水稻雄性核不育突变体ms7的遗传分析及基因定位

doi:10.16819/j.1001-7216.2022.210302

中国水稻科学 ›› 2022, Vol. 36 ›› Issue (1): 27-34.DOI: 10.16819/j.1001-7216.2022.210302

水稻雄性核不育突变体ms7的遗传分析及基因定位

杨晋宇¹^,^#, 白琛²^,³^,^#, 丁小惠²^,³, 申红芳¹, 王磊¹, 应杰政¹^,^*(), 鄂志国¹^,^*()

¹中国水稻研究所水稻生物学国家重点实验室, 杭州 310006
²中国科学院植物研究所植物分子生理学重点实验室, 北京100093
³中国科学院大学,北京100049

收稿日期:2021-03-03 修回日期:2021-04-19 出版日期:2022-01-10 发布日期:2022-01-10
通讯作者: 应杰政,鄂志国
作者简介:第一联系人：
^#共同第一作者;
基金资助:
浙江省自然科学基金资助项目(LY17G030031);国家自然科学基金面上项目(32072050);国家自然科学基金面上项目(71773140);中央级公益性科研院所基本科研业务费专项(CPSIBRF-CNRRI-202129)

Genetic Analysis and Gene Mapping of a Male Sterile Mutant ms7 in Rice

YANG Jinyu¹^,^#, BAI Chen²^,³^,^#, DING Xiaohui²^,³, SHEN Hongfang¹, WANG Lei¹, YING Jiezheng¹^,^*(), E Zhiguo¹^,^*()

¹State Key Laboratory of Rice Biology, China National Rice Research Institute, Hangzhou 310006, China
²Key Laboratory of Plant Molecular Physiology, Institute of Botany, Chinese Academy of Sciences, Beijing 100093, China
³University of Chinese Academy of Sciences, Beijing 100049, China

Received:2021-03-03 Revised:2021-04-19 Online:2022-01-10 Published:2022-01-10
Contact: YING Jiezheng, E Zhiguo
About author:First author contact:
^#These authors contributed equally to the work;

摘要/Abstract

摘要：

【目的】通过对水稻雄性不育突变体的研究,可以鉴定更多与育性或花粉发育相关的基因,有助于解析水稻雄性生殖发育的整个调控网络。【方法】常规种植条件下,突变体ms7 (male sterile 7)与对照种植于浙江富阳和海南陵水,比较它们的育性及主要农艺性状差异,利用混池关联分析和图位克隆方法进行目标基因定位。【结果】整个生育期,突变体ms7生长速率与野生型一致,成熟期的株高、分蘖数、叶数、叶大小、穗长和每穗颖花数等性状与野生型相比也没有显著差异,但ms7结实率为0,表现为完全雄性不育,花药瘦小且颜色发白,半薄切片显示绒毡层降解推迟,花粉镜检呈染败。遗传分析表明花粉败育受单个隐性基因控制,定位于第7染色体上BSA11与YD7045之间1.17 Mb的范围内。【结论】本研究为水稻雄性不育基因ms7的克隆和功能研究打下了基础。

关键词: 水稻, 雄性不育, 混池关联分析, 基因定位

Abstract:

【Objective】Genes related to pollen development and fertility can be identified through the study on male sterile mutants, which is helpful to analyze the whole regulatory network of male reproductive development in rice. 【Method】The fertility and agronomic traits of the ms7 (male sterile 7) mutant and its wild type were compared under conventional planting conditions in Fuyang, Zhejiang and Lingshui, Hainan. The sterile gene was mapped by bulked segregant analysis and map-based cloning.【Result】During the whole growth period, the growth rate of ms7 mutant was the same as that of wild type, and there was no significant difference in plant height, tiller number, leaf number, leaf size, panicle length and spikelets per panicle between ms7 and its wild type. However, ms7 exhibited slender and white anthers with inactive pollen grains which couldn’t be stained with I₂-KI, and no seeds were produced. Observation results of anther cross-sections exhibited that tapetum programmed cell death (PCD) was delayed. Genetic analysis showed that pollen abortion was controlled by a single recessive nuclear gene, which was mapped to a 1.17 Mb region between BSA11 and YD7045 on chromosome 7. 【Conclusion】It will contribute to the further molecular cloning and functional analysis of male sterile gene ms7 in rice.

Key words: rice, male sterile, bulked segregant analysis, gene mapping

杨晋宇, 白琛, 丁小惠, 申红芳, 王磊, 应杰政, 鄂志国. 水稻雄性核不育突变体ms7的遗传分析及基因定位[J]. 中国水稻科学, 2022, 36(1): 27-34.

YANG Jinyu, BAI Chen, DING Xiaohui, SHEN Hongfang, WANG Lei, YING Jiezheng, E Zhiguo. Genetic Analysis and Gene Mapping of a Male Sterile Mutant ms7 in Rice[J]. Chinese Journal OF Rice Science, 2022, 36(1): 27-34.

图/表 7

图1 突变体ms7及其野生型中恢161的表型 A―灌浆期的植株形态;B―ms7和野生型的结实情况;C―ms7和野生型的颖花形态;D―ms7和野生型的花粉碘化钾染色。

Fig. 1. Phenotype of ms7 and its wild type Zhonghui 161. A, ms7 and its wild type Zhonghui 161 (WT) at grain filling stage; B, Panicle of ms7 and WT at the harvest stage; C, Spikelet of ms7 and WT; D, I2-KI staining of ms7 and WT pollens.

图2 野生型中恢161和突变体ms7不同发育时期花药半薄切片观察 A~F为野生型花药在8a至12时期的切片;G~L为ms7突变体花药在8a至12时期的切片。 Ep―表皮;En―内壁;T―绒毡层;Dy―二分体;Tds―四分体;Msp―小孢子;BP―二胞花粉;MP―成熟花粉粒;iMP―未成熟花粉粒。标尺为20 μm。

Fig. 2. Contrast of slice of anther between the ms7 mutant and its wild type. A-F, Wild-type anthers from stages 8a to 12; G-L, ms7 anthers from stages 8a to 12. Ep, Epidermis; En, Endothecium; T, Tapetum; Dy, Dyad cell; Tds, Tetrads; Msp, Microspore; BP, Biceullar pollen; MP, Mature pollen; iMP, Immature pollen. Bar=20 μm.

表1 ms7中雄性不育性状的遗传分析

Table 1 Genetic analysis of the male-sterile trait in ms7.

群体Group	可育植株数No. of fertile plants	不育植株数 No. of sterile plants	卡方值 χ²
F₂-32	240	72	0.6154
F₂-33	232	73	0.1847
F₂-34	313	106	0.0199
F₂-35	273	76	1.9341

表2 SNP数据统计表

Table 2. Statistics of SNP data.

样品 Sample	SNP位点数 SNP number	转换的SNP数 Transition number	颠换的SNP数 Transversion number	T_i/T_v	杂合SNP数 Heterozygosity number	纯合SNP数 Homozygosity number
16F4Mix	3 383 944	2 408 546	969 917	2.48	2 098 068	1 285 876
16S4	3 383 383	2 408 846	968 983	2.49	2 069 857	1 313 526
IR9667	2 714 614	1 929 831	781 109	2.47	1 517 729	1 196 885
ms7	3 014 721	2 147 993	863 049	2.49	549 661	2 465 060

图3 SNP指数在染色体上的分布曲线是变异位点在染色体上所对应的Index值;红、绿和蓝三根线分别是95%、99%和99.9%置信区间阈值线。

Fig. 3. Distribution of SNP-index on the chromosomes. The curve is the index value corresponding to the variation site on the chromosomes; the confidence interval thresholds from bottom to top are 95%, 99% and 99.9%, respectively.

表3 8个InDel标记的引物信息

Table 3. Sequences and physical position of the eight InDel markers.

引物名称 Primer name	差异位点 Position	上游引物 Forward primer(5'-3')	下游引物 Reverse primer(5'-3')
YD7036	16 913 833	CCAGGGGATCTAAACAGG	TTGGACCATCACATAAGC
BSA11	16 969 331	TGCCAATGTCGATTCCAAGAG	TCCAGCCTCTGTATACTTGCA
BSA6	17 002 663	CCCTCACCTTCTGCAAGCTA	CGGTCTTTGAGGAGGTATCGA
BSA36	17 512 757	AACAGAAAGCAACCAACCGC	TTCTTGGAGGTCAGGCAAGA
YD7013	17 777 239	TGGTCTGCACTGCCTTAA	GGATGGACGGATGGGATA
YD7017	17 995 826	ATCTTTCCCGATTGAGCT	CAGGCTTCATTCAAGACAA
YD7045	18 137 137	AGGCTAAGGCGAGGAGAA	GCATACCAGCAAGGACGA
BSA47	18 400 602	TTCATTCTTCTACCTTTGCCATCGG	TCTCTCTCACACTGGTCTCCTCCTC

图4 重组单株的基因型组成和育性表现

Fig 4. Genotypic composition and fertility performance of recombinant plants.

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水稻雄性核不育突变体ms7的遗传分析及基因定位

Genetic Analysis and Gene Mapping of a Male Sterile Mutant ms7 in Rice

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