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Molecular genetic mapping of quantitative trait loci for milling quality in rice (Oryza sativa L.)
Journal of Cereal Science 40 (2004) 109–114 www.elsevier.com/locate/jnlabr/yjcrs
Molecular genetic mapping of quantitative trait loci for milling quality in rice (Oryza sativa L.) Yanjun Donga,*, E. Tsuzukia, Dongzhi Lina, H. Kamiuntena, H. Teraoa, M. Matsuoa, Shihua Chengb a
Agricultural Faculty, Miyazaki University, Miyazaki City 889-2192, Japan b China National Rice Research Institute,Hangzhou 310006, China
Received 16 April 2003; revised 5 September 2003; accepted 26 April 2004
Abstract Identification and mapping of genomic regions controlling quantitative trait loci (QTLs) was undertaken to determine the genomic regions associated with milling traits in rice to facilitate breeding of new rice varieties with high milling quality. The recombinant inbred (RI) population used was derived from cross of a japonica variety, ‘Asominori’, with an indica variety, ‘IR24’ through 289 RFLP markers. Three milling traits, namely, brown rice percentage (BRP), milled rice percentage (MRP), and milled head rice percentage (MHP), which are the main indicators of milling quality in rice, were estimated for each RI line and their parental varieties. Continuous distributions and transgressive segregations of three milling traits were observed in the RI population, showing that the three traits were quantitatively inherited. Two QTLs (q BRP-9 and q BRP-10) for BRP were identified and mapped to chromosomes 9 and 10, and explained 7.2 and 21.3% of the total phenotype variation, respectively. Two QTLs (qMRP-11 and qMRP-12) governing MRP were detected and mapped to chromosomes 11 and 12, accounted for 12.2 and 7.7% of total phenotype variation, respectively. In addition, three QTLs (qMHP-1, qMHP-3 and qMHP-5) controlling MHP were observed and mapped to chromosomes 1, 3 and 5, and explained 16.0, 22.1 and 8.7% of the total phenotype variation, respectively. Among them, five QTLs (q BRP-9, q BRP-10, qMRP-11, qMHP-3 and qMHP-5) from japonica parent, Asominori, and two QTLs (qMRP-12, qMHP-1) from indica IR24 can improve milling quality in rice. The results and the tightly linked molecular markers that flank the QTL will be useful in breeding for improvement of milling quality in rice. q 2004 Elsevier Ltd. All rights reserved. Keywords: BRP, brown rice percentage; cM, centiMorgan; CIM, composite interval mapping; MHP, milled head-rice percentage; MRP, milled rice percentage; QTL, quantitative trait locus; RFLP, restriction fragment length polymorphism; RI, recombination inbred
1. Introduction Rice is a major cereal crop feeding more than 50% of the world’s population. Demand for high quality rice has always been a major factor in rice marketing in developed countries and is becoming more important in developing countries as the economic status of the population increases. Although rice quality depends on many attributes of the rice grain and is also related to preference among different cultures and habits, its major elements include milling performance, appearance, cooking, eating and nutrient quality. Among these, milling characteristics, especially the recovery of milled head rice is of primary concern. Head rice * Corresponding author. E-mail addresses: [email protected], dongjapan@hotmail. com (Y. Dong). 0733-5210/$ - see front matter q 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.jcs.2004.04.008
yield, which is the main determinant of market price, is directly related to brown and milled rice yield. Together these parameters are the indicators of the milling quality of rice. The recent advances in high-density marker linkage maps in rice (Causse et al., 1994; Harushima et al., 1998) have provided a powerful tool for elucidating the genetic basis of quantitatively inherited traits. In the past, numerous quantitative trait loci (QTLs) associated with yield and its components, and other agronomic traits in rice have been identified and mapped using DNA molecular makers (Yano and Sasaki, 1997). However, to our knowledge, genetic analysis of QTLs associated with milling quality, indicated by brown rice percentage (BRP), milled rice percentage (MRP) and milled head-rice percentage (MHP), has not been conducted The objectives of this study were to identify QTLs for three related milling traits in rice using
110
Y. Dong et al. / Journal of Cereal Science 40 (2004) 109–114
recombinant inbred (RI) lines from a japonica/indica cross and so to provide basic information for breeding new rice varieties with higher milling quality through DNA molecular-marker techniques.
replicated three times. Average values for each line were used for QTL analyses.
2. Materials and methods
A composite interval mapping (CIM) analysis method was applied to trait average and marker data to more
2.4. QTL analysis
2.1. Plant material The RI lines in this study, kindly provided by Professor A. Yoshimura of Agricultural faculty of Kyushu University, Japan were developed by single seed descent from the progeny of a cross of japonica variety, ‘Asominori’, with indica variety, ‘IR24’. 165 F6 lines were randomly selected from 227 original F2 individual plants, of which, 71 lines were used for mapping The restriction fragment length polymorphism (RFLP) map covering 1275 cM and possessing 375 RFLP markers in entire rice chromosomes was constructed from the F6 and F7 generations (Tsunematsu et al., 1996). Previously, the same RI population was used successfully for mapping QTLs for many important agronomic traits (Dong et al., 2002, 2003a,b; Sasahara et al., 1999; Yamazaki et al., 1999, 2000; Yoshimura et al., 1998). In this study, we used a subset of 289 RFLP markers without overlapping, for all loci from the original genetic map (Tsunematsu et al., 1996) to map QTLs for milling quality in rice, for which the average interval distance between pairs of markers was 4.4 cM. 2.2. Cultivation and harvest The RI lines together with their parents, ‘Asominori’ and ‘IR24’, were used in this study. Seeds were sown in nursery seedling boxes on the 10th of April 2002. After 30 days seedlings were transplanted to a paddy field at the Experimental Station of Miyazaki University, Japan with two replications. Field management followed the normal methods. In the experiment, all rice-grains from each RI line were harvested 40 d after heading and dried at 40 8C for 72 h and stored at room temperature for at least 2 months before measuring milling quality. 2.3. Measurement of milling traits All rough rice with the same degree of maturity for each RI line and their parents was selected manually and used for investigating milling quality. Rough rice samples (50 g) from each RI line were de-hulled in a Yanmar ST50 mill (Yanmar Co. Ltd, Japan) under the same conditions to measure BRP. The brown rice was milled in a NBS200A mill (Satake Co. Ltd, Japan) to determine MRP and MHP, respectively. The milled head rice was manually separated with a set of screens, and milled grains possessing more than four-fifth of the whole rice grain, constituted milled head rice. Values of BRP, MRP and MHP were based on total rough rice weight. All measurements for each line were
Fig. 1. Frequency distributions of brown rice percentage (BRP), milled rice percentage (MRP) and milled head-rice percentage (MHP), respectively, in RI population from the cross between Asominori and IR24. White, black arrows indicate the average values of IR24 and Asominori, respectively.
Y. Dong et al. / Journal of Cereal Science 40 (2004) 109–114
precisely identify the QTL locations (Zeng, 1994). The CIM analysis was performed using a QTL Cartographer computer program software version 1.13 g (Wang et al., 1999). Specifically, the analyses were calculated using forward regression, with a walk speed of 2 cM, and a window size of 10 cM. A locus with a LOD threshold value of more than 2.0 was set to be a putative QTL. In addition, additive effects and percentage of variation explained by an individual QTL were also estimated. The QTL were named as suggested McCouch et al. (1997).
3. Results 3.1. Distribution of milling traits in the segregating RI population The average values of the three milling traits studied, namely, BRP, MRP and MRP, of both parents (Asominori, IR24) and the frequency distributions of RI population are presented in Fig. 1. Although the differences in between the two parents, Asominori (80.62%) and IR24 (79.50%) were rather small, significant differences were observed in the RI population. For MRP and MHP, the two parents, Asominori and IR24, showed rather large differences, especially for MHP in Asominori (59.26%) and IR24 (42.46%). In addition, continuous phenotypic variation and transgressive segregation for the three traits were observed in the RI population. Accordingly, it could be concluded that the three milling traits studied are quantitatively inherited and data obtained in the experiment are effective for genetic mapping quantitative trait loci (QTLs). 3.2. QTLs for brown rice percentage (BRP) Two QTLs governing BRP were identified and mapped to chromosomes 9 and 10 (Table 1 and Fig. 2) and tentatively named for q BRP-9 and q BRP-10, respectively. The q BRP-9, located between XNpb108 and C570 on chromosome 9, showed an additive effect of 0.61% on
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the trait with a LOD value of 3.44 and explained 7.2% of the total phenotype variation. Another QTL, q BRP-10 (LOD ¼ 3.76) was detected between C1166 and R3285 of chromosome 10, and accounted for 21.3% of total variation. In addition, two loci (q BRP-9 and q BRP-10) from the japonica parent, Asominori, increased the values of BRP. 3.3. QTLs for milled rice percentage (MRP) Two QTLs (Table 1 and Fig. 2) for MRP were detected and mapped to chromosomes 11 and 12, and tentatively designated as qMRP-11 and qMRP-12, respectively. The qMRP-11 with additive effect of 1.40% on the trait and LOD value of 3.83 was located near XNpb257 on chromosome 11 and explained 12.2% of total phenotype variation. The qMRP-12 with LOD value of 2.50 was detected between C104A and XNpb193 on chromosome 12 and explained 7.7% of total variation. In addition, qMRP-11 from Asominori and qMRP-12 from IR24 increased the values of MRP. 3.4. QTLs milled head-rice percentage (MHP) Three QTLs (Table 1 and Fig. 2) controlling MHP in rice were detected and mapped to chromosomes 1, 3 and 5, and tentatively designated as qMHP-1, qMHP-3 and qMHP-5, respectively. The qMHP-3, located between R19 and G1316 on chromosome 3, showed the largest additive effect with a LOD value of 6.17 and increased additive effects of 5.8% on the trait from Asominori and explained 22.1% of the total phenotype variation. Another QTL, qMHP-1 (LOD ¼ 2.57) was detected near C112 on chromosome 1 and accounted for 16.0% of total variation. The remaining QTL, qMHP-5, detected between C1268 and R1553 on chromosome 5, with a LOD value of 2.05, and explained 8.7% of total phenotype variation. In addition, the Asominori alleles both qMHP-3 and qMHP-5 from Asominori, and qMHP-1 from IR24 contributed to the increases in MRP values.
Table 1 Quantitative trait loci (QTLs) controlling three milling traits in rice based on CIM methods (Wang et al., 1999) using RI lines from a cross between Asominori and IR24 Milling traits
Name of QTLs
Chromosome number
Distance (cM)
Closest markera
Peak LOD Value
Additive effectsb
Variationc (%)
BRP
qBRP-9 qBRP-10 qMRP-11 qMRP-12 qMHP-1 qMHP-3 qMHP-5
9 10 11 12 1 3 5
61.2 31.4 52.8 78.8 0.10 71.4 62.6
XNpb108 C1166 XNpb257 C104A C112 G1316 C1268
3.44 3.76 3.83 2.50 2.57 6.17 2.05
0.61 0.80 1.40 21.29 25.01 5.80 4.28
7.2 21.3 12.2 7.7 16.0 22.1 8.7
MRP MHP
a b c
The marker is the most closely linked to QTL detected. Positive values of additive effects indicate Asominori alleles are in the direction of increasing traits. The percentage of explained phenotypic variation.
112 Y. Dong et al. / Journal of Cereal Science 40 (2004) 109–114 Fig. 2. Chromosomal locations of QTLs for milling quality in RI population from the cross between Asominori and IR24. Shaded (Chr.9 and 10), white (Chr.11 and 12) and black (Chr.1, 3 and 5) bars indicate interval markers of putative QTL for BRP, MRP and MHP, respectively, based on CIM method. Black arrowheads indicate the location of peak LOD for putative QTLs detected.
Y. Dong et al. / Journal of Cereal Science 40 (2004) 109–114
4. Discussion In this study, we report the results of QTL mapping for three associated milling traits in rice, using the RI lines derived from japonica ‘Asominori’ and indica ‘IR24’ with 289 RFLP markers. This is the same set of RI lines used previously to analyze QTLs for days to heading (Yoshimura et al., 1998), ovicidal response to whitebacked planthopper (Yamazaki et al., 1999), ovicidal response to brown planthopper (Yamazaki et al., 2000), vascular bundle system and spike morphology (Sasahara et al., 1999), steamed rice shape (Dong et al., 2002), hulled-rice shape (Dong et al., 2003a) and pre-harvest sprouting (Dong et al., 2003b). The results from these analyses showed that the RI population is very useful for identification of rice QTLs. Our analyses detected a total of seven QTLs associated with milling quality. Specifically, two QTLs (q BRP-9 and q BRP-10) for BRP) on chromosomes 9 and 10, two QTLs (qMRP-11and qMRP-12) for milled rice percentage (MRP) on chromosomes 11 and 12, and three QTLs (qMHP-1, qMHP-3 and qMHP-5) for MHP on chromosome 1, 3, and 5 were observed, respectively. Among these, five QTLs (q BRP-9, q BRP-10, qMRP-11, qMHP-3 and qMHP-5) from the japonica parent, Asominori, two QTLs (qMRP-12 and qMHP-1) from the indica parent IR24 can improve milling quality in rice. In addition, it was noted that all QTLs identified for the three milling traits were independent of one another since they were located on different chromosomes. This suggests that improving one milling trait through marker-assisted breeding will not affect the other two milling traits. MHP is the most important milling trait among three traits studied since milled head rice contains most of the edible rice grain components and is the main determinant of market price. Longer rice grains tend to break more easily during milling, suggesting that genomic regions governing rice grain size could be tightly linked to, or the same as, some QTLs for MHP. Dong et al. (2003a) reported two QTLs for hulled-rice length, located on chromosomes 3, 10 and three QTLs for the hulled-rice width on chromosomes 2, 3 and 5, respectively, using the same RI population. In comparing the genomic positions of those QTLs identified by Dong et al.(2003a) for hulled-rice size with seven QTLs detected for milling quality in this study, qMHP-3, located on chromosome 3, with largest effects on MHP was not only tightly linked to one QTL for grain-length, but also allelic to the one QTL for grain width in rice identified by Dong et al. (2003a). However, six other QTLs for milling quality were independent of, or distant from, the genomic regions for grain shape. Theoretically, one of the most effective approaches to increase MHP values would be to transfer the qMHP-3 locus since it has the largest genetic effect (Table 1) on the trait. However, this may be counterproductive since the genomic region covering qMHP-3, has the effect of decreasing grain size (Dong et al., 2003a), which can lead to decrease in rice yield. Thus, medium-long
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and slender rice is preferable in breeding practice. Additionally, the closely linked DNA molecular markers that flank the two QTLs (qMHP-1 and qMHP-5), independent of grain size QTL, they can be very useful for markerassisted breeding to increase MHP. Of course, the molecular markers flanking four other QTLs (q BRP-9 and q BRP-10 for BRP, and qMRP-11and qMRP-12 for MRP) are available to improve milling traits through marker-assisted breeding. In addition, further study on genes associated with milling quality in rice through more abundant markers such as SSRs derived from the rice genome sequence are needed.
Acknowledgements This research was supported by a grant from the Iijima Memorial Foundation for the Promotion of Food Science and Technology of Japan. We are greatly indebted to Professor A. Yoshimura (Plant Breeding Laboratory, Agricultural Faculty of Kyushu University, Japan) for kindly providing materials, molecular data and valuable advices and Japan Society for the Promotion of Science (JSPS) providing postdoctoral fellowships to the first author.
References Causse, M.A., Fultion, T.M., Cho, Y.G., Ahn, S.N., Chunwongse, J., Wu, K., Xiao, J., Yu, Z., Ronald, P.C., Harrington, S.E., Second, G., McCouch, S.R., Tanksley, S.D., 1994. Saturated molecular map of the rice genome based on an interspecific backcross population. Genetics 138, 1251–1274. Dong, Y., Zheng, Y.F., Tsuzuki, E., Terao, H., 2002. Quantitative trait loci controlling steamed-rice shape in a recombinant inbred population. International Rice Research Note 27, 19–20. Dong, Y., Tsuzuki, E., Kamiunten, H., Terao, H., Dongzhi, L., 2003a. Molecular mapping of QTLs for hulled-rice shape using recombinant inbred population. Oryza 38, 9–12. Dong, Y., Tsuzuki, E., Kamiunten, H., Terao, H., Lin, D.Z., Matsuo, M., Zheng, Y.F., 2003b. Identification of quantitative trait loci associated with pre-harvest sprouting resistance in rice (Oryza sativa L.). Field Crops Research 81, 133–139. Harushima, Y., Yano, M., Shomura, A., Sato, M., Shimano, T., Kuboki, Y., Yamamoto, T., Lin, S.Y., Antonio, B.A., Parco, A., Kajiya, H., Huang, N., Yamamoto, K., Nagamura, Y., Kurata, N., Khush, G.S., Sasaki, T., 1998. A High-density rice genetic linkage map with 2275 markers using a single F2 population. Genetics 148, 479–494. McCouch, S.R., Cho, Y.G., Yano, M., Paul, E., Blinstrub, M., 1997. Report on QTL nomenclature. Rice Genetics Newsletter 14, 11–13. Sasahara, H., Fukuta, Y., Fukuyama, T., 1999. Mapping of QTLs for vascular bundle system and spike morphology in rice Oryza sativa L. Breeding Science 49, 75 –81. Tsunematsu, H., Yoshimura, A., Harushima, Y., Nagamura, Y., Kurata, N., Yano, M., Iwata, N., 1996. RFLP framework map using recombinant inbred lines in rice. Breeding Science 46, 279 –284. Wang, S.C., Zeng, B.Z., Basten, C.J. QTL cartographer windows version 1.13 g (Programed, 30 Aug.1999). http://statgen.ncsu.edu/qtlcart/ cartographer.hml.
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Yamazaki, M., Tsunematstu, H., Yoshimura, A., Iwata, N., Yasui, H., 1999. Quantitative trait locus mapping of ovicidal response in rice (Oryza sativa L.) against whitebacked planthopper (Sogatella furcifera Horvath). Crop Science 39, 1178–1183. Yamazaki, M., Yoshimura, A., Yasui, H., 2000. Mapping of quantitative trait loci of ovicidal response to brown planthopper (Nilaparvata lugen Stal) in rice (Oryza sativa L.). Breeding Science 50, 291–296.
Yano, M., Sasaki, T., 1997. Genetic and molecular dissection of quantitative traits in rice. Plant Molecular Biology 35, 145–153. Yoshimura, A., Okamoto, M., Nagamine, T., Tsunematsu, H., 1998. Rice QTL analysis for days to heading under the cultivation of Ishigaki island. Breeding Science 48(suppl 1), 73. Zeng, B.Z., 1994. Precision mapping of quantitative trait loci. Genetics 136, 1457–1468.
Molecular genetic mapping of quantitative trait loci for milling quality in rice (Oryza sativa L.) Yanjun Donga,*, E. Tsuzukia, Dongzhi Lina, H. Kamiuntena, H. Teraoa, M. Matsuoa, Shihua Chengb a
Agricultural Faculty, Miyazaki University, Miyazaki City 889-2192, Japan b China National Rice Research Institute,Hangzhou 310006, China
Received 16 April 2003; revised 5 September 2003; accepted 26 April 2004
Abstract Identification and mapping of genomic regions controlling quantitative trait loci (QTLs) was undertaken to determine the genomic regions associated with milling traits in rice to facilitate breeding of new rice varieties with high milling quality. The recombinant inbred (RI) population used was derived from cross of a japonica variety, ‘Asominori’, with an indica variety, ‘IR24’ through 289 RFLP markers. Three milling traits, namely, brown rice percentage (BRP), milled rice percentage (MRP), and milled head rice percentage (MHP), which are the main indicators of milling quality in rice, were estimated for each RI line and their parental varieties. Continuous distributions and transgressive segregations of three milling traits were observed in the RI population, showing that the three traits were quantitatively inherited. Two QTLs (q BRP-9 and q BRP-10) for BRP were identified and mapped to chromosomes 9 and 10, and explained 7.2 and 21.3% of the total phenotype variation, respectively. Two QTLs (qMRP-11 and qMRP-12) governing MRP were detected and mapped to chromosomes 11 and 12, accounted for 12.2 and 7.7% of total phenotype variation, respectively. In addition, three QTLs (qMHP-1, qMHP-3 and qMHP-5) controlling MHP were observed and mapped to chromosomes 1, 3 and 5, and explained 16.0, 22.1 and 8.7% of the total phenotype variation, respectively. Among them, five QTLs (q BRP-9, q BRP-10, qMRP-11, qMHP-3 and qMHP-5) from japonica parent, Asominori, and two QTLs (qMRP-12, qMHP-1) from indica IR24 can improve milling quality in rice. The results and the tightly linked molecular markers that flank the QTL will be useful in breeding for improvement of milling quality in rice. q 2004 Elsevier Ltd. All rights reserved. Keywords: BRP, brown rice percentage; cM, centiMorgan; CIM, composite interval mapping; MHP, milled head-rice percentage; MRP, milled rice percentage; QTL, quantitative trait locus; RFLP, restriction fragment length polymorphism; RI, recombination inbred
1. Introduction Rice is a major cereal crop feeding more than 50% of the world’s population. Demand for high quality rice has always been a major factor in rice marketing in developed countries and is becoming more important in developing countries as the economic status of the population increases. Although rice quality depends on many attributes of the rice grain and is also related to preference among different cultures and habits, its major elements include milling performance, appearance, cooking, eating and nutrient quality. Among these, milling characteristics, especially the recovery of milled head rice is of primary concern. Head rice * Corresponding author. E-mail addresses: [email protected], dongjapan@hotmail. com (Y. Dong). 0733-5210/$ - see front matter q 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.jcs.2004.04.008
yield, which is the main determinant of market price, is directly related to brown and milled rice yield. Together these parameters are the indicators of the milling quality of rice. The recent advances in high-density marker linkage maps in rice (Causse et al., 1994; Harushima et al., 1998) have provided a powerful tool for elucidating the genetic basis of quantitatively inherited traits. In the past, numerous quantitative trait loci (QTLs) associated with yield and its components, and other agronomic traits in rice have been identified and mapped using DNA molecular makers (Yano and Sasaki, 1997). However, to our knowledge, genetic analysis of QTLs associated with milling quality, indicated by brown rice percentage (BRP), milled rice percentage (MRP) and milled head-rice percentage (MHP), has not been conducted The objectives of this study were to identify QTLs for three related milling traits in rice using
110
Y. Dong et al. / Journal of Cereal Science 40 (2004) 109–114
recombinant inbred (RI) lines from a japonica/indica cross and so to provide basic information for breeding new rice varieties with higher milling quality through DNA molecular-marker techniques.
replicated three times. Average values for each line were used for QTL analyses.
2. Materials and methods
A composite interval mapping (CIM) analysis method was applied to trait average and marker data to more
2.4. QTL analysis
2.1. Plant material The RI lines in this study, kindly provided by Professor A. Yoshimura of Agricultural faculty of Kyushu University, Japan were developed by single seed descent from the progeny of a cross of japonica variety, ‘Asominori’, with indica variety, ‘IR24’. 165 F6 lines were randomly selected from 227 original F2 individual plants, of which, 71 lines were used for mapping The restriction fragment length polymorphism (RFLP) map covering 1275 cM and possessing 375 RFLP markers in entire rice chromosomes was constructed from the F6 and F7 generations (Tsunematsu et al., 1996). Previously, the same RI population was used successfully for mapping QTLs for many important agronomic traits (Dong et al., 2002, 2003a,b; Sasahara et al., 1999; Yamazaki et al., 1999, 2000; Yoshimura et al., 1998). In this study, we used a subset of 289 RFLP markers without overlapping, for all loci from the original genetic map (Tsunematsu et al., 1996) to map QTLs for milling quality in rice, for which the average interval distance between pairs of markers was 4.4 cM. 2.2. Cultivation and harvest The RI lines together with their parents, ‘Asominori’ and ‘IR24’, were used in this study. Seeds were sown in nursery seedling boxes on the 10th of April 2002. After 30 days seedlings were transplanted to a paddy field at the Experimental Station of Miyazaki University, Japan with two replications. Field management followed the normal methods. In the experiment, all rice-grains from each RI line were harvested 40 d after heading and dried at 40 8C for 72 h and stored at room temperature for at least 2 months before measuring milling quality. 2.3. Measurement of milling traits All rough rice with the same degree of maturity for each RI line and their parents was selected manually and used for investigating milling quality. Rough rice samples (50 g) from each RI line were de-hulled in a Yanmar ST50 mill (Yanmar Co. Ltd, Japan) under the same conditions to measure BRP. The brown rice was milled in a NBS200A mill (Satake Co. Ltd, Japan) to determine MRP and MHP, respectively. The milled head rice was manually separated with a set of screens, and milled grains possessing more than four-fifth of the whole rice grain, constituted milled head rice. Values of BRP, MRP and MHP were based on total rough rice weight. All measurements for each line were
Fig. 1. Frequency distributions of brown rice percentage (BRP), milled rice percentage (MRP) and milled head-rice percentage (MHP), respectively, in RI population from the cross between Asominori and IR24. White, black arrows indicate the average values of IR24 and Asominori, respectively.
Y. Dong et al. / Journal of Cereal Science 40 (2004) 109–114
precisely identify the QTL locations (Zeng, 1994). The CIM analysis was performed using a QTL Cartographer computer program software version 1.13 g (Wang et al., 1999). Specifically, the analyses were calculated using forward regression, with a walk speed of 2 cM, and a window size of 10 cM. A locus with a LOD threshold value of more than 2.0 was set to be a putative QTL. In addition, additive effects and percentage of variation explained by an individual QTL were also estimated. The QTL were named as suggested McCouch et al. (1997).
3. Results 3.1. Distribution of milling traits in the segregating RI population The average values of the three milling traits studied, namely, BRP, MRP and MRP, of both parents (Asominori, IR24) and the frequency distributions of RI population are presented in Fig. 1. Although the differences in between the two parents, Asominori (80.62%) and IR24 (79.50%) were rather small, significant differences were observed in the RI population. For MRP and MHP, the two parents, Asominori and IR24, showed rather large differences, especially for MHP in Asominori (59.26%) and IR24 (42.46%). In addition, continuous phenotypic variation and transgressive segregation for the three traits were observed in the RI population. Accordingly, it could be concluded that the three milling traits studied are quantitatively inherited and data obtained in the experiment are effective for genetic mapping quantitative trait loci (QTLs). 3.2. QTLs for brown rice percentage (BRP) Two QTLs governing BRP were identified and mapped to chromosomes 9 and 10 (Table 1 and Fig. 2) and tentatively named for q BRP-9 and q BRP-10, respectively. The q BRP-9, located between XNpb108 and C570 on chromosome 9, showed an additive effect of 0.61% on
111
the trait with a LOD value of 3.44 and explained 7.2% of the total phenotype variation. Another QTL, q BRP-10 (LOD ¼ 3.76) was detected between C1166 and R3285 of chromosome 10, and accounted for 21.3% of total variation. In addition, two loci (q BRP-9 and q BRP-10) from the japonica parent, Asominori, increased the values of BRP. 3.3. QTLs for milled rice percentage (MRP) Two QTLs (Table 1 and Fig. 2) for MRP were detected and mapped to chromosomes 11 and 12, and tentatively designated as qMRP-11 and qMRP-12, respectively. The qMRP-11 with additive effect of 1.40% on the trait and LOD value of 3.83 was located near XNpb257 on chromosome 11 and explained 12.2% of total phenotype variation. The qMRP-12 with LOD value of 2.50 was detected between C104A and XNpb193 on chromosome 12 and explained 7.7% of total variation. In addition, qMRP-11 from Asominori and qMRP-12 from IR24 increased the values of MRP. 3.4. QTLs milled head-rice percentage (MHP) Three QTLs (Table 1 and Fig. 2) controlling MHP in rice were detected and mapped to chromosomes 1, 3 and 5, and tentatively designated as qMHP-1, qMHP-3 and qMHP-5, respectively. The qMHP-3, located between R19 and G1316 on chromosome 3, showed the largest additive effect with a LOD value of 6.17 and increased additive effects of 5.8% on the trait from Asominori and explained 22.1% of the total phenotype variation. Another QTL, qMHP-1 (LOD ¼ 2.57) was detected near C112 on chromosome 1 and accounted for 16.0% of total variation. The remaining QTL, qMHP-5, detected between C1268 and R1553 on chromosome 5, with a LOD value of 2.05, and explained 8.7% of total phenotype variation. In addition, the Asominori alleles both qMHP-3 and qMHP-5 from Asominori, and qMHP-1 from IR24 contributed to the increases in MRP values.
Table 1 Quantitative trait loci (QTLs) controlling three milling traits in rice based on CIM methods (Wang et al., 1999) using RI lines from a cross between Asominori and IR24 Milling traits
Name of QTLs
Chromosome number
Distance (cM)
Closest markera
Peak LOD Value
Additive effectsb
Variationc (%)
BRP
qBRP-9 qBRP-10 qMRP-11 qMRP-12 qMHP-1 qMHP-3 qMHP-5
9 10 11 12 1 3 5
61.2 31.4 52.8 78.8 0.10 71.4 62.6
XNpb108 C1166 XNpb257 C104A C112 G1316 C1268
3.44 3.76 3.83 2.50 2.57 6.17 2.05
0.61 0.80 1.40 21.29 25.01 5.80 4.28
7.2 21.3 12.2 7.7 16.0 22.1 8.7
MRP MHP
a b c
The marker is the most closely linked to QTL detected. Positive values of additive effects indicate Asominori alleles are in the direction of increasing traits. The percentage of explained phenotypic variation.
112 Y. Dong et al. / Journal of Cereal Science 40 (2004) 109–114 Fig. 2. Chromosomal locations of QTLs for milling quality in RI population from the cross between Asominori and IR24. Shaded (Chr.9 and 10), white (Chr.11 and 12) and black (Chr.1, 3 and 5) bars indicate interval markers of putative QTL for BRP, MRP and MHP, respectively, based on CIM method. Black arrowheads indicate the location of peak LOD for putative QTLs detected.
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4. Discussion In this study, we report the results of QTL mapping for three associated milling traits in rice, using the RI lines derived from japonica ‘Asominori’ and indica ‘IR24’ with 289 RFLP markers. This is the same set of RI lines used previously to analyze QTLs for days to heading (Yoshimura et al., 1998), ovicidal response to whitebacked planthopper (Yamazaki et al., 1999), ovicidal response to brown planthopper (Yamazaki et al., 2000), vascular bundle system and spike morphology (Sasahara et al., 1999), steamed rice shape (Dong et al., 2002), hulled-rice shape (Dong et al., 2003a) and pre-harvest sprouting (Dong et al., 2003b). The results from these analyses showed that the RI population is very useful for identification of rice QTLs. Our analyses detected a total of seven QTLs associated with milling quality. Specifically, two QTLs (q BRP-9 and q BRP-10) for BRP) on chromosomes 9 and 10, two QTLs (qMRP-11and qMRP-12) for milled rice percentage (MRP) on chromosomes 11 and 12, and three QTLs (qMHP-1, qMHP-3 and qMHP-5) for MHP on chromosome 1, 3, and 5 were observed, respectively. Among these, five QTLs (q BRP-9, q BRP-10, qMRP-11, qMHP-3 and qMHP-5) from the japonica parent, Asominori, two QTLs (qMRP-12 and qMHP-1) from the indica parent IR24 can improve milling quality in rice. In addition, it was noted that all QTLs identified for the three milling traits were independent of one another since they were located on different chromosomes. This suggests that improving one milling trait through marker-assisted breeding will not affect the other two milling traits. MHP is the most important milling trait among three traits studied since milled head rice contains most of the edible rice grain components and is the main determinant of market price. Longer rice grains tend to break more easily during milling, suggesting that genomic regions governing rice grain size could be tightly linked to, or the same as, some QTLs for MHP. Dong et al. (2003a) reported two QTLs for hulled-rice length, located on chromosomes 3, 10 and three QTLs for the hulled-rice width on chromosomes 2, 3 and 5, respectively, using the same RI population. In comparing the genomic positions of those QTLs identified by Dong et al.(2003a) for hulled-rice size with seven QTLs detected for milling quality in this study, qMHP-3, located on chromosome 3, with largest effects on MHP was not only tightly linked to one QTL for grain-length, but also allelic to the one QTL for grain width in rice identified by Dong et al. (2003a). However, six other QTLs for milling quality were independent of, or distant from, the genomic regions for grain shape. Theoretically, one of the most effective approaches to increase MHP values would be to transfer the qMHP-3 locus since it has the largest genetic effect (Table 1) on the trait. However, this may be counterproductive since the genomic region covering qMHP-3, has the effect of decreasing grain size (Dong et al., 2003a), which can lead to decrease in rice yield. Thus, medium-long
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and slender rice is preferable in breeding practice. Additionally, the closely linked DNA molecular markers that flank the two QTLs (qMHP-1 and qMHP-5), independent of grain size QTL, they can be very useful for markerassisted breeding to increase MHP. Of course, the molecular markers flanking four other QTLs (q BRP-9 and q BRP-10 for BRP, and qMRP-11and qMRP-12 for MRP) are available to improve milling traits through marker-assisted breeding. In addition, further study on genes associated with milling quality in rice through more abundant markers such as SSRs derived from the rice genome sequence are needed.
Acknowledgements This research was supported by a grant from the Iijima Memorial Foundation for the Promotion of Food Science and Technology of Japan. We are greatly indebted to Professor A. Yoshimura (Plant Breeding Laboratory, Agricultural Faculty of Kyushu University, Japan) for kindly providing materials, molecular data and valuable advices and Japan Society for the Promotion of Science (JSPS) providing postdoctoral fellowships to the first author.
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