR标记。

关键词: 甘蓝型油菜, 白花, 测序, 候选区间, SSR

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

陈雪,王瑞,井付钰,张胜森,贾乐东,段谋正,吴宇. 基于二代测序的甘蓝型油菜白花基因候选区间定位及连锁标记验证[J]. 中国农业科学, 2020, 53(6): 1108-1117.

Xue CHEN,Rui WANG,FuYu JING,ShengSen ZHANG,LeDong JIA,MouZheng DUAN,Yu WU. Location and Linkage Markers for Candidate Interval of the White Petal Gene in Brassica napus L. by Next Generation Sequencing[J]. Scientia Agricultura Sinica, 2020, 53(6): 1108-1117.

0
/ / 推荐

导出引用管理器 EndNote|Reference Manager|ProCite|BibTeX|RefWorks

链接本文: https://www.chinaagrisci.com/CN/10.3864/j.issn.0578-1752.2020.06.003

https://www.chinaagrisci.com/CN/Y2020/V53/I6/1108

图/表 7

图1

表1

图2

图3

表2

图4

图5

参考文献 31

[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.

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

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