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Acta Agronomica Sinica ›› 2022, Vol. 48 ›› Issue (6): 1325-1332.doi: 10.3724/SP.J.1006.2022.14072

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

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 Online:2022-06-12 Published:2021-10-21
  • Contact: LIU Zhong-Song E-mail:530611402@qq.com;zsliu48@hunau.net
  • Supported by:
    National Natural Science Foundation of China(U20A2029)

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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

Table 1

Primers used for amplification of Bju.TT8"

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

Fig. 1

TT8 alleles in Brassica juncea A: BjuA09.TT8 alleles on chromosome A09; B: BjuA09.TT8 electrophoretogram; C: BjuB08.TT8 alleles on chromosome B08; D: the SNP mutation of BjuB.TT8.b5 allele; E: BjuB08.TT8 electrophoretogram; M: 1 kb DNA ladder; YS: yellow seed; BS: brown/black seed."

Table 2

Frequency statistics of different haplotype combinations of TT8 gene in Brassica juncea"

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

Fig. 2

Cluster analysis of TT8 genes in Brassica species and Arabidopis thaliana Sequences were aligned using the Muscle program in Mega-X, where Gap Open was set to -6600.00. The phylogenetic tree was constructed using Neighbor-Joining method under the default parameters. bootstrap values less than 50 are not shown. NCBI gene name and serial number are AthTT8 (NC 003075.7), BraA.TT8 (NC 024803.2), BnaA.TT8 (027765.2), BnaC.TT8 (NC 027775.2), and BolC.TT8 (NC 027756.1), respectively."

Table 3

Transposable elements in TT8 alleles of Brassica juncea"

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 — — — —

Fig. 3

Geographic distributions of different TT8 haplotpyes in Brassica juncea Accessions with different haplotypes are counted by country or by province within China. The different colors in the pie chart represent 14 different haplographs, each representing the proportion of different haplots in the area. The size of pie stands for the number of accessions detected, and the larger the pie, the more accessions detected. The pies at the bottom summarizes the distribution of the different haplotypes on different continents, and the numbers in brackets indicate the number of accessions detected. AS: Asia; EU: Europe; NA: North America; OA: Oceania; AF: Africa; SA: South America."

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