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

doi:10.16819/j.1001-7216.2024.221203

Abstract

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

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

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.

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.

Figures/Tables 4

References 23

[1]	包劲松. 稻米淀粉品质遗传与改良研究进展[J]. 分子植物育种, 2007(6): 1-20.
	Bao J S. Progress on inheritance and improvement of rice starch quality[J]. Molecular Plant Breeding, 2007(6): 1-20. (in Chinese with English abstract)
[2]	高维维, 陈思平, 王丽平, 陈立凯, 郭涛, 陈志强. 稻米蒸煮品质性状与分子标记关联研究[J]. 中国农业科学, 2017, 50(4): 599-611.
	Gao W W, Chen S P, Wang L P, Chen L K, Guo T, Chen Z Q. Association analysis of rice cooking quality traits with molecular markers[J]. Scientia Agricultura Sinica, 2017, 50(4): 599-611. (in Chinese with English abstract)
[3]	Fan C C, Yu X Q, Xing Y Z, Xu C G, Luo L J, Zhang Q F. The main effects, epistatic effects and environmental interactions of QTLs on the cooking and eating quality of rice in a doubled-haploid line population[J]. Theoretical and Applied Genetics, 2005, 110(8): 1445-1452.
[4]	Bao J S, Sun M, Zhu L H, Corke H. Analysis of quantitative trait loci for some starch properties of rice: Thermal properties, gel texture and swelling volume[J]. Journal of Cereal Science, 2004, 39(3): 379-385.
[5]	Umemoto T, Yano M, Satoh H, Shomura A, Nakamura Y. Mapping of a gene responsible for the difference in amylopectin structure between japonica-type and indica-type rice varieties[J]. Theoretical and Applied Genetics, 2002, 104(1): 1-8.
[6]	Gao Z Y, Zeng D L, Cui X, Zhou Y H, Yan M X, Huang D N, Li J Y, Qian Q. Map-based cloning of the ALK gene, which controls the gelatinization temperature of rice[J]. Science in China: Series C, 2003, 46(6): 661-668.
[7]	Gao Z Y, Zeng D L, Cheng F M, Tian Z X, Guo L B, Su Y, Yan M X, Jiang H, Dong G J, Han B, Li J Y, Qian Q. ALK, the key gene for gelatinization temperature, is a modifier gene for gel consistency in rice[J]. Journal of Integrative Plant Biology, 2011, 53(9): 756-765.
[8]	Kharabian-Masouleh A, Waters D L E, Reinke R F, Ward R, Henry R J. SNP in starch biosynthesis genes associated with nutritional and functional properties of rice[J]. Scientific Reports, 2012, 2: 557.
[9]	Cuevas R P, Daygon V D, Corpuz H, Nora L, Reinke R, Waters D, Fitzgerald M. Melting the secrets of gelatinisation temperature in rice[J]. Functional Plant Biology, 2010, 37(5): 439-447.
[10]	Waters D L E, Henry R J, Reinke R F, Fitzgerald M A. Gelatinization temperature of rice explained by polymorphisms in starch synthase[J]. Plant Biotechnology Journal, 2006, 4(1): 115-122.
[11]	Umemoto T, Aoki N, Lin H, Nakamura Y, Inouchi N, Sato Y, Yano M, Hirabayashi H, Maruyama S. Natural variation in rice starch synthase IIa affects enzyme and starch properties[J]. Functional Plant Biology, 2004, 31(7): 671-684.
[12]	Bao J S, Corke H, Sun M. Nucleotide diversity in starch synthase IIa and validation of single nucleotide polymorphisms in relation to starch gelatinization temperature and other physicochemical properties in rice[J]. Theoretical and Applied Genetics, 2006, 113(7): 1171-1183.
[13]	Zhou Y, Zheng H Y, Wei G C, Zhou H, Han Y N, Bai X F, Xing Y Z, Han Y P. Nucleotide diversity and molecular evolution of the ALK gene in cultivated rice and its wild relatives[J]. Plant Molecular Biology Reporter, 2016, 34(5): 1-8.
[14]	Ririe K M, Rasmussen R P, Wittwer C T. Product differentiation by analysis of DNA melting curves during the polymerase chain reaction[J]. Analytical Biochemistry, 1997, 245(2): 154-160.
[15]	Lipsky R H, Mazzanti C M, Rudolph J G, Xu K, Vyas G, Bozak D, Radel M Q, Goldman D. DNA melting analysis for detection of single nucleotide polymorphisms[J]. Clinical Chemistry, 2001, 47(4): 635-644.
[16]	Gundry C N, van Dersteen J G, Reed G H, Pryor R J, Chen J, Wittwer C T. Amplicon melting analysis with labeled primers: A closed-tube method for differentiating homozygotes and heterozygotes[J]. Clinical Chemistry, 2003, 49(3): 396-406.
[17]	Koeyer D D, Douglass K, Murphy A, Whitney S, Nolan L, Song Y, Jong W D. Application of higher solution DNA melting for genotyping and variant scanning of diploid and autotetraploid potato[J]. Molecular Breeding, 2010, 25(1): 67-90.
[18]	Wittwer C T. High-resolution DNA melting analysis: Advancements and limitations[J]. Human Mutation, 2009, 30(6): 857-859.
[19]	赵均良, 张少红, 刘斌. 应用高分辨率熔解曲线技术分析水稻分子标记基因型[J]. 中国农业科学, 2011, 44(18): 3701-3708.
	Zhao J L, Zhang S H, Liu B. Application of high- resolution melting curve analysis for molecular marker genotyping in rice[J]. Scientia Agricultura Sinica, 2011, 44(18): 3701-3708. (in Chinese with English abstract)
[20]	陈竹锋, 金名捺, 刘金成, 李早霞, 唐晓艳. 基于HRM体系开发抗稻瘟病基因Pi2特异性分子标记[J]. 分子植物育种, 2017, 15(3): 938-943.
	Chen Z F, Jin M N, Liu J C, Li Z X, Tang X Y. Development of a HRM-based functional marker for rice blast resistance gene Pi2[J]. Molecular Plant Breeding, 2017, 15(3): 938-943. (in Chinese with English abstract)
[21]	Luo W L, Guo T, Yang Q Y, Wang H, Liu Y Z, Zhu X Y, Chen Z Q. Stacking of five favorable alleles for amylase content, fragrance and disease resistance into elite lines in rice (Oryza sativa) by using four HRM-based markers and a linked gel-based marker[J]. Molecular Breeding, 2014, 34(3): 805-815.
[22]	罗文龙, 郭涛, 周丹华, 陈海英, 王慧, 陈志强, 刘永柱. 利用基于HRM的功能标记分析水稻Wx和fgr的基因型[J]. 湖南农业大学学报, 2013, 39(6): 597-603.
	Luo W L, Guo T, Zhou D H, Chen H Y, Wang H, Chen Z Q, Liu Y Z. Analysis of rice genotypes rice Wx and fgr by HRM-based functional marker[J]. Journal of Hunan Agricultural University, 2013, 39(6): 597-603. (in Chinese with English abstract)
[23]	肖鹏, 邵雅芳, 包劲松. 稻米糊化温度的遗传与分子机理研究进展[J]. 中国农业科技导报, 2010, 12(1): 23-30.
	Xiao P, Shao Y F, Bao J S. Research progress on genetic and molecular mechanism of starch gelatinization temperature of rice grain[J]. Journal of Agricultural Science and Technology, 2010, 12(1): 23-30. (in Chinese with English abstract)

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

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