高粱双粒突变体Dgs的遗传分析与基因定位

doi:10.13560/j.cnki.biotech.bull.1985.2021-1294

摘要/Abstract

摘要：

穗粒数对作物产量至关重要,对高粱双粒突变体基因进行定位,为下一步的基因克隆和功能分析奠定基础,为高粱产量性状的调控和解析提供依据。利用体视镜观察野生型和双粒突变体花器官表型,通过构建单粒材料晋粱5号和双粒突变体杂交的F₂群体,分析突变体的遗传规律,采用BSA-seq测序与图位克隆相结合的方法进行连锁分析及基因定位。结果表明,与野生型相比,双粒突变体雌蕊和雄蕊数目增加,最终结一壳双粒;遗传分析表明双粒性状受1对显性基因控制;基因定位显示目的基因位于高粱第6染色体Indel2930和SSR7060标记之间,物理距离为404 kb。Dgs是一个全新的高粱双粒突变基因。

关键词: 高粱, 双粒突变体, 花器官, 基因定位, 遗传分析

Abstract:

Grain number per panicle is very important for crop yield. In order to identify and clone the gene controlling grain number per panicle,a double-grain mutant gene of sorghum[Sorghum bicolor（L.）Moench]was located,for laying a foundation for gene cloning and functional analysis and providing evidences for the regulation and analysis of sorghum yield traits. The flower organ phenotypes of wild-type sorghum plant and double-grain mutant were observed by stereoscopy. A F₂ population was constructed from the cross between S. bicolor Jin 5 and the double-grain mutant to analyze the inheritance pattern of the mutant and gene mapping. BSA-seq sequencing and map cloning were used for linkage analysis and gene mapping. The results showed that,compared with wild type,the mutant mainly showed an increase in the number of pistil and stamen,ultimately two kernels in one pair of glumes. Genetic analysis indicated that the double-grain trait was controlled by a pair of dominant genes. Gene mapping showed that the target gene was located between markers Indel2930 and SSR7060 on the chromosome 6 of S. bicolor with physical distance of 404 kb. Dgs is a novel sorghum double-grain mutant gene.

Key words: sorghum, double-grain mutant, flower organ, gene mapping, genetic analycis

周诗晨, 仪治本, 王馨翊, 杨晓颖, 孙丽娜, 栾维江, 梁闪闪. 高粱双粒突变体Dgs的遗传分析与基因定位[J]. 生物技术通报, 2022, 38(7): 171-177.

ZHOU Shi-chen, YI Zhi-ben, WANG Xin-yi, YANG Xiao-ying, SUN Li-na, LUAN Wei-jiang, LIANG Shan-shan. Genetic Analysis and Gene Mapping of Sorghum Double-grain Mutant Dgs[J]. Biotechnology Bulletin, 2022, 38(7): 171-177.

图/表 7

图1 野生型晋粱5号和突变体Dgs小花及籽粒的表型 A：野生型晋粱5号的小花;B：突变体Dgs小花;C：野生型晋粱5号的籽粒;D：突变体Dgs籽粒

Fig.1 Phenotype of the floret and grain of wild-type S. bicolor Jin 5 and mutant Dgs A：Flower of wild-type S. bicolor Jin 5. B：Flower of mutant Dgs. C：Grain of wild-type S. bicolor Jin 5. D：Grain of mutant Dgs

表1 晋粱5号×Dgs F2群体表型统计分析

Table 1 Statistical analysis of phenotype of F2 population from S. bicolor Jin 5×Dgs

杂交组合 Cross	双粒单株个体数 Number of double-grain individuals	单粒单株个体数 Number of single-grain individuals	总数 Total	χ²（3∶1）	P
晋梁5号 × 双粒突变体 S. bicolor Jin 5 × Dgs	313	117	430	1.119	0.290

表2 测序数据质量情况

Table 2 Statistics from the qualities of the sequencing

样品 Sample	初始测序reads数目 Raw reads	过滤后的reads数 Clean reads	过滤后碱基数 Clean base	Q20/%	Q30/%	GC含量 GC content/%
双粒突变体 Dgs	39 005 403	38 995 403	11 679 957 024	97.83	93.6	43.28
晋粱5号 Jin5	38 666 782	38 656 413	11 579 578 194	97.74	93.44	43.86
单粒混池/ Single-grain bulk（S bulk）	148 822 039	148 768 510	44 563 912 284	97.61	93.15	43.25
双粒混池/ Double-grain bulk（D bulk）	156 721 594	156 668 065	46 930 613 822	97.58	93.26	43.67

图2 Dgs双粒突变体基因初步定位

Fig. 2 Preliminary gene mapping of Dgs double grain mutant

图3 部分F2代隐性单株的PCR产物凝胶电泳图 A：InDel2930标记检测;B：InDel6958标记检测;M：双粒突变体亲本;WT：晋粱5号亲本;1-22：F2代单粒单株。▲：交换单株

Fig. 3 PCR agarose gel electrophoresis of partial F2 reces-sive individuals A：Marker detection of InDel2930. B：Marker detection of InDel6958. M：Parent of the double-grain mutant Dgs. WT：Parent of Jin 5. 1-22：Single-seed individuals in F2 generation. ▲：Recombinant line

表3 Dgs连锁分析的主要引物信息

Table 3 Information of primers used for linkage analysis of Dgs

标记名称 Maker	正向引物 Forward primer（5'-3'）	反向引物 Reverse primer（5'-3'）
Indel2930	CGACGCCGGGATTTCCATTT	TTGGTCCACATCCGAGCAGG
Indel6958	ATTTACGGGTAGCGAGCACT	GGCCGTATCCTCAATCCTGT
SSR7060	GCACACCACCGAGTCAGC	CTCCTCCACCTGCAGCAT
SSR1018	ACCTGTAAATTCCATGCATCGT	CAGAAGGTCAACTGTTTCTCCC
SSR2629	CAAATAACAGCCCCCAAAGG	CTGCTAAGGCGTGCTTTATT
SSR9952	CGCAAATGACGAGTCTTGGT	AGTCGGATCTGATCTAATCGCA
SSR8023	GTGTACGTGACGCGGTGAAA	GTCGTCTCCCTGGTATGTATGG

图4 Dgs的遗传连锁图谱

Fig. 4 Genetic linkage map of the Dgs gene

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