Annals of the Sri Lanka Department of Agriculture. 2008.10:01-08.
USE OF dCAPS MARKERS IN GRAIN QUALITY IMPROVEMENT OF INDICA RICE VARIETIES A.S.M.T. ABAYAWICKRAMA1 and TOYOAKI ANAI2 1 Rice Research and Development Institute, Batalagoda, Ibbagamuwa, Sri Lanka 2 Laboratory of Genetics and Biotechnology, Faculty of Agriculture, Saga University, Japan
ABSTRACT The waxy (Wx) gene is known to encode the Granule-Bond Starch Synthase (GBSS), responsible for the synthesis of amylose in the rice endosperm. In cultivated rice, two wild type alleles, Wxa and Wxb are known. The Wxa allele is predominant in non-waxy Indica cultivars, whereas the Wxb allele is common in the nonwaxy Japonica cultivars. Protein level of Wxa is 10 fold higher than that of Wxb. The low level expression of Wxb results from the inefficient splicing of intron-1 present in the untranslated leader region due to a G to T mutation at the 5` splice site. With the objective of identifying molecular marker of Wx gene, hybridization was performed between Indica varieties, Tetep and K-16 with Japonica variety Reihou. Then molecular differentiation of Wx gene of these hybrid parents, F 1 and F2 generations were determined by derived Cleaved Amplified Polymorphic Sequence (dCAPS) marker. A mismatch primer was used to generate a restriction site in the Wxa allele (AGGT) but not in the Wxb allele (AGTT) with the restriction enzyme, Sty1, that recognizes the 5`CCAAGG 3â€™ as the cleavage sequence. Strains having the AGGT sequence (Indica cultivar) digested into two fragments (133 bp and 29 bp) and displayed a 133 bp fragment in the polyacrylamide gel, while strain carrying AGTT (Japonica cultivar) represented by a longer undigested fragment (162 bp). dCAPS analysis could also detect heterozygotes that would not be revealed by sequencing analysis. F 2 plants having homozygous Wxb allele are desired in backcross breeding programs to improve the grain quality of Indica rice. KEYWORDS: Amylose content, dCAPS marker, Indica rice, waxy gene.
INTRODUCTION Rice (Oryza sativa L.) is an important food for more than half of the worldâ€™s population and is a model plant for other cereal species (Terada et al., 2002). Rice accumulates large amount of starch in the endosperm of the grain and the major components of the starch are amylose and amylopectin. The ratio of amylose to total starch, measured as the amylose content, varies from cultivar to cultivar. Amylose is the most important grain constituent that influences the rice end-use quality-eating and cooking. Therefore, control of the amylose content of starch is a major objective in rice breeding (Itoh et al., 2003). Based on amylose content of milled rice, germplasm is commonly categorized into several amylose classes or quality types: waxy (0-2%), very low amylose (3-9%), low amylose (10-19%), intermediate amylose (20-24%) and high amylose (>24%) (Bergman et al., 2001). High level of amylose is associated with dry, fluffy rice that separate when cooked, which leads to poor
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grain quality. In contrast, low-amylose rice is moist and sticky. Intermediate amylose rice is preferred in most rice-growing areas of the world, except where low-amylose Japonica varieties are grown (Lang and Buu, 2004). The waxy (Wx) gene is known to encode the Granule-Bond Starch Synthase (GBSS), responsible for the synthesis of amylose in the rice endosperm (Fig. 1). This gene is present in cereals and expressed in endosperm, pollen and embryo sac. In cultivated rice, two wild type alleles, Wxa and Wxb are known. The Wxa allele is predominant in non-waxy Indica cultivars, whereas the Wxb allele is common in the non-waxy Japonica cultivars. Protein level of Wxa is 10 fold higher than that of Wxb. The low level expression of Wxb was shown to result from the inefficient splicing of intron-1 present in the untranslated leader region due to a G to T mutation at the 5` splice site (Isshiki et al., 1998). Excision of intron-1 from the Wx transcript is highly related to the control of the amylose content in the rice endosperm. Cytosol
Sucrose Sucrose synthase
UDP-glucose pyrophosphorylase Glucose 1-P
APD Glucose pyrophosphorylase ADP Glucose
Figure 1. Diagrammatic representation of the biosynthesis pathway of starch in rice endosperm (Myers et al., 2000). GBSS = Granule-Bond Starch Synthase * = starch synthase branching and de-branching enzymes
Since the amylose content affects the quality of rice grains, studies of Wx locus are not only of interest in understanding gene regulation but also they are important for agricultural applications (Hirano and Sano, 1991). DNA polymorphism is the basis to develop molecular markers that are widely used in gene mapping and are allowed to select particular characters in
USE OF dCAPS MARKERS IN INDICA RICE 3
plant improvement programme (Bao et al., 2006). It is important to develop molecular marker technologies that can be directly used in breeding programmes. With the objective of identifying molecular marker of Wx gene, hybridization was performed between Indica varieties, Tetep and K-16 with Japonica variety Reihou. Molecular differentiation of Wx gene (Wxa and Wxb) of these hybrid parents, F 1 and F2 generations was determined by derived Cleaved Amplified Polymorphic Sequence (dCAPS) marker. The detection of this one-base splicing mutation is important in identifying elite lines with desired genotype in grain quality improvement programs, without relying on laborious sequencing procedures (Yamanaka et al., 2004 and Sato and Nishio, 2003). MATERIALS AND METHODS Hybridization Hybridization was carried out between Indica varieties Tetep and K-16, and Japonica variety Reihou. Reihou was emasculated and used as the female parent. Pollination was done for 2-3 consecutive days to ensure successful pollination. Emasculated flowers were covered with paper bags to avoid crossings by contaminated pollen. F1 seeds were harvested and advanced into F2 generation. DNA sequencing In order to determine the sequence alignment of interest, PCR amplification and sequence analysis were performed with the primer set of F1 (5`CGC TTC TCT TCT CTC TCC CGT CCC GT3`) and R1 (5`TGG CGA GAG ACA TGA TTT AAC GAG AG3`) which amplifies the fragment containing the first exon-intron junction of the waxy gene. Amplified DNA fragment was electrophoresed on 1.5% agarose gel (Sigma), with 5x gel loading buffer. The DNA fragment of the size about 600 bp was cut from the gel and the DNA was extracted from the gel using Gel Extraction Kit-500 (QIAEX II) following the manufactures` protocol with slight alterations. The purified DNA fragment was cloned into pGEM-T Easy vector system using the ‘original TA cloning kit` from Promega. The vector (pGEM-T Easy) containing the inserted DNA fragment was then transformed into the laboratory strain of Escherichia coli, XL-10 Gold. The transformed E.coli was spread on LB agar plates containing 50 mg/µl carbenicillin, 0.4 mg/l Xgal (5-bromo-4-chloroindoly1-ß-galactoside) and 200 mM IPTG (isopropyl ßD-thiogalactopyranoside). The plates were incubated at 37°C overnight, and positive colonies were selected and confirmed for the presence of insertion by performing PCR with the same set of primers. Positive colonies were grown
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in 5 ml LB medium and plasmids were extracted and purified using the FlexiPrepTM kit (Amersham Biosciences) according to the manufactures` protocol with slight modifications. The concentration of purified plasmids were adjusted to 200 ng/µl and used for the sequencing. For the DNA sequencing experiment, samples were prepared using ABI PRISM® BigDyeTM Terminator V3.1 Ready Reaction Cycle Sequencing Kit (Applied Biosystems). For each reaction 3 µl of cloned plasmid (200 ng/µl), 1.3 µl of 1x Ready Reaction mix, 1.35 µl of 5x sequencing buffer and 1 µl of 1 µM sequencing primers (M13 or RV-P) were used and the reaction volume was adjusted to 10 µl by adding sterile MilliQ H2O. The PCR amplification was done using a program of 30 sec at 95 °C for one cycle, 15 sec. at 95°C and 4 min. at 60°C for 30 cycles. Amplified product was purified using 125 mM EDTA 5 µl and 95% ethanol 65 µl in a reaction mixture of 100 µl. Then the sample was centrifuged at 15000 rpm for 5 min. and DNA pellet was washed with 70% ethanol. 30 µl of 5x sequencing buffer (BigDye Terminator) was added to each sample. Before adding samples to the sequencing analyzer, DNA was denatured at 95°C for 5 min. dCAPS marker Molecular differentiation of Wxa and Wxb was identified using dCAPS marker. DNA was extracted from fresh leaves of Indica varieties Tetep and K-16, and Japonica variety Reihou, and five F1 plants, crossed between Indica and Japonica. To determine the inheritance pattern of Wx gene, F2 segregating population were screened by the dCAPS marker. In order to determine the one-base substitution by dCAPS analysis, PCR primer set F1 (5`TGT TGT TCA TCA GGA AGA ACA TCT CCA AG3`) and R1 (5`TTA ATT TCC AGC CCA ACA CC3`) were used. This will generate a cleavage site for Sty1, especially in the Wxa at the first exon-intron junction of the waxy gene. PCR amplification was performed using 1ng of purified DNA, 2 mM dNTPs, 1 µl, PCR buffer, 1 µM of each primer and 1.25U Taq polymerase. A total reaction of 35 cycles was programmed for 30 sec. at 95°C, 30 sec. at 60°C and 1 min. at 72°C in a BIORAD DNA thermal cycler. Further, the above PCR product was digested with Sty1 endonuclease at 37°C for overnight. After digestion, each digest was electrophoresed in a 15% polyacrylamid gel. RESULTS Since a single-base substitution at the donor site of the first intron of the Wx gene present, a mismatch primer was used to generate a
USE OF dCAPS MARKERS IN INDICA RICE 5
restriction site in the Wxa allele (AGGT) but not in the Wxb allele (AGTT). Restriction enzyme, Sty1 identifies the restriction site of CCAAGG which is only present in Wxa amplified sequence. Wxa – GAAGAACATCTGCAAG* GTATACAT (AGGT) Wxb – GAAGAACATCTGCAAG* TTATACAT (AGTT) (*=5` splice site of intron-1) Strains having the AGGT sequence (Wxa) digested into two fragments (133 bp and 29 bp) and displayed a 133 bp fragment in the polyacrylamide gel, while strain carrying AGTT (Wxb) was represented by a longer undigested fragment (162 bp). M
Figure 2. Molecular differentiation of Wxa and Wxb alleles by dCAPS analysis. M=100 bp ladder, 1-4=different F1s (crossed between Tetep and Reihou), 5=F1 (crossed between K-16 and Reihou), T=Tetep and R=Reihou.
dCAPS analysis could also detect heterozygotes that would not be revealed by sequencing analysis. Figure 2 shows the heterozygocity of Wx gene in different F1s (lane 1-5) crossed between Indica and Japonica varieties. Presence of both Wxa and Wxb alleles in each F1 plant was indicated by two bands, where the cleaved Wxa allele was at the lower position while the uncleaved Wxb allele at the upper positions. The results obtained from sequencing analysis showed single-base substitution at the first intron-exon junction of Wx allele in Indica and Japonica varieties (homozygous varieties), but not in their respective F1s (Table 1). Table 1. Sequencing analysis of first exon-intron junction of the waxy gene. Strain
1. 2. 3.
(F1) (F1) (F1)
Allele Wxa / Wxb Wxa / Wxb Wxa / Wxb
Indica/Japonica Indica/japonica Indica/japonica Indica/japonica
Sequence G G G
6 ABAYAWICKRAMA AND ANAI 4. (F1) 5. (F1) Tetep Reihou K16
Wxa / Wxb Wxa / Wxb Wxa Wxb Wxa
Indica/japonica Indica/japonica Indica Japonica Indica
G G G T G
In order to determine the inheritance pattern of Wx gene, 20 F2 plants derived from 4F1 plants (crossed between Tetep and Reihou) were screened by the dCAPS marker. F2 plants showed segregation to the Wx allele (Fig. 3). Plants 1, 2, 5, 7, 9, 10, 12, 19 and 20 showed the heterozygocity for Wx allele while plants; 3, 11 and 14 possessed Wxb allele and plants; 4, 6, 8, 13, 15, 16, 17 and 18 possessed Wxa allele. In rice grain quality improving programs, especially for Indica rice, it is important to use dCAPS markers to identify plants with desired Wx allele.
8 9 10
11 12 13 14 15
16 17 18 19 20 M
bp 200 100
Figure 3. Molecular differentiation of Wxa and Wxb alleles by dCAPS analysis in the F 2 generation. M=100bp ladder, 1-5=F2 plants derived from 1(F 1) plant, 6-10= F2 plants derived from 2(F1) plant, 11-15= F2 plants derived from 3(F1) plant and 16-20= F2 plants derived from 4(F1) plant.
DISCUSSION Among the molecular marker systems currently available, dCAPS marker that identifies Single Nucleotide Polymorphism (SNP) present in a particular gene is suitable for routine application in breeding programs. Analysis of dCAPS, utilizes the Polymerase Chain Reaction (PCR) which requires only small quantities of DNA. dCAPS marker is an accurate, quick and successful tool for analysing large number of samples without the need of direct sequence determination. In grain quality improvement programs, identification of Wx gene is immensely important at the seedling stage for further selection and hybridization. Use of dCAPS marker in backcross breeding programmes, especially in grain quality improvement of Indica rice, will facilitate the capturing of plants with Wxb allele with Indica genotype. In addition, dCAPS allows identifying heterozygous locus in segregating populations.
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According to Figure 2, dCAPS marker detected the waxy allele SNP as accurate as the sequencing analysis. Therefore, this marker can be used as an effective and suitable tool for large-scale analysis. Furthermore, the expression of the Wxb allele affects by the temperature during seed development. The Wxb gene activates in response to cool temperatures. Hence, the level of Wx protein increases at cool temperatures, resulting in higher content of amylose in mature seeds. However, the temperature has no effect on Wxa in protein expression in the endosperm and pollen (Hirano and Sano, 2000). In addition, at Wx locus, another locus, called dull (du), controls amylose synthesis (Isshiki et al., 2000). A series of du mutants have been found as dull phenotypes in mature seeds and these seeds have been shown to contain less amylose. The genes responsible for these mutations are independent of the Wx locus. A biochemical analysis suggested that the low amylose contents in du mutants were caused by reduced levels of Wx protein. CONCLUSIONS Identification of SNP at the untranslated leader region of the 5` splice site of the Wx gene, allowed to discriminate Wxa and Wxb alleles. Therefore, the dCAPS marker can effectively be used in identification of Wx allele in grain quality improvement programmes. ACKNOWLEDGEMENTS The financial aid provided by the Ministry of Education, Culture and Sports in Japan is greatly appreciated. REFERENCES Bao, J.S., H. Corke and M. Sum. 2006. Microsatellites, single nucleotide polymorphisms and a sequence tagged site in starch-synthesizing genes in relation to starch physicochemical properties in nonwaxy rice (Oryza sativa L.). Theoretical and Applied Genetics 113:1185-1196. Bergman, C.J., J.T. Delgado, A.M. McClung and R.G. Fjellstrom. 2001. An improved method of using a microsatellite in rice waxy gene to determine amylase class. Cereal Chemistry 78(3):257-260. Hirano, H. and Y. Sano. 1991. Molecular characterization of the waxy locus of rice (Oryza sativa). Plant Cell Physiology 32(7):989-997. Hirano, H. and Y. Sano. 2000. Comparison of waxy gene regulation in the endosperm and pollen in Oryza sativa L. Genes Genetics 75:245-249. Isshiki, M., K. Morino, M. Nakajima, R.J. Okagaki, S.R. Wessler, T. Izawa and K. Shimamoto. 1998. A naturally occurring functional allele of the rice waxy locus has a GT to TT mutation at 5` splice site of the first intron. The Plant
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