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作物学报 ›› 2022, Vol. 48 ›› Issue (6): 1325-1332.doi: 10.3724/SP.J.1006.2022.14072

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

芥菜种子颜色调控基因TT8的等位变异及其地理分布分析

张钰坤1,2(), 陆赢1,2, 崔看3, 夏石头3, 刘忠松1,2,*()   

  1. 1湖南农业大学农学院 / 作物基因工程湖南省重点实验室, 湖南长沙 410128
    2国家油料作物改良中心湖南分中心, 湖南长沙 410128
    3湖南农业大学生物科学技术学院, 湖南长沙 410128
  • 收稿日期:2021-04-24 接受日期:2021-09-09 出版日期:2022-06-12 网络出版日期:2021-10-21
  • 通讯作者: 刘忠松
  • 作者简介:E-mail: 530611402@qq.com
  • 基金资助:
    国家自然科学基金项目(U20A2029)

Allelic variation and geographical distribution of TT8 for seed color in Brassica juncea Czern. et Coss.

ZHANG Yu-Kun1,2(), LU Ying1,2, CUI Kan3, XIA Shi-Tou3, LIU Zhong-Song1,2,*()   

  1. 1Crop Gene Engineering Key Laboratory of Hunan Province / College of Agronomy, Hunan Agricultural University, Changsha 410128, Hunan, China
    2Hunan Branch of National Oilseed Crops Improvement Center, Changsha 410128, Hunan, China
    3College of Bioscience and Biotechnology of Hunan Agricultural University, Changsha 410128, Hunan, China
  • Received:2021-04-24 Accepted:2021-09-09 Published:2022-06-12 Published online:2021-10-21
  • Contact: LIU Zhong-Song
  • Supported by:
    National Natural Science Foundation of China(U20A2029)

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摘要：

在芸薹属植物中TT8基因是种子颜色的调控基因。异源四倍体芥菜TT8基因有2个拷贝, 分别位于A09与B08染色体上。本研究针对不同拷贝设计特异引物, 对749份芥菜进行扩增、测序或酶切检测, 发现BjuA09.TT8和BjuB08.TT8各有7个和6个等位变异。与野生型相比, BjuA09.TT8.a1-a5和BjuB08.TT8.b1-b4等位基因都发生了大片段插入, 而BjuA09.TT8.a6出现了删除, 但BjuB08.TT8.b5仅在第7外显子处有1个碱基替换。使用Repeatmasker软件对等位基因序列与甘蓝型油菜注释库进行重复序列比对, 发现BjuA09.TT8.a1-a4和BjuB08.TT8.b1-b4等位基因主要含有I类转座子, 少量为II类转座子及Helitron类转座子插入。统计还发现, BjuA09.TT8.a4-BjuB08.TT8.b5单倍型为主要的黄籽单倍型, 占所分析黄籽材料的89.49% (247/276), 其次为BjuA09.TT8.a4-BjuB08.TT8.b3单倍型, 占6.52%。地理分布分析结果显示, 中国尤其是新疆芥菜黄籽突变等位基因频率显著高于世界其他地区, 这与历史记载相印证, 说明芥菜黄籽性状可能起源于中国新疆。本研究结果为油菜黄籽育种选用优异基因资源提供了依据。

关键词: 芥菜, 黄籽, TT8, 等位变异, 转座子, 种质资源, 地理分布

Abstract:

The gene TT8 regulates seed color in Brassica species. Two TT8 copies, designated as BjuA09.TT8 and BjuB08.TT8, were cloned from the chromosomes A09 and B08 of the allotetraploid B. juncea, which had 7 and 6 alleles in worldwide 749 B. juncea accessions, respectively. Compared with the wild type, BjuA09.TT8.a1-a5 and BjuB08.TT8.b1-b4 alleles carried large insertions, while BjuA09.TT8.a6 and BjuB08.TT8.b5 had a deletion and a base substitution at exon 7, respectively. Comparison of the allele sequences with the annotated library of swede rapeseed (B. napus) using the software Repeatmasker revealed that BjuA09.TT8.a1-a4 and BjuB08.TT8.b1-b4 alleles contained class I transposons, a few class II transposons, and Helitron-like transposon insertions. The haplotype analysis showed that BjuA09.TT8.a4-BjuB08.TT8.b5 was the major yellow seed haplotype, accounting for 89.49% (247/276) of the yellow seed accessions detected, followed by the haplotype BjuA09.TT8.a4- BjuB08.TT8.b3, amounting for 6.52%. Analysis of geographical origin of yellow seed accessions revealed that there were had a higher frequency of yellow seed mutation especially in Xinjiang region, China, than the other parts of the world, suggesting possible origin of yellow seed mustard in Xinjiang, China together with historical records. This study provides a basis for the selection of superior genetic resources for breeding yellow seed rapeseed.

Key words: Brassica juncea, yellow seed, TT8, allelic variation, transposable elements, genetic resources, geographical distribution

表1

芥菜TT8基因全长扩增所用引物及其序列"

引物名称
Primer name		 引物序列
Primer sequence (5'-3')		 预期区间
Predicted interval
ANF CCAATCAGTGGATGACACGG 扩增BjuA09.TT8全长序列
The amplification of the BjuA09.TT8 full-length sequence
BNF ACCTGCAGGATACATCTCAC 扩增BjuB08.TT8全长序列
The amplification of the BjuB08.TT8 full-length sequence
4R CTGATAATGTAGCATAGACGACGCTA 扩增Bju.TT8等位基因共用的R端
The reverse primer sequence commonly used for the amplification of Bju.TT8 alleles
18T ANF acccggggatcctctagaga CCAATCAGTGGATGACACGG BjuA09.TT8全长克隆 Full-length cloning of BjuA09.TT8
18T BNF acccggggatcctctagaga ACCTGCAGGATACATCTCAC BjuB08.TT8全长克隆 Full-length cloning of BjuB08.TT8
18T 4R catgcctgcaggtcgacgat CTGATAATGTAGCATAGACGACGCTA Bju.TT8全长克隆共用的R端
The reverse primer sequence common to full-length cloning of two Bju.TT8 copies

图1

芥菜TT8等位基因变异 A: 芥菜BjuA09.TT8等位变异; B: BjuA09.TT8等位基因变异电泳图; C: 芥菜BjuB08.TT8等位变异; D: BjuB08.TT8.b5等位基因的SNP突变位点; E: BjuB.TT8等位基因变异电泳图; M: 1 kb DNA ladder; YS: 黄籽; BS: 褐籽。"

表2

芥菜TT8基因不同单倍型组合的频率统计"

BjuA09.TT8
allele		 BjuB08.TT8
allele		 种皮颜色
Seed color		 代表性材料
Representative accession		 品种总数量
Number of accessions		 中国所占数量
Number of Chinese accessions
A B BS 紫叶芥 Purple-leaf mustard 369 164
A b1 BS 黔西马尾油菜Qianxiyoucai 3 2
A b2 BS BOJ18-19 1 1
A b4 BS PI 347618 2 0
A b5 BS 普安苦油菜 Pu'ankuyoucai 46 16
a2 B BS CR 2729 7 1
a4 B BS PBR 97 42 13
a6 B BS CR 2493 3 2
a1 b5 YS PI 458929 2 1
a2 b5 YS PI 531271 4 2
a3 b5 YS 玉溪马桥高株 Yuximaqiao tall 2 1
a4 b5 YS 四川黄籽 Sichuan yellow 247 132
a4 b3 YS 和田油菜 Hetianyoucai 18 11
a5 b5 YS 会理高足黄油菜 Huili tall yellow 3 2

图2

芸薹属植物和拟南芥TT8基因聚类分析 使用MEGA-X中的Muscle程序对序列进行比对, 其中Gap Open设置为-6600.00。利用Neighbor-Joining方法构建系统进化树, 参数为默认值。不显示小于50%的值。NCBI基因名称及序列编号: AthTT8 (NC 003075.7)、BraA.TT8 (NC 024803.2)、BnaA.TT8 (027765.2)、BnaC.TT8 (NC 027775.2)、BolC.TT8 (NC 027756.1)。"

表3

芥菜TT8等位基因包含的转座子分析结果"

Bju.TT8
allele		 基因长度Gene length (bp) SNP/插入缺失位置
Location of SNP/insertion or deletion		 插入删除长度Length of insertion or deletion (bp) 转座子种类/家族
TEs class/family		 重复序列数目
Number of
repeats		 涉及序列长度
Occupied
(bp)		 序列比例Percentage of sequence (%)
A 3551 — — — — — —
a1 9203 +2551 bp Exon6 +5652 LINE/L1 2 4718 51.27
a2 7320 +1246 bp Intron 4 +3769 LTR;
LTR/Cppia;
LTR/Gypsy;
RC/Helitron		 2
2
1
1		 109
2495
11
560		 1.49
34.08
0.15
7.65
a3 5667 +1246 bp Intron4/
+3047 bp Exon 7		 +849
+1267		 DNA/CMC-Enspm;
LTR/Copia		 1
1		 927
1263		 16.36
22.29
a4 4818 +3047 bp Exon7 +1267 LTR/copia 1 1263 26.21
Bju.TT8
allele		 基因长度Gene length (bp) SNP/插入缺失位置
Location of SNP/insertion or deletion		 插入删除长度Length of insertion or deletion (bp) 转座子种类/家族
TEs class/family		 重复序列数目
Number of
repeats		 涉及序列长度
Occupied
(bp)		 序列比例Percentage of sequence (%)
a5 3859 +3090 bp Exon7 +308 — — — —
a6 2970 +455 bp Exon2 -581 — — — —
B 2768 — — — — — —
b1 9416 +803 bp Intron2 +6648 LINE/L1
DNA/Maverick		 4
1		 4756
138		 50.51
1.47
b2 4457 +1388 bp Intron5 +1689 SINE/tRNA
DNA/hAT-Ac
LINE/L1
RC/Helitron		 1
2
1
1		 27
1562
97
113		 0.61
35.05
2.18
2.54
b3 4076 +1958 bp Exon6 +1308 LINE/L1 1 112 2.75
b4 2985 +2390 bp Exon7 +217 LINE/L1 1 253 8.48
b5 2768 +2742 bp Exon7 C/T — — — —

图3

芥菜不同TT8单倍型材料的地理分布 统计不同国家以及中国不同省区各种单倍型材料的种子颜色。饼图中的不同颜色代表14种不同的单倍型, 每个饼图表示不同单倍型在该区域的比例。每个饼图的大小代表样本数量的多少, 饼图越大代表样本量越多。底部的饼图总结了14种单倍型在6大洲的分布情况, 括号里的数字表示被分析的种质数量。AS: 亚洲; EU: 欧洲; NA: 北美洲; OA: 大洋洲; AF: 非洲; SA: 南美洲。"

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