Developing and Validating the Functional Marker of Rice Waxy Gene, M-Wx 
Lijun Gao1,2 
,
Meng Zhou1,2 ,
Rentian Chen1 ,
Hanliang Gao1 ,
Qun Yan1 ,
Weiyong Zhou2 ,
Guofu Deng1,2 
,
Meng Zhou1,2 ,
Rentian Chen1 ,
Hanliang Gao1 ,
Qun Yan1 ,
Weiyong Zhou2 ,
Guofu Deng1,2
1. Guangxi Academy of Agricultural Sciences, Nanning, 530007, P.R. China
2. Guangxi Key Lab for Crop Improvement and Biotechnology, Nanning, 530007, P.R. China
Author Correspondence author
Rice Genomics and Genetics, 2012, Vol. 3, No. 10 doi: 10.5376/rgg.2012.03.0010
Received: 15 Aug., 2012 Accepted: 19 Oct., 2012 Published: 25 Oct., 2012
2. Guangxi Key Lab for Crop Improvement and Biotechnology, Nanning, 530007, P.R. China
Author Correspondence author
Rice Genomics and Genetics, 2012, Vol. 3, No. 10 doi: 10.5376/rgg.2012.03.0010
Received: 15 Aug., 2012 Accepted: 19 Oct., 2012 Published: 25 Oct., 2012
© 2012 BioPublisher Publishing Platform
This is an open access article published under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Preferred citation for this article:
Gao et al., 2012, Developing and Validating the Functional Marker of Rice Waxy Gene, M-Wx, Vol.3, No.10 61-65 (doi: 10.5376/rgg.2012.03.0010)
Abstract
It would be an important significance of developing molecular markers that could effectively track rice amylose content in the breeding program of high-quality rice varieties. In this study the dominant functional marker M-Wx was developed based on the SNP difference that can cause Wx gene function changes. The effectiveness of the functional marker M-Wx was validated by detecting the rice genotypes of 72 parents and amylose contents. The results showed that 228 bp and 425 bp bands were detected in 43 of 72 parents, genotype assigning to TT, which the amylose content were less than 15.6%, whereas the remaining 29 parents only appeared a 425 bp control band, genotype assigning to GG, which the amylose content were much more than 20.1%. The results of this study indicated the marker M-Wx should accurately detect the level of the amylose content in rice, which and can be applied to improve varieties and enhance rice quality in marker-assisted selection.
Keywords
Rice; Waxy gene; Amylose; Functional marker
Waxy gene (waxy, Wx) is a key gene coding amylose synthase that was successfully cloned in 1990 (Wang et al., 1990), which encode starch granule-bound starch synthase to control the synthesis of amylose in rice endosperm.
In recent years, studies have shown that the level of amylose content in different varieties was determined by the splicing efficiency of the first intron in Wx gene (Wang, 1995), however, the splicing efficiency of the first intron was regulated by which the intron+1 nucleotide is the wild type G or mutant type T (Cai et al., 1998).
Based on the above principle, Cai et al (2002) invented a PCR-AccI molecular markers of the Wxgene, which the marker-genotypes amplified by PCR after digestion with AccI enzyme and electrophoresis with 2% agarose gel presented a 403 bp band for the GG genotype and 460 bp band for the TT genotype; whereas the GT heterozygous genotype presented two bands of 403 bp and 460 bp in length. The 63 rice parental cultivars were detected by using the marker, the results showed that the amylose content of rice varieties with GG genotype were about between 20% and 32%, while the amylose content of rice varieties with TT genotype were less than 18%.
Subsequently, Mao et al (2004) developed the specific amplification of one-step PCR dominant marker that can amplify the first intron plus G base in normal, however, this marker required high annealing temperature in the PCR amplification. If the annealing temperature was increased to 67℃, the amplification specificity cold be increased. Therefore, the drawback was strict requirement that would result in failure of PCR amplification causing G nucleotide undetectable.
Amylose content is an important factor to affect the cooking and eating quality. Therefore, it has an important significance of developing effective Wx markers, but the above two functional markers had obvious disadvantages, one required the digestion enzymes resulting in higher costs and complicated operation, which was not conducive to high-throughput molecular marker-assisted breeding; while another marker was the dominant marker designed based on SNP, but the annealing temperature is too high to be adjusted. If the annealing temperature tends to a little low, either G or T in the locus could be amplified, whereas the annealing temperature tends to high, it could be difficult to amplify the target band.
In consideration of the above problems, we attempted to design of a functional marker with a positive control, M-WX, based on the SNP. The designed marker can be normally amplified at 55℃ of the annealing temperature, and the base difference can be detected by electrophoresis in 1.2% agarose gel. Through detecting the relationship between amylose content and genotypes in 72 rice parental germplasms, the effectiveness of the functional marker M-Wx was validated, which might provide some theoretical basis and practical guidance for applying to amylose breeding.
1 Results and analysis
1.1 Primer design
Since single nucleotide mutation can cause the functional change of Waxy genes, the WXT was designed based on the difference position of G-T that can specifically amplify the mutant T, and a fault base match was introduced at the fourth position of the 3' end together in order to strengthen specificity. The forward primer WxF and reverse prime WxR were designed for the upstream and downstream, respectively (Table 1). While the genotype TT or GT exist in rice DNA template, PCR amplification will generate two bands of 228 bp and 428 bp in length, whereas in the GG genotype, only 425 bp band appears as the same as the reference.
1.2 Correlation between the functional marker M-Wx detection and genotype amylose content
In order to validate the correlation between the functional marker M-Wx detection and genotype amylose content, we selected 72 rice parental germpl- asms that are genetically stable and homozygous Wx gene. By using the marker M-Wx, the results found that the 72 materials were amplified their PCR product with a rate of 100% and distinguishable bands in 1.2% agarose gel electrophoresis (Figure 1 and Figure 2). The 43 ​​of 72 rice parental materials generated two bands of 228 bp and 425 bp in length, genotype was assigned to TT, which the amylose content were less than 15.3% with the average of 12.5%​​; while 29 of 72 rice parental materials only generated a band of 425 bp in length, genotype was assigned to GG, which the amylose content were more than 20.7% with the average of 22.3% (Table 2).
The results showed that the genotype detected with M-Wx marker should be exactly in line with the phenotype of amylose content in 72 rice parental materials. 42 of non-glutinous rice parental materials with TT genotype detected with M-Wx contain 9.1%~15.3% of rice amylose content, while 29 of rice parental materials with GG genotype detected with M-Wx contain 20.1%~25.6% of rice amylose content.
Since M-Wx can effective track a SNP mutation with G-> T that occurs in the slicing site in 5' end of the first intron in waxy gene, thus M-Wx marker becomes an effective functional markers. The testing results revealed that the rice waxy gene Wx tremendous impact on amylose content, the materials with TT genotype average less 9.8% of amylose content than that with GG genotype.
Since single nucleotide mutation can cause the functional change of Waxy genes, the WXT was designed based on the difference position of G-T that can specifically amplify the mutant T, and a fault base match was introduced at the fourth position of the 3' end together in order to strengthen specificity. The forward primer WxF and reverse prime WxR were designed for the upstream and downstream, respectively (Table 1). While the genotype TT or GT exist in rice DNA template, PCR amplification will generate two bands of 228 bp and 428 bp in length, whereas in the GG genotype, only 425 bp band appears as the same as the reference.
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1.2 Correlation between the functional marker M-Wx detection and genotype amylose content
In order to validate the correlation between the functional marker M-Wx detection and genotype amylose content, we selected 72 rice parental germpl- asms that are genetically stable and homozygous Wx gene. By using the marker M-Wx, the results found that the 72 materials were amplified their PCR product with a rate of 100% and distinguishable bands in 1.2% agarose gel electrophoresis (Figure 1 and Figure 2). The 43 ​​of 72 rice parental materials generated two bands of 228 bp and 425 bp in length, genotype was assigned to TT, which the amylose content were less than 15.3% with the average of 12.5%​​; while 29 of 72 rice parental materials only generated a band of 425 bp in length, genotype was assigned to GG, which the amylose content were more than 20.7% with the average of 22.3% (Table 2).
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The results showed that the genotype detected with M-Wx marker should be exactly in line with the phenotype of amylose content in 72 rice parental materials. 42 of non-glutinous rice parental materials with TT genotype detected with M-Wx contain 9.1%~15.3% of rice amylose content, while 29 of rice parental materials with GG genotype detected with M-Wx contain 20.1%~25.6% of rice amylose content.
2 Discussions
The present study used functional marker M-Wx developed based on the waxy gene to 72 rice materials, which can generate clear amplified bands in the regular PCR conditions by a 1.2% agarose gel electrophoresis that can accurately identify the parents through the GG genotype or TT genotype. Compared to previous published two functional markers, this new marker would be easy to operate and low cost. It should be suitable to detect large batch of materials in rice breeding program.
So far, in the parents of the three-line hybrid rice cultivars, the maintainer line of the three-line hybrid rice has high amylose content, which would be one of the major factors led to poor quality of three-line hybrid rice. Using the maintainer line with genotype TT of the waxy gene Wx such as Yixiang B and Yue-feng B as materials can be applied to develop low amylose maintainer line by means of the marker M-Wx.
Our research group applied this functional marker M-Wx in breeding practices for three-line hybrid rice, obtaining a number of low-amylose CMS, one of them was named 62A that combined the hybrid combination with excellent performance. The hybrid combination, 60A/Fuhui 838 was passed in the provincial selecting trial of the Guangxi Region, permitting the combi- nation enter re-trial (unpublished).
In consideration of the breeding practices in our research group, M-Wx marker has high rate to make the test samples generate clear bands, which should meet the requirements of the large quantities in rice breeding detection.
3 Materials and methods
3.1 Materials used in this research
3.1 Materials used in this research
The 72 rice parent materials with genetic stability popular used in Guangxi province were employed as the experimental materials (Table 2).
3.2 Determination of amylose content in rice parents
The tested materials as early rice planted in the experimental field of Guangxi Academy of Agricultural Sciences in 2010, and harvested at the stage of maturity. The content of rice amylose was examined according to National standards (GB/T15683-1995) in the Guangxi Key Laboratory of Crop Genetic Improvement Biotechnology.
3.3 Rice genomic DNA extraction
The leaves of 72 rice parent materials were sampled in 20 days later after transplanting, and placed in -20℃ refrigerator. The genomic DNA was extracted by the CTAB method of Murray et al (1980).
3.4 PCR amplification
PCR reaction system in the 10 uL volume containing 1.0 uL 10× Buffer, 0.2 uL dNTP, 0.5 uL 4 mol/L each three primers , 0.1 uL Taq polymerase, 1.0 uL template DNA, adding ddH2O up to 10 uL. The configuration of the reaction system placed on ice until the PCR reaction temperature rose to 94℃, then transferring PCR plate in PCR machine for amplification in order to reduce the formation of primer dimers. PCR procedures started a cycle for pre-denaturation 5 min at 94℃, then carried out 35 cycles following up 94℃ for 30 s, 55℃ for 30 s, and 72℃ for 45 s and last cycle for extension at 72℃ for 10 min. PCR product was identified in a 1.2% agarose gel electrophoresis, stained with ethidium bromide, and observed and took photos under ultraviolet light.
Cai X.L., Liu Q.Q., Tang S.Z., Cu M.H., Wang Z.Y., 2002, Development of molecular marker for screening the rice cultivars with intermediate amylose content in Oryza sativa subsp. indica, Plant Physiology and Molecular Biology, 28(2): 137-144
Acknowledgement
This project was jointly supported by the special project of Guangxi “Bagui scholars”; The National Science & Technology Pillar Program during the 12th Five-year Plan Period (2012AA- 101201-2); International S&T Cooperation Program of China (2012DF131220); Guangxi Scientific and technological project during the 12th Five-year Plan Period (20100005-2); Guangxi Natural Science Found (2010GXNSFA013085); Guangxi Academy of Agricultural Sciences Found (201013).
ReferencesCai X.L., Liu Q.Q., Tang S.Z., Cu M.H., Wang Z.Y., 2002, Development of molecular marker for screening the rice cultivars with intermediate amylose content in Oryza sativa subsp. indica, Plant Physiology and Molecular Biology, 28(2): 137-144
Cai X.L., Wang Z.Y., Xing Y.Y., Xing Y.Y., Zhang J.L., Hong M.M., 1998, Aberrant splicing of intron 1 leads to the heterogeneous 5' UTR and decreased expression of waxy gene in rice cuLtivars of intermediate amylose content. Plant Journal, 14(4): 459-46
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Wang Z.Y., Zheng F.Q., Shen G.Z., Gao J.P., Snustad P.S., Li M.G., zhang J.L., Hong M.H., 1995, The amylose content in rice endosperm is related to the post-transcriptional reguLation of the waxy gene, The Plant Journal, 7(4): 613-622
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