Research Report
Development of a New japonica Rice Variety Nan-jing 46 with Good Eating Quality by Marker Assisted Selection
Cailin Wang Yadong Zhang Zhen Zhu Tao Chen Ling Zhao Jing Lin Lihui Zhou
Institute of Food Crops, Jiangsu Academy of Agricultural Sciences, Jiangsu High Quality Rice R&D Center, Nanjing Branch of China National Center for Rice Improvement, Nanjing, 210014
Corresponding author: ; Authors
Journal of Energy Bioscience 2010, Vol 1 No 1 DOI: 10.5376/JEB.2010.01.0001
Received: Sep. 1, 2010
Accepted: Oct. 14, 2010
Published: Oct. 19, 2010
This article was first published in the Molecular Plant Breeding (Regular Print Version), and here was authorized to redistribute 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:
Wang et al 2009, Development of a New japonica Rice Variety Nan-jing 46 with Good Eating Quality by Marker Assisted Selection, 7(6): 1070-1076
Abstract In order to developing a new japonica rice variety with both good eating quality and high yield, we employed the high yield japonica rice “Wu-xiang-jing 14” as the maternal parent was crossed to the good quality japonica rice “Kantou 194” with low-amylose content and translucent endosperm controlled by Wx-mq. By comparing the DNA sequence of Wx-mq gene and its allelic genes Wx-b, Wx-a, wx in chromosome 6,. two mutated bases occurring in exon 4 and exon 5 were found in Wx-mq. The change of G-A in exon 4 just produced a recognition site for NIaⅢ digestion, so a cleaved amplified polymorphic sequences (CAPS) marker was designed, which could identify the Wx-mq homozygous genotype and heterozygous genotype. This marker was used for marker assisted selection (MAS) of low-amylose content in F5 and F6 lines derived from the combination of Wu-xiang-jing 14/Kantou 194. The investigation result of endosperm appearance was completely coincident with that of molecular detection, and the efficiency of MAS with the CAPS marker was up to 100%. As a result, “Nan-jing 46”, a new japonica rice variety with good eating quality and high yield, was successfully developed.
Keywords Marker assisted selection, Good eating quality, Rice, Cleaved amplified polymorphic sequences (CAPS)
Background
As an important objective of rice breeding, rice quality has been paid more and more attention by breeders and consumers in recent years. Since 2000, quality has become the most important target in rice improvement among the three objectives (yield, quality and resistance) in Jiangsu Province. Since then, great achievements have been made in rice quality improvement. During 2000-2008, a total of 56 japonica rice varieties with grain quality above the third class of National Standard were approved to release to farmers in Jiangsu Province, including 8 varieties of the first class, 17 varieties of the second class and 31 varieties of the third class. However, most of the varieties in China still could not satisfy the requirements of consumers in China, especially in eating quality. These varieties have no competitiveness in the international market (Wang et al., 2008b).
In contrary, eating quality has been emphasized by breeders in Japan, and lots of good-eating varieties are developed. ‘Koshihikari’ is the best example. It is well-known for its appearance and good eating quality. Many studies indicate that the amylose content (AC) of endosperm is one of the key factors determining cooking and eating quality in rice (Matsuo et al., 1990; Hushibuchi, 1992). Low-amylose content leads to soft texture, oleosus appearance, glossiness and good glutinousness. Conversely, high-amylose content results in hard texture, unpolished appearance and poor viscosity after cooking (Zhu et al., 2004). Thus, amylose content is used as a major indicator for eating quality improvement in rice.
Low-amylose rice is a medium type between glutinous and non-glutinous rice. With the decrease of amylose content, the appearance of endosperm tends to be mist, milky white and translucence (Figure 1). For this reason the low-amylose rice is also called semi-glutinous rice (Zhu et al., 2004). This kind of rice is favored by consumers for its good eating quality, which owns the softness of glutinous rice, the elasticity of non-glutinous rice, and characterized by soft texture and excellent puffing ability when cooked. It is particularly suitable for processing, and for making mixed rice and puffing food, such as Sushi, light meal or other kinds of ready-to-eat food.
Figure 1 Comparison of the phenotype of endosperm
Note: Left: Glutinous; Middle: Among semi-glutinous; Right: Non-glutinous rice
So far, at least 14 low-amylose genes have been reported in rice (Heu, 1986; Yano et al., 1988; Heu and Kim, 1989; Kaushik and Khush, 1991; Sato, 2002; Koh et al., 1997; Suto et al., 1996; Sato et al., 2001). These genes can be classified into two types according to allelic relationship with Wx locus. Among them, Wx-mq gene resulting in translucent endosperm is ascribed to allelic to Wx locus. Utilizing this mutation in rice, breeders in Japan have developed some commercial varieties with low-amylose content and good-eating quality, such as ‘Milky Queen’ (Suto et al., 1996; Sato et al., 2001), ‘Kantou 194’ and ‘New-hikari’ (Tomit et al., 2007).
In order to breed new excellent rice varieties, high-yield variety ‘Wu-xiang-jing 14’ and good quality variety ‘Kantou 194’ with Wx-mq gene were used the female and male parent to make combination, respectively. After selection with molecular marker for several generations, ‘Nan-jing 46’, a translucent endosperm japonica rice variety with good taste, high production and resistance to rice stripe disease (RSV) was bred, and it was approved to release to farmers by the crop variety certification committee of Jiangsu in January, 2008 (Certificate Number is SU SHEN DAO 200814).
1 Results
1.1 The breeding process of Nan-jing 46
The breeding process of Nan-jing 46 was shown in Figure 2. High-yield variety ‘Wu-xiang-jing 14’ (original name: 99-15) with genotype of Wx-bWx-b and good taste variety ‘Kantou 194’ with genotype of Wx-mqWx-mq were used as female and male parents respectively to make cross combination in winter of 1999 at Hainan province. F1 plants were grown at Nanjing in 2000 and F2 seeds were harvested at Hainan in the same year. The F3 population was planted at Nanjing in 2001 and harvested by bulk method. Then, the F4 population was planted at Nanjing in 2002. From F4 generations, individual selection was carried out and the selected individuals were planted in plots (F5). From F5 generation, individual selection was only carried out in the homozygous plots with Wx-mqWx-mq genotype, which were identified by MAS at tiller stage. In 2005, a selected line (F7) with Wx-mqWx-mq genotype and stable characters in good quality, high yield and resistance to rice stripe disease was designated tentatively as ‘Ning 5047’. Then it was recommended for regional trial of medium-maturing late japonica variety in Jiangsu province in 2006. In next year, it was continually tested in regional trial and productivity trial. Finally, it was approved to release to farmers by the Crop Variety Certification Committee of Jiangsu and denominated ‘Nan jing 46’ in January, 2008 (Certificate Number: SU SHEN DAO 200814) (Wang et al., 2008a).
1999, Hainan Wu-xiang-jing 14(99-15/Kantou 194)
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2000, Nanjing F1
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2000, Hainan F2 Mixed harvesting excellent plants
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2001, Nanjing F3 Mixed harvesting excellent plants
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2002, Nanjing F4 Selective harvesting excellent plants.
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2003, Nanjing F5 MAS for excellent plants with Wx-mq homozygous genotype
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2004, Nanjing F6 MAS for excellent plants with Wx-mq homozygous genotype
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2005, Nanjing F7 Good eating quality, high production and resistance to rice stripe disease rice line-Ning 5047
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2006, Jiangsu F8 Multi-sites test, regional trial in Jiangsu province
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2007, Jiangsu F9 Regional and productivity trial in Jiangsu province
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2008, Nanjing Nan-jing 46
Figure 2 Breeding process of Nan-jing 46
1.2 Identification of the CAPS marker directly related to Wx-mq gene
The nucleotide sequences of Wx-mq gene for translucent endosperm had been characterized by Sato et al. (2002), which have two mis-sense mutations occurring in exon 4 and exon 5 compared with that in Wx-b. To make clear whether these mutations was also in Wx-a and wx gene sequences, the DNA sequences comparison from this segment was done. The result indicated that these mutated bases were only found in Wx-mq. The base substitutions continued to be analyzed by the software: dCAPS Finder 2.0 (http://helix.wustl.edu/dcaps/dcaps.html). Luckily, the change of G-A in exon 4 just produced a recognition site for NIaⅢ digestion, so a CAPS marker was designed for detecting this variation by Primer Premier 5.0 (Figure 3).
Figure 3 Strategy of CAPS marker designed for detecting Wx-mq mutation in Wx locus
Note: Black boxes represent exons in Wx gene. Single arrows and white boxes indicate the position of the primers and missense mutations in exon 4, respectively
1.3 Results of molecular marker assisted selection
To make clear the band types of Wx-mq gene in translucent endosperm, 10 rice materials including 6 varieties and 4 F1 materials were used for molecular detection. The electrophoresis result of digested products after amplification indicates that products of AA homozygous genotype (Wx-mqWx-mq) in exon 4 produced 215bp and 170bp bands, when digested completely by NIaⅢ (Milky Queen, Kantou 194, Nan-jing 46, Milky Queen/Kantou and Kantou 194/Milky Queen); while those with GG homozygous genotype (without Wx-mq gene) couldn’t be digested and showed only one band of 385bp in gel (Su-yu-nuo, Ju-feng-zhan and Te qing). The CAPS marker was co-dominant, So PCR products amplified from F1 plants with heterozygous genotype of GA (Wx-mqWx-b or Wx-mqwx) were cleaved partially by NiaⅢ and showed the above three bands after digestion (Kantou 194/Ju-feng-zhan and Kantou 194/Su-yu-nuo) (Figure 4).
Figure 4 Detection of Wx-mq genotype with the CAPS marker
Note: M: 100 bp ladder marker; 1: Milky Queen; 2: Kantou 194; 3: Nan jing 46; 4: Milky Queen / Kantou 194; 5: Kantou 194 / Milky Queen; 6: Kantou 194 / Ju-feng-zhan; 7: Kantou 194 / Su-yu-nuo; 8: Su-yu-nuo; 9: Ju-feng-zhan; 10: Te qing. 1~5 represent homozygote genotypes of Wx-mq gene, 6~7 are heterozygous genotypes with Wx-mq gene, 8~10 represent genotypes without Wx-mq gene
Figure 5 Detection of Wx-mq genotype with the CAPS marker for F5 lines derived from Wu-xiang-jing 14/Kantou 194
Note: M: 100 bp ladder marker; 1~3: Kantou 194, Wu-xiang-jing 14, Wu-xiang-jing 14 / Kantou 194; 5, 6, 7, 14, 15, 20, 22: Genotypes without Wx-mq gene; 4, 10, 11, 12, 18, 19: Heterozygotes with Wx-mq gene; 8, 9, 13, 16, 17, 21: Homozygotes with Wx-mq gene
Figure 6 Detection of Wx-mq genotype with the CAPS marker for F6 lines derived from Wu-xiang-jing 14/Kantou 194
Note: M: 100 bp ladder marker; 1~3: Kantou 194, Wu-xiang-jing 14, Wu-xiang-jing 14/Kantou 194; 4~11: Homozygotes with Wx-mq gene; 12~22: Genotypes without Wx-mq gene
Based on the band characters of Wx-mq gene, lines of F5, F6 derived from Wu-xiang-jing 14/Kantou 194 were detected with molecular marker at tillering stage. All the three band types were detected in the 30 rice lines tested. Among them, 6 lines were homozygotes of Wx-mq with two bands of 215bp and 170bp, 6 lines were heterozygotes of Wx-mq with three bands of 385bp, 215bp and 170bp, and 18 lines were genotypes without Wx-mq gene with only one band of 385bp (Figure 5). These results were completely consistent with the investigation results of endosperm appearance. Subsequently, 11 semi-glutinous plants in homozygous plots with Wx-mq gene and 13 non-glutinous plants in homozygous plots without Wx-mq gene were selected to generate F6 lines. MAS was also performed at tillering stage of F6 generation. The results showed that two bands of 215 bp and 170 bp were detected in all the 11 plots derived from the 11 semi-glutinous plants. And only one band of 385 bp was detected in 12 plots derived from the 12 non-glutinous plants except for one plot showed two bands of 215bp and 170bp (Figure 6). The identification result of endosperm appearance was also completely consistent with that of MAS. The above results indicated that this CAPS marker was perfectly effective in assisted selection for Wx-mq gene. The efficiency of molecular selection with the CAPS marker for Wx-mq gene in lines of F5, F6 derived from Wu-xiang-jing 14/Kantou 194 was up to 100% (Table 1).
Table 1 Efficiency of assistant selection for Wx-mq genotype with the CAPS marker in F5 and F6 lines derived from Wu-xiang-jing 14/Kantou 194
Genotype / Generation / No. of plants / No. of plants coincident with identification of endosperm / Coincidence rate (%)Wx-mqWx-mq / F5
F6 / 6
12 / 6
12 / 100
100
Wx-mqWx-b / F5
F6 / 6
- / 6
- / 100
-
Wx-bWx-b / F5
F6 / 18
12 / 18
12 / 100
100
After two years selection with molecular marker, a good eating-quality and high-yield rice line with stable characters in agronomical traits and resistance to rice stripe disease was bred, designated tentatively as ‘Ning 5047’. The plant height of ‘Ning 5047’ was 98~102 cm with 8~10 panicles per plant. The panicle was erect and 16 cm long with 140~150 grains per panicle. Its seed-setting rate was 90%~92% and 1000-grain weight was 25~26 g. Furthermore, the identification result of Food Quality Inspection and Testing Center (Wuhan), Ministry of Agriculture indicated that its quality could reach the second-class in national standard. It combined the fragrance of Wu-xiang-jing 14 and low-amylose content of Kantou 194, so the cooked rice had excellent taste with glossy appearance, good glutinousness and flexibility. ‘Ning 5047’ took the first place continuously in contest of taste and evaluation for palatability in Jiangsu Province in both 2006 and 2007 and was honoured as ‘the best rice for eating’ (Wang et al., 2008a). In August 2007, ‘Ning 5047’ won excellence award in the national contest of taste and evaluation for palatability in Shenyang, and became the only variety in southern rice region which ascend into the top ten rice varieties. The composite score of ‘Ning 5047’ was 81.5 points, just 4.21 points lower than that of ‘Koshihikari’, which is considered the best eating quality rice in Japan, and 11.5 points higher than that of ‘Wu-yu-jing 3’, which is well known for good eating quality in Jiangsu (Sun, 2007). Furthermore, this variety was also outstanding in other characters, such as higher and stable yield performance, resistance to rice stripe disease. Its grain yield could exceed 9 ton per hectare in normal cultivation conditions, and reach up to 10.5 ton per hectare in high yield cultivation. In the regional trial in Jiangsu province, its average yield was 9.12 ton per hectare in two years, which was 5.6% higher than that of the check variety ‘Wu-yun-jing 7’. In 2007, the average yield was 8.86 ton per hectare in productivity trial, 3.0% higher than the check variety (Wang et al., 2008a).