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
Author    Correspondence author
Rice Genomics and Genetics, 2010, Vol. 1, No. 3   doi: 10.5376/rgg.2010.01.0003
Received: 22 Oct., 2010    Accepted: 21 Nov., 2010    Published: 25 Dec., 2010
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Wang et al., 2009, Development of a New japonica Rice Variety Nan-jing 46 with Good Eating Quality by Marker Assisted Selection, Molecular Plant Breeding, 7(6): 1070-1076


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 NIaâ…¢ 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.

Marker assisted selection; Good eating quality; Rice; Cleaved amplified polymorphic sequences (CAPS)

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

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 1999 at Hainan province. F1 plants were grown at respectively to make cross combination in winter of 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).

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 ( Luckily, thechange of G-A in exon 4 just produced a recognition site for NIaâ…¢ 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

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 215 bp and 170 bp bands, when digested completely by NIaâ…¢ (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 385 bp 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 Niaâ…¢ 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

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 215 bp and 170 bp, 6 lines were heterozygotes of Wx-mq with three bands of 385 bp, 215 bp and 170 bp, and 18 lines were genotypes without Wx-mq gene with only one band of 385 bp (Figure 5). 

Figure 5 Detection of Wx-mq genotype with the CAPS marker for F5 lines derived from Wu-xiang-jing 14/Kantou 194

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-gluti-nous 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 215 bp and 170 bp (Figure 6). 

Figure 6 Detection of Wx-mq genotype with the CAPS marker for F6 lines derived from Wu-xiang-jing 14/Kantou 194

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

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).

2 Discussion
Molecular marker is a detectable special DNA fragment or allelic variation in organism. The polymorphism revealed by molecular marker can directly reflect the variation of DNA in genomes. It offers many advantages over other categories of markers such as morphological, cytological or biochemical markers. For example, it is characterized by high accuracy, which is not limited by plant position, season and environment, and it is large in quantity, which can cover the whole genome. Moreover, DNA marker is ‘neutral’, which has no effect on phenotype and no inevitable linkage with undesirable characters. Furthermore, some DNA markers are co-dominant and show a mass of genetic information (Fang et al., 2002). Consequently, crop breeders pay more and more attention to MAS. In recent years, some great achievements of marker-assisted breeding have been obtained regarding resistance to rice blast, quality breeding, utilization of yield-enhancing QTLs, especially resistance to bacterial blight (Feng, 2006).

With the rapid development of molecular biology, a great many important genes or QTLs have already been mapped on chromosomes, and some molecular markers closely linked have also been screened. These markers should be very useful for indirect selection in rice breeding. However, only a few varieties or lines have been bred by MAS so far. The reason can be explained in such aspects. Firstly, major genes or loci discovered aren’t enough for marker-assisted breeding. Secondly, the genetic distance between gene and marker is too far to select effectively in rice breeding. Finally, the basic research is not correlated with breeding. It is well known that populations used for gene mapping always derive from indica-japonica crosses, but markers in these population show no polymorphism in breeding population and cann’t be applied in breeding practice. Therefore, the best method resolving this problem is to develope markers based on the sequence variation of target gene.

The CAPS marker in this study was designed from the allelic diversity in Wx locus, and applied to detect a mis-sense mutation (G-A) in exon 4. It was directly related to mutated gene of translucent endosperm and could distinguish different genotype of Wx-mq, so its selection efficiency was up to 100%. Our breeding practice has proved that the selection efficiency of molecular marker directly related to gene itself is very ideal.

Certainly, translucent endosperm is a simple character for selection. It can be recognized easily by mature seed. The superiority of CAPS marker in this study is that it can distinguish Wx-mq genotypes in heterozygote from that in homozygote at seedling stage. However, to those characters which cann’t be identified by phenotype or expressed only in special conditions, the advantage of MAS seem more significant in breeding.

3 Materials and Methods
3.1 Materials
Japonica variety ‘Wu-xiang-jing 14’ with high yield was bred by Wujin Rice and Wheat Experiment Station in Changzhou City, Jiangsu province, good taste japonica variety ‘Kantou 194’ with Wx-mq gene and resistance to rice stripe disease (RSV) was introduced from Japan. The plant lines of F4~F7 derived from the combination of Wu-xiang-jing 14/Kantou 194 were used as materials in this study. Kantou 194 and Milky Queen with Wx-mq gene, Te qing, Ju-feng-zhan and Su-yu-nuo carrying Wx-a, Wx-b and wx genes, respectively, and the F1 hybrid seeds derived from the crosses between Kantou 194 and four varieties mentioned above were used for verifying the genotypes of endosperm for Wx-mq gene with the molecular marker.

3.2 Development of the breeding lines and their cultivation

Wu-xiang-jing 14 was used as female parent and crossed with Kantou 194 as male parent. F2 and F3 seeds were harvested by bulk method. Individual selection was carried out from the F4 generation. All experiment materials were sowed on 15th to 18th in May at the experimental field of Institute of Food Crops, Jiangsu Academy of Agricultural Sciences (JAAS), and then transplanted after a month. The number of plants for F2, F3 and F4 population were 600, 1 000 and 2 000, respectively. From the F5 generations, 40~50 plants were cultivated in a plot. The plant spacing was 17 cm×17 cm, and a blank row was kept between every plot. Field management was applied periodically as required.

3.3 Total DNA extraction from leaves
Leaf samples were taken from rice plant for every experimental plot of F4~F5 lines in 30 days after transplant. Genomic DNA was isolated from leaves following a method as described by Dellaporta et al. (1983). The detailed steps of DNA extraction were as follows: Place 0.5~2.0 g plant leaves in a mortar and add an excess of liquid nitrogen. When the nitrogen evaporates, grind the leaf tissue thoroughly into fine powder and transfer the powder from the mortar into a 1.5 mL Eppendorf tube. Add 600 μL DNA extraction buffer (20% SDS, 1 mol/L Tris-HCl, 0.5 mol/L EDTA, 5 mol/L NaCl) and mix thoroughly. Incubate the tube at 65°C in a water bath for 30 min, intermittently invert the tube several times gently. Add 1/4 volume of 5 mol/L potassium acetate, and incubate tubes on ice for at least 30 min. Add 300~400 μL chloroform-isoamyl alcohol mixture (24:1), oscillated on a shaking table at 120 r/min for 30 min. Centrifuge at 8 000~10 000 r/min for 15 min at 4°C. Transfer the supernatant (400 μL) to a new tube. Add an equal volume of chloroform-isoamyl alcohol mixture (24:1) and oscillate on a shaking table at 80~90 r/min for 30 min. Centrifuge at 8 000 r/min for 15 min at 4°C, and then transfer the supernatant (400 μL) to a new tube. Precipitate the DNA by adding 2 volume of ice-cold ethanol and intermittently invert the tube several times gently. Centrifuge at 12 000 g for 6 min at 4°C. Discard the ethanol and wash the DNA pellet with 200 μL ice-colded 70% ethanol. Air dry and dissolve the DNA in 100~200 μL TE buffer and store at -20°C until using.

3.4 PCR amplification, NIaâ…¢ digestion and electrophoresis detection
PCR amplification used a pair of CAPS primers (forward sequences: 5’-TGTGGCTGAGGTAGGAGCA-3’; reverse sequences: 5’-AACGCATCTGGTTGTCTTTGT-3’). A 10 μL PCR reaction mixture was composed of DNA (10 ng/μL) 1 μL, Primers (4 pmol/μL) 0.8 μL, 10×Buffer 1 μL, MgCl2 (2 mmol/L) 0.6 μL, dNTP (2.5 m mmol /L) 0.2 μL, Taq (5 U/μL) 0.1 μL, and ddH2O 6.3 μL. Reaction was performed in MJ Reseach PTC-200 as follows: initial denaturation at 94°C for 10 min; then 35 cycles of 94°C for 30 s, 52.2°C for 30 s, 72°C for 1 min, with a final extension step at 72°C for 7 min, then stored at 12°C until using.

A 20 μL reaction system digested by restriction endonuclease NIaâ…¢ was composed of PCR production 2 μL, endonuclease NIaâ…¢ 2 μL (5 U/μL), 10×NEB Buffer 2 μL, 100×BSA 0.2 μL, ddH2O 13.8 μL. Reaction was performed in MJ Reseach PTC-200 at 37°C for 4 h, and digestion products were visualized in 1% agarose gels and stained with ethidium bromide.

3.5 Identification of the characteristics of endosperm
To evaluate the effect of molecular marker assisted selection, endosperm appearance of individuals in the homozygous plot of Wx-mq genotype detected by MAS were identified. If endosperms in all plants show mist trait, milky white and translucence in appearance, we can conclude that this plot was homozygous genotype of Wx-mqWx-mq.

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