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Acta Agron Sin ›› 2017, Vol. 43 ›› Issue (11): 1715-1723.doi: 10.3724/SP.J.1006.2017.01715

• RESEARCH NOTES • Previous Articles     Next Articles

Development of PCR Functional Markers for Multiple Alleles of Wx and Their Application in Rice

MAO Ting1,2,LI Xu2,LI Zhen-Yu2,*,XU Zheng-Jin1,*   

  1. 1 Rice Research Institute, Shenyang Agricultural University, Shenyang 110866, China; 2 Liaoning Province Saline and Alkaline Land Utilization and Research Institute, Panjin 124010, China
  • Received:2017-02-03 Revised:2017-07-23 Online:2017-11-12 Published:2017-08-01
  • Contact: 李振宇, E-mail: lyskjb@126.com; 徐正进, E-mail: xuzhengjin@126.com E-mail:chinamaoting1985@163.com
  • Supported by:

    This study was supported by the Cultivation Plan for Youth Agricultural Science Technology Innovative Talents of Liaoning Province (2015035, 2015036) and the Natural Science Foundation of China (31371587, 31430062).

Abstract:

Amylose content (AC) is one of the most important factors impacting rice eating quality, which was mainly determined by Wx locus. Multiple alleles, such as Wxa, Wxb, Wxin, and Wxmw contribute to the variation of AC in rice varieties. Selecting rice varieties with low-medium AC through Marker-assisted selection (MAS) is an important way to improve rice eating quality. However, distinguishing the Single Nucleotide Polymorphisms (SNP) of Wx alleles requires the DNA sequencing or restriction enzyme digestion, which is time consuming and labor-intensive. In this study, we developed effective MAS markers by tetra-primer ARMS-PCR technology utilizing functional SNPs corresponding to the different alleles, moreover we put forward corresponding solving schemes on low amplification efficiency and inaccuracy extension through adjusting the location of deliberate mismatch bases introduced. We easily differentiated the multiple alleles of Wx utilizing the markers Waxygt-ARMS2 and Waxyac-ARMS2, showing that these markers have significant application value with the features of rapid operation and low cost. We further analyzed the genotype distribution of Wx allele in Liaoning Province rice varieties, and found all of the forty tested varieties carried the Wxb allele with a narrow genetic diversity. The results of this study could provide theoretical basis and technical support on both new rice varieties breeding and rice eating quality improvement utilizing Wx alleles corresponding to low-medium AC by MAS in Liaoning Province.

Key words: Rice, Wx, Tetra-primer ARMS-PCR, Amylose content, Rice eating quality improvement

[1] Li Y B, Fan C C, Xing Y Z, Yun P, Luo L J, Yan B, Peng B, Xie W B, Wang G W, Li X H, Xiao J H, Xu C G, He Y Q. Chalk5 encodes a vacuolar H+-translocating pyrophosphatase influencing grain chalkiness in rice. Nat Genet, 2014, 46: 398–404
[2] Zeng D L, Tian Z X, Rao Y C, Dong G J, Yang Y L, Huang L C, Leng Y J, Xu J, Sun C, Zhang G H, Hu J, Zhu L, Gao Z Y, Hu X M, Guo L B, Xiong G S, Wang Y H, Li J Y, Qian Q. Rational design of high-yield and superior-quality rice. Nat Plants, 2017, 3: 17031
[3] 吴长明, 孙传清, 付秀林, 王象坤, 李自超, 张强. 稻米品质性状与产量性状及籼粳分化度的相互关系研究. 作物学报, 2003, 29: 822–828
Wu C M, Sun C Q, Fu X L, Wang X K, Li Z C, Zhang Q. Study on the relationship between quality, yield characters or indica-japonica differentiation in rice (Oryza sativa L.). Acta Agron Sin, 2003, 29: 822–828 (in Chinese with English abstract)
[4] 王才林, 陈涛, 张亚东, 朱镇, 赵凌, 林静. 通过分子标记辅助选择培育优良食味水稻新品种. 中国水稻科学, 2009, 23: 25–30
Wang C L, Chen T, Zhang Y D, Zhu Z, Zhao L, Lin J. Breeding of a new rice variety with good eating quality by marker assisted selection. Chin J Rice Sci, 2009, 23: 25–30 (in Chinese with English abstract)
[5] 隋炯明, 李欣, 严松, 严长杰, 张蓉, 汤述翥, 陆驹飞, 陈宗祥, 顾铭洪. 稻米淀粉 RVA 谱特征与品质性状相关性研究. 中国农业科学, 2005, 38: 657–663
Sui J M, Li X, Yan S, Yan C J, Zhang R, Tang S Z, Lu J F, Chen Z X, Gu M H. Studies on the rice RVA profile characteristics and its correlation with the quality. Sci Agric Sin, 2005, 38: 657–663 (in Chinese with English abstract)
[6] Sano Y. Differential regulation of waxy gene expression in rice endosperm. Theor Appl Genet, 1984, 68: 467–473
[7] Ayres N M, McClung A M, Larkin P D, Bligh H F J, Jones C A, Park W D. Microsatellites and a single-nucleotide polymorphism differentiate apparent amylose classes in an extended pedigree of US rice germ plasm. Theor Appl Genet, 1997, 94: 773–781
[8] Mikami I, Aikawa M, Hirano H Y, Sano Y. Altered tissue-specific expression at the Wx gene of the opaque mutants in rice. Euphytica, 1999, 105: 91–97
[9] Wang Z Y, Zheng F Q, Shen G Z, Gao J P, Snustad D P, Li M G, Zhang J L, Hong M M. The amylose content in rice endosperm is related to the post-transcriptional regulation of the waxy gene. Plant J, 1995, 7: 613–622
[10] Yamanaka S, Nakamura I, Watanabe K N, Sato Y I. Identification of SNPs in the waxy gene among glutinous rice cultivars and their evolutionary significance during the domestication process of rice. Theor Appl Genet, 2004, 108: 1200–1204
[11] 朱霁晖, 张昌泉, 顾铭洪, 刘巧泉. 水稻Wx基因的等位变异及育种利用研究进展. 中国水稻科学, 2015, 29: 431–438
Zhu J H, Zhang C Q, Gu M H, Liu Q Q. Progress in the allelic variation of Wx gene and its application in rice breeding. Chin J Rice Sci, 2015, 29: 431–438 (in Chinese with English abstract)
[12] Mikami I, Uwatoko N, Ikeda Y, Yamaguchi J, Hirano H Y, Suzuki Y, Sano Y. Allelic diversification at the wx locus in landraces of Asian rice. Theor Appl Genet, 2008, 116: 979–989
[13] 倪晖. 水稻Wx基因不同等位变异的效应及其育种应用的初步研究. 扬州大学硕士学位论文, 江苏扬州, 2013
Ni H. A Preliminary Analysis of the Effect of different Wx Alleles on Rice Grain Quality. MS Thesis of Yangzhou University, Yangzhou, China, 2013 (in Chinese with English abstract)
[14] 刘巧泉, 蔡秀玲, 李钱锋, 汤述翥, 龚志云, 于恒秀, 严长杰, 王宗阳, 顾铭洪. 分子标记辅助选择改良特青及其杂交稻米的蒸煮与食味品质. 作物学报, 2006, 32: 64–69
Liu Q Q, Cai X L, Li Q F, Tang S Z, Gong Z Y, Yu H X, Yan C J, Wang Z Y, Gu M H. Molecular marker-assisted selection for improving cooking and eating quality in Teqing and its hybrid rice. Acta Agron Sin, 2006, 32: 64–69 (in Chinese with English abstract)
[15] 蔡秀玲, 刘巧泉, 汤述翥, 顾铭洪, 王宗阳. 用于筛选直链淀粉含量为中等的籼稻品种的分子标记. 植物生理与分子生物学报, 2002, 28: 137–144
Cai X L, Liu Q Q, Tang S Z, Gu M H, Wang Z Y. Development of a molecular marker for screening the rice cultivars with intermediate amylose content in Oryza sativa subsp. indica. J Plant Physiol Mol Biol, 2002, 28: 137–144 (in Chinese with English abstract)
[16] 姚姝, 陈涛, 张亚东, 朱镇, 赵凌, 赵庆勇, 周丽慧, 王才林. 分子标记辅助选择聚合水稻暗胚乳突变基因Wx-mq和抗条纹叶枯病基因Stv-bi. 中国水稻科学, 2010, 24: 341–347
Yao S, Chen T, Zhang Y D, Zhu Z, Zhao L, Zhao Q Y, Zhou L H, Wang C L. Pyramiding of translucent endosperm mutant gene Wx-mq and rice stripe disease resistance gene Stv-bi by marker-assisted selection in rice (Oryza sativa). Chin J Rice Sci, 2010, 24: 341–347 (in Chinese with English abstract)
[17] 田志喜, 严长杰, 钱前, 严松, 谢会兰, 王芳, 徐洁芬, 刘贵富, 王永红, 刘巧泉, 汤述翥, 李家洋, 顾铭洪. 水稻淀粉合成相关基因分子标记的建立. 科学通报, 2010, 55: 2591–2601
Tian Z X, Yan C J, Qian Q, Yan S, Xie H L, Wang F, Xu J F, Liu F G, Wang Y H, Liu Q Q, Tang S Z, Li J Y, Gu M H. Development of gene-tagged molecular markers for starch synthesis-related genes in rice. Chin Sci Bull, 2010, 55: 2591–2601 (in Chinese)
[18] 刘兴顺, 程牛亮, 梁小波, 赵彩虹. 四引物扩增受阻突变体系聚合酶链反应在SNP基因分型中的研究. 山西医科大学学报, 2008, 39: 483–485
Liu X S, Cheng N L, Liang X B, Zhao C H. Study on tetra-primer amplification refractory mutation system-polymerase chain reaction in SNP genotyping. J Shanxi Med Univ, 2008, 39: 483–485 (in Chinese with English abstract)
[19] Ye S, Dhillon S, Ke X, Collins A R, Day I N. An efficient procedure for genotyping single nucleotide polymorphisms. Nucleic Acids Res, 29: E88
[20] 陈涛, 骆名瑞, 张亚东, 朱镇, 赵凌, 赵庆勇, 周丽慧, 姚姝, 于新, 王才林. 利用四引物扩增受阻突变体系PCR技术检测水稻低直链淀粉含量基因Wx-mq. 中国水稻科学, 2013, 27: 529–534
Chen T, Luo M R, Zhang Y D, Zhu Z, Zhao L, Zhao Q Y, Zhou L H, Yao S, Yu X, Wang C L. Detection of Wx-mq gene for low-amylose content by tetra-primer amplification refractory mutation system PCR in rice. Chin J Rice Sci, 2013, 27: 529–534 (in Chinese with English abstract)
[21] Kim S R, Ramos J, Ashikari M, Virk P S, Torres E A, Nissila E, Hechanova S L, Mauleon R, Jena K K. Development and validation of allele-specific SNP/indel markers for eight yield-enhancing genes using whole-genome sequencing strategy to increase yield potential of rice, Oryza sativa L. Rice, 2016, 9: 12
[22] Andersen J R, Lübberstedt T. Functional markers in plants. Trends Plant Sci, 2003, 11: 554–560
[23] Hu B, Wang W, Ou S J, Tang J Y, Li H, Che R H, Zhang Z H, Chai X Y, Wang H R, Wang Y Q, Liang C Z, Liu L C, Piao Z Z, Deng Q Y, Deng K, Xu C, Liang Y, Zhang L H, Li L G, Chu C C. Variation in NRT1.1B contributes to nitrate-use divergence between rice subspecies. Nat Genet, 2015, 47: 834–838
[24] 潘存红, 王子斌, 马玉银, 殷跃军, 张亚芳, 左示敏, 陈宗祥, 潘学彪. InDel和SNP标记在水稻图位克隆中的应用. 中国水稻科学, 2007, 21: 447–453
Pan C H, Wang Z B, Ma Y Y, Yin Y J, Zhang Y F, Zuo S M, Chen Z X, Pan X B. InDel and SNP markers and their application in map-based cloning of rice genes. Chin J Rice Sci, 2007, 21: 447–453 (in Chinese with English abstract)
[25] Sun J, Liu D, Wang J Y, Ma D R, Tang L, Gao H, Xu Z J, Chen W F. The contribution of intersubspecific hybridization to the breeding of super-high-yielding japonica rice in northeast China. Theor Appl Genet, 2012, 125: 1149–1157
[26] 陈温福, 徐正进, 张龙步, 张文忠, 马殿荣. 北方粳型稻超高产育种理论与实践. 中国农业科学, 2007, 40: 869–874
Chen W F, Xu Z J, Zhang L B, Zhang W Z, Ma D R. Theories and practices of breeding japonica rice for super high yield. Sci Agric Sin, 2007, 40: 869–874 (in Chinese with English abstract)

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