利用QTL-Seq结合分子标记定位粳稻垩白粒率控制位点qChalk8

doi:10.16819/j.1001-7216.2024.231207

摘要/Abstract

摘要：

【目的】垩白是影响稻米外观品质的重要性状。本研究旨在利用QTL-Seq和分子标记作图定位粳稻垩白粒率调控相关的QTL。【方法】利用高垩白粒率粳稻材料拉木加和低垩白粒率粳稻品种水晶3号构建F₂分离群体，将两份独立的F₂群体分别种植于河南原阳和海南三亚，两个群体单株垩白粒率考种后分别选取极端个体混池进行QTL-Seq分析，然后使用分子标记作图对稳定的QTL进行验证。【结果】两个F₂群体的QTL-Seq分析发现8号染色体存在一个较为稳定的QTL位点qChalk8。进一步通过分子标记作图将该QTL定位于17.6－18.5 Mb，该QTL位点的LOD值为7.08，表型贡献率为15.9%。【结论】利用QTL-Seq和分子标记作图定位了粳稻垩白粒率相关QTL-qChalk8，为粳稻垩白调控基因进一步的精细定位和基因克隆奠定了基础。

关键词: 水稻, 垩白, 垩白粒率, QTL-Seq, QTL定位

Abstract:

【Objective】Chalkiness is an important characteristic that affects the appearance quality of rice grains. This study aims to use QTL-Seq and molecular marker mapping methods to locate QTLs related to chalky rice rates in japonica rice.【Method】F₂ segregating populations were constructed using the japonica germplasm Lamujia with a high chalky rice rate and the japonica variety Shuijing 3 with a low chalky rice rate. Two independent F₂ populations were planted in Yuanyang, Henan Province, and Sanya, Hainan Province, respectively. The single-plant chalky rice rate was examined, and extreme individuals were pooled for QTL-Seq analysis. Reliable QTLs were then subjected to verification using molecular marker mapping.【Result】QTL-Seq analysis of the two F₂ populations revealed the existence of a stable chalky rice rate QTL on chromosome 8, designated as qChalk8. Further molecular marker mapping confined the QTL to a genomic region between 17.6 Mb and 18.5 Mb. The LOD score for qChalk8 was 7.08, and qChalk8 could explain 15.9% of the phenotypic variation.【Conclusion】QTL-Seq and molecular marker mapping successfully identified qChalk8 related to chalky rice rates in japonica rice. The localization of qChalk8 establishes a foundation for further fine mapping and gene cloning of the chalkiness regulatory gene in japonica rice.

Key words: rice, grain chalkiness, chalky rice rate, QTL-Seq, QTL mapping

杜彦修, 孙文玉, 袁泽科, 张倩倩, 李富豪, 李俊周, 孙红正. 利用QTL-Seq结合分子标记定位粳稻垩白粒率控制位点qChalk8[J]. 中国水稻科学, 2024, 38(6): 665-671.

DU Yanxiu, SUN Wenyu, YUAN Zeke, ZHANG Qianqian, LI Fuhao, LI Junzhou, SUN Hongzheng. Mapping of qChalk8 Controlling Chalky Rice Rate in japonica Rice by Combining QTL-Seq with Molecular Markers[J]. Chinese Journal OF Rice Science, 2024, 38(6): 665-671.

图/表 6

表1 本研究中分子标记定位作图所用多态性引物及序列

Table 1. Primers used in molecular marker mapping in this study

引物名称Primer	位置 Location	正向序列 Forward sequence	反向序列 Reverse sequence
8_0.8Mb	Chr8_837961	CCGGCATTTTGACAGGAAGTG	ACCTCTCTCTCTTTCTCTCTCCC
8_3.5Mb	Chr8_3532447	TCCAGATTCGTGATATTAGGATGAGT	GGAGGATCAGAGAGACCATGC
8_4.5Mb	Chr8_4537532	TCGTCGGGATTATTCCGAGC	ACACCGCCACCAATCAAAGA
8_7.7Mb	Chr8_7747072	CGGTCCATTGCACACCCTTA	ACATAACACATGCACCATGGA
8_9.4Mb	Chr8_9385739	TTGATGGGAGTGCCTGAAGA	TCACTGGTTACTATTGGTCACGT
8_15.8Mb	Chr8_15858062	GTAAGAAGCCAGAGCCGGAG	CTAAGAGAAGAGCGGGGTGC
8_16.5Mb	Chr8_16560856	ACAACCAACCCGGCTTGTTA	ACCATCCATTTGGCCTTGTT
8_16.9Mb	Chr8_16906251	CGACGAGGGGTCTTACCAAG	CTTCTCCCGATCCGCTCAAC
8_17.6Mb	Chr8_17683064	ACTTGCAACAACGATTGACCT	AAGCCACCAACCTTATTGCA
8_18.5Mb	Chr8_18507050	TGCTTTTCGCTTGGGCTAGA	GTTCCACTCTGCTACGGCTT
8_19.6Mb	Chr8_19583199	TAAACCCTTCAGTCCACGGC	ATGAGCTGGGGTATCGACCT
8_21.8Mb	Chr8_21807790	TCGCTCGCTTCCTGAAGTTT	GAAAAGCTCTGGCCAAACCG

表2 亲本水晶3号和拉木加垩白和籽粒粒型性状测定

Table 2. Measurement of chalkiness rate and grain shape traits of Shuijing 3 and Lamujia

性状 Trait	水晶3号 Shuijing 3	拉木加 Lamujia
垩白粒率 Chalky rice rate (%)	8.1±0.5	91.9±0.3
粒长Grain length (mm)	4.8±0.0	4.9±0.0
粒宽 Grain width (mm)	2.7±0.0	2.8±0.0
长宽比 Length-width ratio	1.8±0.0	1.7±0.0

图1 水晶3号和拉木加及其正反交F1在不同生态条件下的垩白粒率 A: 水晶3号和拉木加在海南三亚和河南原阳的垩白粒率表型；B: 正反交F1在海南三亚和河南原阳的垩白粒率。SLF1为水晶3号×拉木加正交F1，LSF1为拉木加×水晶3号反交F1。

Fig. 1. Chalky rice rates of Shuijing 3, Lamujia and their reciprocal cross F1 in two environments A, Chalky rice rate of Shuijing 3 and Lamujia in Sanya, Hainan Province and Yuanyang, Henan Province; B, Chalky rice rate of reciprocal cross F1 in Sanya and Yuanyang. SLF1 is Shuijing 3×Lamujia F1 and LSF1 is Lamujia×Shuijing 3 F1.

图2 海南和河南两地F2群体的垩白粒率频次分布 A：海南三亚F2群体；B：河南原阳F2群体。虚线分别表示高垩白混池和低垩白混池。

Fig. 2. Histogram of chalky rice rate distribution of Hainan and Henan F2 populations A, F2 population of Sanya, Hainan; B, F2 population of Yuanyang, Henan. The dashed line rectangles indicate high chalkiness and low chalkiness bulks.

图3 海南与河南F2群体垩白极端个体混池QTL-Seq测序ΔSNP指数分析 A: 海南F2群体垩白极端个体混池的ΔSNP指数，红线和蓝线分别代表95%、99%置信区间; B: 河南群体垩白极端表型个体混池的ΔSNP指数; C: 海南群体高垩白混池（蓝点）和低垩白混池（绿点）变异位点的SNP指数; D: 河南群体高垩白混池（蓝点）和低垩白混池（绿点）变异位点的SNP指数。

Fig. 3. ΔSNP-index analysis of extreme individuals bulks of Hainan and Henan F2 populations A, ΔSNP-index of extreme individuals bulks of Hainan F2 population, the red line indicates 95% confidence interval and the blue line indicates 99% confidence interval; B, ΔSNP-index of extreme individuals bulks of Henan F2 population; C, SNP-index of high chalkiness bulk (blue dots) and low chalkiness bulk (green) in Hainan population; D, SNP-index of high chalkiness bulk (blue dots) and low chalkiness bulk (green) in Henan population.

图4 8号染色体垩白粒率与分子标记遗传连锁分析

Fig. 4. Genetic linkage map of chalky rice rate and molecular markers on chromosome 8

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