基于HRM技术开发水稻糊化温度基因ALK功能标记

doi:10.16819/j.1001-7216.2024.221203

中国水稻科学 ›› 2024, Vol. 38 ›› Issue (1): 106-110.DOI: 10.16819/j.1001-7216.2024.221203

• 实验技术 • 上一篇

基于HRM技术开发水稻糊化温度基因ALK功能标记

王军¹^,³, 周晶², 陶亚军¹^,³, 李文奇¹^,³, 朱建平¹^,³, 范方军¹^,³, 王芳权¹^,³, 许扬¹^,³, 陈智慧¹^,³, 蒋彦婕¹^,³, 李霞¹^,³, 杨杰¹^,³^,^*()

¹江苏省农业科学院粮食作物研究所 / 农业农村部淮河下游种质创制重点实验室，南京 210014
²扬州大学生物科学与技术学院，江苏扬州 225009
³扬州大学 / 江苏省粮食作物现代产业技术协同创新中心，江苏扬州 225009

收稿日期:2022-12-09 修回日期:2023-02-16 出版日期:2024-01-10 发布日期:2024-01-16
通讯作者: * email: yangjie168@aliyun.com
基金资助:
江苏省重点研发计划项目(BE2020339);江苏省种业振兴揭榜挂帅项目(JBGS[2021]008)

Development of HRM-based Functional Marker for Gelatinization Temperature Gene ALK in Rice

WANG Jun¹^,³, ZHOU Jing², TAO Yajun¹^,³, LI Wenqi¹^,³, ZHU Jianping¹^,³, FAN Fangjun¹^,³, WANG Fangquan¹^,³, XU Yang¹^,³, CHEN Zhihui¹^,³, JIANG Yanjie¹^,³, LI Xia¹^,³, YANG Jie¹^,³^,^*()

¹Institute of Food Crops, Jiangsu Academy of Agricultural Sciences / Key Laboratory of Germplasm Innovation in Downstream of Huaihe River, Ministry of Agriculture and Rural Affairs, Nanjing 210014, China
²College of Bioscience and Biotechnology, Yangzhou University, Yangzhou 225009, China
³Jiangsu Co-Innovation Center for Modern Production Technology of Grain Crops, Yangzhou University, Yangzhou 225009, China

Received:2022-12-09 Revised:2023-02-16 Online:2024-01-10 Published:2024-01-16
Contact: * email: yangjie168@aliyun.com

摘要/Abstract

摘要：

【目的】糊化温度是影响稻米蒸煮食味品质的重要指标，ALK是控制水稻糊化温度的主效基因，开发可快速高通量鉴定ALK基因型的功能标记有助于提高水稻品质改良的效率。【方法】根据ALK基因4198位、4329位和4330位存在的单碱基差异，设计基于HRM技术检测的基因功能标记。【结果】通过PCR检测结合测序分析，筛选了ALK基因2个功能区域的功能标记ALKH4、ALKH5。利用ALKH4、ALKH5对81份籼稻品种、279份粳稻品种进行了ALK基因型检测，结果发现，16份粳稻和19份籼稻为G-GC基因型；51份粳稻为A-GC基因型；212份粳稻和62份籼稻为G-TT基因型。【结论】基于HRM技术开发的基因功能标记ALKH4、ALKH5可以快速高通量鉴定控制糊化温度ALK不同基因型，具有重要的应用价值。

关键词: 水稻, 糊化温度, ALK, 功能标记, HRM技术

Abstract:

【Objective】 Gelatinization temperature is an important parameter in rice eating quality which is mainly controlled by the ALK gene. Fast and high-throughput gene functional markers for ALK can improve selection efficiency in quality improvement. 【Method】 According to single nucleotide polymorphism in the functional region of ALK alleles, we developed the gene functional marker based on high resolution melting (HRM) technology. 【Result】 Through PCR amplification and sequencing test, we selected gene markers ALKH4 and ALKH5 in two functional regions of ALK. ALKH4 and ALKH5 were employed to genotype 81 indica varieties and 279 japonica varieties. The results showed that only 16 japonica varieties and 19 indica varieties carrying G-GC genotype were identified as well as 51 japonica varieties carrying A-GC genotype; while 212 japonica varieties and 62 indica varieties fell into G-TT genotype. 【Conclusion】 The HRM-based functional markers ALKH4 and ALKH5 could distinguish different genotypes of gelatinization temperature ALK locus in a fast and high-throughput manner, and have significance in breeding practice.

Key words: rice, gelatinization temperature, ALK, functional marker, HRM technology

王军, 周晶, 陶亚军, 李文奇, 朱建平, 范方军, 王芳权, 许扬, 陈智慧, 蒋彦婕, 李霞, 杨杰. 基于HRM技术开发水稻糊化温度基因ALK功能标记[J]. 中国水稻科学, 2024, 38(1): 106-110.

WANG Jun, ZHOU Jing, TAO Yajun, LI Wenqi, ZHU Jianping, FAN Fangjun, WANG Fangquan, XU Yang, CHEN Zhihui, JIANG Yanjie, LI Xia, YANG Jie. Development of HRM-based Functional Marker for Gelatinization Temperature Gene ALK in Rice[J]. Chinese Journal OF Rice Science, 2024, 38(1): 106-110.

图/表 4

表1 基于序列差异设计的功能标记和测序引物

Table 1. Functional markers and sequencing primers designed based on nucleotide polymorphism.

引物名称 Primer name	引物序列 Primer sequence(5′-3′)	片段大小 Expected size /bp	扩增位点 Amplification site
ALKH-1F	TGGCGTACGGCACCGTCCC	104	A/G
ALKH-1R	GTCGAACGTCCACCCGAGG
ALKH-2F	GCCGCACAAGCTCATCGAG	104	TT/GC
ALKH-2R	TGAGGTCCTGCGACATGCC
ALKH-3F	TCGGCGGGCTGAGGGA	69	A/G
ALKH-3R	CGAACGTCCACCCGAGGC
ALKH-4F	CACCGTCCCCGTCGTG	73	A/G
ALKH-4R	GGTGTCCTCGAACGGGTC
ALKH-5F	CGTACCGCAAGTACAAGGAG	66	TT/GC
ALKH-5R	AGCTGAGGTCCTGCGACA
ALKH-6F	TCGAGACGTACCGCAAGTACAA	82	TT/GC
ALKH-6R	GCGTGGTCCCAGCTGAGG
ALKC-1F	GGTGGGGTTCTCGGTGAAG	687	测序 Sequencing
ALKC-1R	GCATCAATGGACATAACAAACAC

图1 12份水稻品种ALK基因HRM功能标记的多态性分析

Fig. 1. HRM polymorphism analysis of ALK functional markers amplification products of 12 rice varieties.

图2 24个F2分离群体的的ALK基因型鉴定

Fig. 2. ALK genotype identification in 24 F2 plants.

图3 部分水稻品种ALK基因功能位点序列 A~D分别为连粳18201、润扬稻1892、扬辐粳1号和F2单株。红色框示ALK基因4198位、4329位和4330位碱基。

Fig. 3. Sequence of ALK gene functional domains for partial rice varieties. A~D, Lianjing 18201, Runyangdao 1892, Yangfujing 1 and F2 plant, respectively. Red box indicates 4198 bp, 4329 bp and 4330 bp of ALK gene.

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基于HRM技术开发水稻糊化温度基因ALK功能标记

Development of HRM-based Functional Marker for Gelatinization Temperature Gene ALK in Rice

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