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作物学报 ›› 2021, Vol. 47 ›› Issue (4): 587-598.doi: 10.3724/SP.J.1006.2021.04115

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

GBS高密度遗传连锁图谱定位甘蓝型油菜粉色花性状

周新桐1,2(), 郭青青1,2,*(), 陈雪1,2, 李加纳1,2, 王瑞1,2,*()   

  1. 1西南大学现代农业科学院, 重庆400715
    2西南大学农学与生物科技学院 / 重庆市油菜工程技术研究中心, 重庆400715
  • 收稿日期:2020-05-30 接受日期:2020-09-13 出版日期:2021-04-12 网络出版日期:2020-12-01
  • 通讯作者: 郭青青,王瑞
  • 作者简介:周新桐, E-mail: wjzxt22@163.com;|郭青青, E-mail: 1833266719@qq.com
  • 基金资助:
    西南大学校创项目(106352020478)

Construction of a high-density genetic map using genotyping by sequencing (GBS) for quantitative trait loci (QTL) analysis of pink petal trait in Brassica napus L.

ZHOU Xin-Tong1,2(), GUO Qing-Qing1,2,*(), CHEN Xue1,2, LI Jia-Na1,2, WANG Rui1,2,*()   

  1. 1Academy of Agricultural Sciences, Southwest University, Chongqing 400715, China
    2College of Agronomy and Biotechnology, Southwest University / Chongqing Engineering Research Center for Rapeseed, Chongqing 400715, China
  • Received:2020-05-30 Accepted:2020-09-13 Published:2021-04-12 Published online:2020-12-01
  • Contact: GUO Qing-Qing,WANG Rui
  • Supported by:
    Innovation Project of Southwest University Students(106352020478)

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摘要：

甘蓝型油菜花瓣颜色是重要的观赏性状, 花瓣颜色的选育和改良已成为材料创制的主要研究方向。目前对甘蓝型油菜粉色花性状定位研究未见报道。本研究以甘蓝型油菜纯系黄花62和甘蓝型油菜纯系粉花77为亲本构建双单倍体(doubled haploid, DH)群体。利用简化基因组测序技术(genotyping-by-sequencing, GBS)筛选出3253个单核苷酸多态性(single nucleotide polymorphism, SNP)标记, 构建了全长1766.06 cM甘蓝型油菜连锁图谱, 标记间平均遗传距离为0.54 cM; 使用WinQTL Cartographer复合区间作图方法对粉色花性状进行数量性状座位(quantitative trait locus, QTL)定位, 检测到位于A07和C03染色体上各1个QTL; 将定位区间内基因与甘蓝和白菜同源基因进行线性比对, 在这些区间中找到了一些存在于3个物种的同源基因; 对粉色花定位区间内基因进行可变剪切分析发现, 2个花色相关基因BnaA07g15980D和BnaA07g17500D在亲本粉色花瓣中发生内含子保留可变剪切。上述研究结果为甘蓝型油菜粉色花相关基因精细定位和分子连锁标记开发提供了更多线索。

关键词: 甘蓝型油菜, 粉色花性状, 遗传连锁图谱, QTL定位

Abstract:

Petal color is an important ornamental trait in B. napus, and the breeding and improvement of petal color have become the main goal in breeding and genetic research. To date, the research about interval location of pink petal trait in B. napus is very less yet. In this study, the genetic basis of petal phenotype was examined in the 62 (yellow petal) and 77 (pink petal) parents as well as 114 individuals comprising the doubled haploid (DH) mapping population. This DH population was examined using genotyping by sequencing (GBS) with 6065 high-density polymorphism single nucleotide polymorphism (SNP) markers to construct a genetic linkage map comprised of 3253 polymorphic markers. The genetic map spanned 1766.06 cM, with an average distance of 0.54 cM between markers. The complete interval mapping method identified two quantitative trait loci (QTL) for petal color located on chromosomes A07 and C03, respectively. Synteny analysis showed that some homologous genes in the interval of B. napus were located in Brassica rapa and Brassica oleracea. Also, eight genes related to flower color were analyzed between inbred line 77 and inbred line 62, the splice junctions of BnaA07g15980D and BnaA07g17500D were belong to intron retention type in pink petal of 77 parent. This study lays a foundation for further research on fine mapping of pink petal trait and molecular marker-assisted selection in Brassica napus L.

Key words: Brassica napus L., pink petal trait, genetic linkage map, QTL mapping

表1

遗传连锁群信息统计"

连锁群
Linkage groups		 标记数量
Number of markers		 遗传距离
Genetic distance
(cM)		 平均遗传距离
Average genetic distance
(cM)		 标记间最大间隔
Maximum spacing between markers (cM)
lg1 69 93.50 1.36 15.24
lg2 217 97.00 0.45 18.52
lg3 146 150.84 1.03 9.17
lg4 153 99.61 0.65 6.43
lg5 220 141.07 0.64 6.66
lg6 256 101.53 0.40 5.51
lg7 160 131.66 0.82 18.37
lg8 275 133.58 0.49 6.73
lg9 222 77.51 0.35 10.69
lg10 78 84.29 1.08 13.89
lg11 63 76.21 1.21 12.50
lg12 349 42.19 0.12 5.53
lg13 214 88.18 0.41 4.97
lg14 410 118.09 0.29 11.33
lg15 72 57.28 0.80 16.00
lg16 147 97.54 0.66 9.47
lg17 103 102.42 0.99 6.44
lg18 66 53.73 0.81 16.87
lg19
总计Total		 33
3253		 19.83
1766.06		 0.60
0.54		 9.65
18.52

图1

连锁群标记分布图 X轴表示连锁群; Y轴表示遗传距离(单位: cM); 蓝色为有效SNP标记。"

图2

甘蓝型油菜亲本花瓣表型观察 A1与A2: 母本62; B1与B2: 父本77。"

图3

甘蓝型油菜DH群体花色表型"

表2

甘蓝型油菜花色性状统计参数"

性状
Trait		 平均数
Average		 中位数
Median		 标准偏差
Standard deviation		 最小值
Minimum		 最大值
Maximum		 范围
Range		 四分位距
Interquartile range		 偏斜度
Skewness		 峰度
Kurtosis		 显著性
Significance
花色PC 24.47 22.89 11.28 6.21 53.04 46.83 12.78 0.85 0.09 0.00

图4

花色性状分布密度直方图 X轴代表花色表型组中值, Y轴代表分布密度。"

图5

WinQTL复合区间方法确定花色QTL连锁群位置 图A中, X轴表示19条染色体, Y轴分别表示LOD值和加性效应a (H1); 横线表示LOD阈值线。图B、图C分别表示在7号染色体和13号染色体检测到的一个花色QTL位点; X轴表示对应染色体上的遗传距离, Y轴分别表示LOD值和加性效应a (H1); 横线表示LOD阈值线。"

表3

WinQTL复合区间作图花色QTL结果"

QTL LOD峰值
LOD peak		 位置
Position (cM)		 左端标记
Left marker		 右端标记
Right marker		 99%置信
区间范围
99% CI (cM)		 加性效应
Additive effect		 贡献率
R2 (%)		 区间基因数量
Number of genes
qFC-chr7-1 6.62 23.8 mk2056 mk2036 8.383-42.019 4.58 14.29 287
qFC-chr13-1 14.01 30.8 mk4000 mk3898 20.305-41.353 6.61 34.02 308

图6

甘蓝型油菜WinQTL复合区间花色QTLs物理区间与甘蓝、白菜同源基因共线性分析"

表4

甘蓝型油菜亲本花色定位区间内基因可变剪切类型"

方法
Method		 亲本Parent 染色体
Chromosome		 定位区间内基因总数
Total number of genes in location interval		 发生可变剪切基因总数
Number of genes with
alternative splicing		 可变剪切出现次数
Number of the alternative splicings
IR ES A3SS A5SS 总计
Total
WinQTL Cart 77 A07 287 26 26 2 5 0 33
62 A07 287 26 25 1 4 2 32
77 C03 308 14 10 0 5 1 16
62 C03 308 11 8 0 5 2 15

图7

两亲本花瓣中花色相关基因可变剪切视图 A: BnaA07g15980D在黄花亲本62花瓣中转录本剪切视图; B: BnaA07g15980D在粉花亲本77花瓣中转录本剪切视图; C: BnaA07g17500D在黄花亲本62花瓣花瓣中转录本剪切视图; D: BnaA07g17500D在粉花亲本77花瓣中转录本剪切视图。灰色五边形为外显子, 白色区域为内含子, 连线表示不同剪接方式。图片包含4个区域, 第1部分为注释文件基因模型, 第2部分是测序结果, 第3部分为测序结果与注释文件比较基因剪切模型(带有代表性的isoform同源蛋白异构体), 第4部分为测序文件中支持外显子reads数目。左下角、右下角数字为该基因的起始位点和终止位点。"

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