水稻杂种偏分离位点SegD8的精细定位

doi:10.16819/j.1001-7216.2023.220203

摘要/Abstract

摘要： 目的通过研究水稻渐渗系群体构建时产生的偏分离现象，可以发现更多与偏分离相关的基因，有助于解析偏分离在水稻中的主要作用及其产生机制。方法应用籼粳组合华占/Koliya构建的一套渐渗系群体，以其后代基因型的分离比作为表型对偏分离位点进行定位。结果在第8染色体上发现一个染色体区段在其后代中的基因型分离比偏离孟德尔分离定律。进一步分析表明该偏分离现象受第8染色体上两个位点控制，分别命名为SegD8A和SegD8B，其中SegD8A为偏分离位点，但其偏分离属性受SegD8B调控。当SegD8A和SegD8B位点都为杂合型时，子代中SegD8A^HH和SegD8A^KK的比例接近1∶4；当SegD8B为纯合华占基因型时，子代中SegD8A^HH和SegD8A^KK的比例接近1∶1；当SegD8B为纯合Koliya基因型时，子代中SegD8A^HH几乎不出现。我们将SegD8A基因定位在一个大小为46.5 kb的区间内，SegD8B基因定位在一个大小为1160.2 kb的区间内。结论本研究为进一步克隆这两个基因，并阐明该偏分离现象奠定了基础。

关键词: 杂交-渐渗, 水稻, 偏分离

Abstract:

【Objective】 This study aims to fine-map and clone genes related to segregation distortion(SD) by using a rice introgressive line population, and thus to explore the molecular mechanism underlying the segregation distortion in rice. 【Method】 A set of introgressive line population was constructed by using an indica/japonica combination Huazhan/Koliya. The gene loci were mapped with different genotype separation ratios of offspring as phenotypes. 【Result】 We identified a segment on chromosome 8 that displayed obvious deviated segregation ratio from Mendelian's separation law. Further analysis revealed that the SD was controlled by two loci on chromosome 8, which were termed as SegD8A and SegD8B, respectively. SegD8A was a segregation locus, but its segregation was regulated by SegD8B. When both SegD8A and SegD8B were heterozygous, the ratio of SegD8A^HH and SegD8A^KK in progeny was close to 1:4. When SegD8B was homozygous Huazhan genotype, the ratio of SegD8A^HH and SegD8A^KK in progenies was close to 1:1. When SegD8B was homozygous Koliya genotype, SegD8A^HH almost did not appear in its progeny. The SegD8A gene was mapped to a 46.5 kb region and the SegD8B gene was mapped to an 1160.2 kb region using the separation ratio of the progeny. 【Conclusion】 The results lay a foundation for further cloning of these two genes and elucidating the partial separation phenomenon.

Key words: hybridization-introgression, rice (Oryza sativa L.), segregation distortion

袁洋, 敖和军, 周仲华, 应杰政, 张健, 倪深. 水稻杂种偏分离位点SegD8的精细定位[J]. 中国水稻科学, 2023, 37(1): 37-44.

YUAN Yang, AO Hejun, ZHOU Zhonghua, YING Jiezheng, ZHANG Jian, NI Shen. Fine-mapping of SegD8 Loci for Rice Hybrid Segregation Distortion[J]. Chinese Journal OF Rice Science, 2023, 37(1): 37-44.

图/表 8

图1 NS139.5的SNP基因型

Fig. 1. SNP microarray genotype of NS139.5.

表1 分子标记在第8染色体上的偏分离

Table 1. Segregation distortion of molecular markers on chromosome 8.

标记 Marker	基因型 Genotype			χ²_(1:2:1)
标记 Marker	HZ/HZ	HZ/BLY	BLY/BLY	χ²_(1:2:1)
CHR8-24	6	35	29	15.11^**
CHR8-22	7	40	23	8.74^*
CHR8-5	8	41	21	6.89^*
CHR8-25	9	36	25	7.37^*

表2 自交群体偏分离株系正常花粉率与结实率

Table 2 Normal pollen rates and setting rates of segregation distortion in inbred populations.

正常花粉率Normal pollen rate/%
92.56
92.35^ns
92.11^ns
92.41^ns
7.88

图2 BC3F2代群体花粉染色图 A―正常子代低温不育; B―偏分离子代低温不育; C―子代正常分离; D―子代偏分离。

Fig. 2. Pictures of pollen stained of BC3F2 population. A, Sterility of normally-segregated offspring at low temperature; B, Sterility of offspring with segregation distortion at low temperature; C, Normal segregation of offspring; D, Segregation distortion of offspring.

表3 群体后代偏分离比率的类型

Table 3. Types of partial segregation ratio of population offspring.

单株 Plant	标记 Marker	基因型 Genotype			分离比Segregation ratio (HZ/HZ∶HZ/BLY∶BLY/BLY)
单株 Plant	标记 Marker	HZ/HZ	HZ/BLY	BLY/BLY	分离比Segregation ratio (HZ/HZ∶HZ/BLY∶BLY/BLY)
NFX9-56-4	CHR8-45	5	26	16	1∶5∶4
NFX9-80-1	CHR8-45	4	24	20
NFX10-128-5	CHR8-45	5	25	18
NFX9-34-1	CHR8-45	1	24	23	0∶1∶1
NFX9-37-6	CHR8-45	0	25	23
NFX10-113-1	CHR8-45	1	20	27
NFX9-26-4	CHR8-45	12	24	12	1∶2∶1
NFX10-130-6	CHR8-45	13	24	10
NFX6-13-6	CHR8-45	21	52	21

图3 SegD8A在水稻第8染色体上的精细定位 A―SegD8A初定位在第8染色体CHR8-45和CHR8-53之间; B―SegD8A精细定位在CHR8-78和SNP8-6之间46.5 kb区间内; C―精细定位区间内包含的开放阅读框(open reading frame, ORFs)和预测基因。

Fig. 3. Fine mapping of SegD8A on chromosome 8 of rice. A, Primary mapping of SegD8A between markers CHR8-45 and CHR8-53 on chromosome 8; B, SegD8A was fine mapped to the interval between markers CHR8-78 and SNP8-6, a 46.5 kb region; C, Open reading frames (ORFs) and predictive genes within the fine-mapped region.

图4 渐渗系在目标区间的基因型组成

Fig. 4. Genotypic compositions of introgression lines in target region.

表4 本研究使用的标记

Table 4. DNA markers used in this study.

标记 Marker	类型 Type	正向引物（5'-3') Forward primer(5'-3')	反向引物（5'-3'） Reverse primer(5'-3')
CHR8-45	InDel	TTAGCATGGAGTTTATTTTTAGTGAT	ACCATGACCACCTTCGCA
CHR8-56	InDel	GACCCTCCTATTTGCCCTTGT	GTTGTTTGTCTTTTGTGGACTGTG
CHR8-78	InDel	CCAAAGAAATTGCCAATCCTAC	CTATGTTGTGTTTCCTGAAATCACA
SNP8-6	SNP	CCAAGCGTTTGCTCACTGC	TACTCTATCCCTTCTTTCTTCGCTT
CHR8-63	InDel	GAGTAACTTTTTTTATGTCGCCAA	ACCTGGTTTCGCAAGTTCG
CHR8-53	InDel	CGAGCAGACCAATCTAAGGGAT	AACAGAAGACCCCAGAGAATCAA
CHR8-71	InDel	GGAAAATAAAAACAAAAGACGGAAC	CATCAGAGGAGGAGTACTTCAGTGA
CHR8-80	InDel	GCTAAACTTTCCTGTAACATCCTGA	GAAAATGGTTTCAATTTCTACCCA
CHR8-29	InDel	ATACGCAGAGCCATATATAAAAGAT	CTACACGCACAAACTATTTCAGTAC
CHR8-86	InDel	AGCAGGCACGGGCGATTA	TGGAGCATTTGGCAGGGTTA

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