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Chilean journal of agricultural research

On-line version ISSN 0718-5839

Chilean J. Agric. Res. vol.75 no.4 Chillán Dec. 2015

http://dx.doi.org/10.4067/S0718-58392015000500002 

RESEARCH

Evaluation of genetic effect on physiochemical properties changes of Wx near isogenic lines of Y58S in rice

Wentao Sheng1, Lijie Zhou2, Jun Wu2, Bin Bai2, and Qiyun Deng2*

1 Nanchang Normal University, Department of Biological Technology, Nanchang 330032, Nanchang, China. *Corresponding author (dqy900@gmail.com).
2Hunan Hybrid Rice Research Center, Changsha 410125, Hunan, China.


The Wx gene primarily influences the physiochemical properties changes of rice (Oryza sativa L.) To improve the grain qualities of the cv. Y58S with low amylose content (AC) value, the genetic effect of Wx gene was explored on quality traits. With the BC3F2 genetic population of Y58S line associated with different AC value as the materials, this study reported the genetic effect of three different Wx alleles (Wxa, Wxin, Wxb) under near-isogenic background. It was shown that Wx had major effects on the quality traits, and the genetic effect value was in the order Wxa > Wxin > Wxb. In addition, during the course of physiochemical properties changes of higher AC rice cultivars determined by Wxa or Wxin allele, accompanied by the increasing of AC, gel consistency and head milled rice recovery would be decreased in combination with the rising of chalky traits, all of which resulted in substantial fluctuations on quality traits (p < 0.01). However, the genetic law was not evident for Wxb allele. Therefore, except for the major gene Wx, the minor genes in the regulative network of starch-synthesis might be utilized for quality improvement.

Key words: Amylose content, Oryza sativa, physiochemical properties, quality improvement, Wx.


INTRODUCTION

The genetic effect evaluation of the major gene is very meaningful for quality improvement of rice (Oryza sativa L.) It was reported that Wx gene was a predominant determinant for the synthesis of amylose content (AC), which caused marked influence on the comprehensive physiochemical traits including AC, gel consistency (GC) and gelatinization temperature (GT) (Tian et al., 2010). In general, the cooking quality is a major criterion in evaluating rice grain trait, which is determined by the starch composition and correlated with the changes of AC, GC, and GT values. The rice grain with higher AC value demonstrates dry, fluffy and separated cooked rice features, representing poor cooking qualities. And in south of China, people tend to have cooked rice associated with intermediate AC, soft GC, and low GT.

Currently, a series of Wx alleles including Wxa, Wxin, Wxb, Wxop, and wx had been confirmed, which could bring about the AC change (Qiao et al., 2012). Mikami et al. (2008) found that these five alleles were directly related to high, intermediate, low, soft, and glutinous rice cultivars individually, by analyzing the genetic rule of AC with Wx isogenic lines of 'T65'. And Wx gene had the pleiotropic roles, which not only controlled the changes of AC and GC, but also influenced the GT value (Tian et al., 2009). Ni et al. (2011) reported that the introduction of Wxb allele into the parent genetic background of the hybrid 'Xieyou 57', resulting in AC reduced to lower level and the whole quality of the hybrids had been greatly optimized. Meanwhile, 'Manawthukha', a conventional high AC value cultivar, was obviously improved on quality traits, by importing Wxm from 'Basmati370'. Furthermore, little negative effect was caused on the yield characteristics (Myint et al., 2009). Thus, findings described herein showed that it might be feasible to improve grain quality by the method of substitution of Wx alleles. However, the study of Li et al. (2014) indicated that a complicated correlation existed on the whole qualities, where the value of chalkiness was associated with the change of AC, GC, and the head rice recovery in different introgressing lines of chalky gene. And we can also see that there was still short of the comprehensive analysis for the relationship between the selection of different Wx alleles and quality traits from the appearance, the milling and cooking qualities. Therefore, through the genetic material established using Wx near isogenic lines (Wxa, Wxin, and Wxb; NILs), the genetic effect would be assessed on Wx alleles and the comprehensive quality traits involving the relationship between AC and Wx genotype. The present study was conducted to provide theoretical clues for the quality improvement of Y58S line with low AC value by utilization of Wx alleles in rice.

MATERIALS AND METHODS

Plant material
Four rice lines were used as the parents in this study: (1) Y58S, the backbone two-line sterile line for a lot of elite hybrids grown in China, was used as the receiver of Wx gene; (2) 'Teqing', 'Starbonnet99', and 'Yuejingsimiao 2' were used as donor of Wx alleles determining high, intermediate, and low AC, respectively. A cross was made between Y58S and three donors, followed by three backcrossing with Y58S as the recurrent parent and one self-pollination. In the generation of BC3F2 population, the materials were classified and the mean values of quality traits were compared according to the Wx genotype.

DNA marker analysis
On the basis of the diversity of (CT)n microsatellite in leader region and the first base G/T site (In1 (G/T)) polymorphism in the first intron of Wx (Ayres et al., 1997; Teng et al., 2012), the genotype of 'Teqing' ((CT)11-G) was Wxa associated with high AC value; the 'Starbonnet99' genotype ((CT)20-G) belonged to Wxin connected with intermediate AC value; 'Yuejingsimiao 2' ((CT)17-T) owned Wxb allele relevant with low AC value; the Y58S genotype ((CT)18-T) was Wxb correlated with low AC value. Utilization of the characteristics of In1 (G/T) site cleaved by restriction endonuclease AccI, the primer Wx-I (F:5'-GTTGGAAGCATCACGAGTTT-3'; R:5'-CGTCGTTGCAGACGAACA-3') was devised to discriminate the genotype of Wxa and Wxin (Cai et al., 2002; Ni et al., 2011). Meanwhile, in order to distinguish the genotype of Wxb between 'Yuejingsimiao 2' and Y58S, the Wxb genotype of 'Yuejingsimiao 2' and Y58S was designated as Wxyy and Wxy, respectively. The polymorphic microsatellite markers found upstream and downstream of intragenic marker RM190, were used to differentiate the genotype of Wxyj and Wxy (Myint et al., 2009).

Measuring quality traits
In the light of the genotype, quality traits were determined using grain sample harvested from the genetic population at physiological maturity of Changsha Experimental Station (27°51' N, 111°53' E) in Hunan Province of south China in 2014. The dried grains were stored naturally in a greenhouse for 3-mo prior to the analysis. The quality traits were evaluated with three replicates following the procedures included in the Agricultural Industry Standard NY/T593-2013 (2013) issued by the Ministry of Agriculture, PR China. Briefly, for the milling quality, the rate of the whole rice after milling was a main parameter used to evaluate quality and efficiency of the milling process. For the appearance trait, it was calculated on the proportion between the area of chalkiness and the grain size. And for the cooking quality, AC of milled rice was measured by the colorimetric method with iodine and potassium iodide (I2-KI) staining. The GC was assessed by measuring the flow length of rice glue. And GT was evaluated on the basis of the alkali spreading value (ASV), such that the higher GT is considered to be lower ASV value. All traits composed of brown rice ratio (BRR), milled rice ratio (MRR), head milled rice recovery (HMRR), milled rice length (MRL), milled rice width (MRW), chalky rice ratio (CRR), chalkiness (Ch), GT, GC, and AC, were subjected to statistical analysis using the SPSS 17.0 software program (SPSS, Chicago, Illinois, USA). ANOVA was performed using all the mean value of data in all traits at one percent probability level.

RESULTS

Genotyping using Wx markers
PCR product size of Wx-I amplification was a 423 bp fragment containing the splicing site in tested materials. Upon digestion with AccI, the amplified product of Wxa or Wxin types produced two fragments of 275 and 148 bp; the PCR product of Wxb type could not be digested and only one fragment of 423 bp was revealed; the heterozygous genotype gave rise to three bands of 423, 275, and 148 bp (Figure 1). Justified by the physical position RM190 (1764661 ~ 1764696 bp), the microsatellite markers RM589 (1380931 ~ 1380978 bp) and RM19350 (1967182 ~ 1967205 bp) had been found to distinguish the genotype between 'Yuejingsimiao 2' and Y58S (International Rice Genome Sequencing Project, 2005). Polymorphisms were identified between alleles based on the number of microsatellite repeat, two kinds of DNA profiles ranging from 148 and 138 bp and 197 and 191 bp between Y58S and 'Yuejingsimiao 2' in RM589 and RM19350, respectively (Figures 2 and 3).


Figure 1. Wx genotype was detected by Wx-I after AccI digestion in BC 3F2 population of Y58S.


1: Y58S; 2: 'Teqing' ('Starbonnet99'); 3 ~ 5: homozygous genotype (Wxa or Wxin) of individual plant in BC3F2 population;
6 ~ 7, 13: homozygous genotype (Wxb) of individual plant;
8 ~ 10, 12, and 15: heterozygous genotype of individual plant; M: 100 bp ladder fragment.

Figure 2. BC3F2 segregation population of Y58S/Yuejingsimiao 2 associated with Wx linked marker RM586.

1: Y58S; 2: 'Yuejingsimiao 2'; 3 ~ 15: segregation individual of BC3F2 population; M: 100 bp ladder fragment.



Figure 3. BC3F2 segregation population of Y58S/Yuejingsimiao 2 associated with Wx linked marker RM19350.



1: Y58S; 2: Yuejingsimiao 2; 3 ~ 15: segregation individual of BC3F2 population; M: 100 bp ladder fragment.
Note: The individual plant was synchronously detected with RM586 and RM19350. For the homozygous genotype (Wxy),
the makers RM586 and RM19350 had the same band type with Y58S; for the homozygous genotype (Wxyj),
the makers RM586 and RM19350 had the same band type with 'Yuejingsimiao 2'; for the heterozygous genotype (Wxy/Wxyj),
the makers RM586 and RM19350 had heterozygous band types between 'Yuejingsimiao 2' and Y58S, respectively.

Comparison of quality traits among different genotypes
Analysis of quality traits from different genotypes is presented in Table 1. The result showed that these substitution lines caused dramatic influence on HMRR, chalky traits, AC, and GC, especially for these materials with Wxa or Wxin genotype. For the BC3F2 population (Y58S/Teqing), as compared to the homozygous dominate Wxa/Wxa and recessive Wxb/ Wxb genotype, the obvious difference in these traits of AC, GC, chalky traits were found for the heterozygous genotype (Wxa/Wxb) (p < 0.01). As for the trait of HMRR, the difference between Wxa/Wxa and Wxa/Wxb genotype did not reach significant level (p < 0.01). It was postulated that they might be associated with higher AC producing similar effect on grain qualities, and the difference of the comprehensive effect were nearly identical for this trait of these materials without significance. For the BC3F2 population (Y58S/Starbonnet99), compared with the homozygous dominate Wxin/Wxin and recessive Wxb/ Wxb genotype, noticeable differences were shown in these traits of AC, GC, chalky traits, and HMRR for the heterozygous genotype (Wxin/Wxb) (p < 0.01). But, the difference among these traits comprising of BRR, MRR, MRL, MRW, and ASV did not reach significant level in the two populations. From data, it was confirmed that the alleles of Wxa and Wxin could have distinct impact on HMRR, chalky traits, GC, and AC, inducing the comprehensive negative effect on quality. For the BC3F2 population (Y58S/'Yuejingsimiao 2'), by comparing with the homozygous genotype between Wxyy/Wxyy and Wxy/ Wxy, no dramatic difference was revealed on the quality characteristics for the heterozygous genotype (Wxyy/Wxy). The result indicated that different types of Wxb alleles had the same genetic effect on the phenotype alteration of the comprehensive grain traits.


Table 1. Quality traits analysis of Wx gene in BC3F2 population of Y58S.



BRR: Brown rice ratio; MRR: milled rice ratio; HMRR: head milled rice recovery; MRL: milled rice length; L/B ratio, milled rice length/width ratio;
CRR: chalky rice ratio; Ch: chalkiness; ASV: alkali spreading value; GC: gel consistency; AC: amylose content.
Data for the three combinations with a column followed by different lower (upper)
case letters indicated significant difference among the three combinations at 5% (1%) probability levels, respectively.


Correlation analysis among different quality traits
For the milling quality, HMRR was significantly positively correlated with MRL, the milled rice length/ width ratio (L/B ratio), ASV and GC (0.794**, 0.771**, 0.805**, 0.952**), which showed that the increasing of MRL, ASV and GC occurred with the raising of HMRR, simultaneously (Table 2). However, HMRR was negatively linked with CRR, Ch, and AC (-0.972**, -0.984**, -0.988**), which revealed that along with the increasing of CRR, Ch, and AC, HMRR would be decreased remarkably. For the appearance quality, MRL was significantly positively related with L/B ratio, ASV and GC (0.910**, 0.941**, 0.699**), and also was negatively associated with CRR, Ch, and AC (-0.693**, -0.750**, -0.753**), which indicated that the increment of MRL and L/B ratio would result in lowering the chalkiness and AC values. For the chalky traits, CRR was significantly positively correlated with Ch and AC (0.980**, 0.960**), which suggested the chalky traits would be increased due to the augment of AC. And CRR was significantly negatively correlated with ASV and GC (-0.732**, -0.943**), which showed that the rice cultivars with low GT and soft GC had the tendency of owning the low chalky traits (Table 2). For the cooking traits, AC was significantly negatively relevant with ASV and GC (-0.795**, -0.975**), which revealed these traits of AC, GC, and GT might be controlled by Wx gene or else in a region very tightly linked to this gene (Table 2). As above mentioned, the present result illustrated a complex paradox existed between these quality traits. Therefore, this complex linkage correlation should be broken for obtaining superior qualities during the course of rice breeding.

Table 2. Coefficient of phenotypic correlation between grain quality characteristics in Wx isogenic lines of Y58S BC3F2 genetic population.



**Significant at 1% probability level. BRR: Brown rice ratio; MRR: milled rice ratio; HMRR: head milled rice recovery;
MRL: milled rice length; L/B ratio: milled rice length/width ratio; CRR: chalky rice ratio; Ch: chalkiness; ASV: alkali spreading value;
GC: gel consistency; AC: amylose content.

DISCUSSION

The Wx gene had been recognized as a major determinant of grain quality, which impacted the wide range of variation on AC value. As it is well known, the inheritance of AC is very complicated due to the influence of modified genes, cytoplasmic effects, the triploid nature of the endosperm, environmental temperature, and genetic background (Tian et al., 2009; Ardashir et al., 2012). To minimize the genetic background affecting the magnitude and quantity of the quality trait, we used Wx NILs in the population of BC3F2 generation, which had three divergent alleles effecting starch properties. Among these combinations, the range of AC values were as follows (Wxa/Wxa > Wxlnl Wxm > Wxb/Wxb); the magnitude of AC variations in different types of Wxb/Wxb genotype was low and nearly the same; the genetic effect of heterozygous genotype was between two homozygous genotypes. It was just accorded with the expression ability of granule binding starch synthetase (GBSS) associated with different Wx genotypes (Mikami et al., 2008). And the study confirmed the complex relationship between AC and different genotypes furthermore.

Up to now, there was some disagreement about the role of Wx gene on GC. Huang et al. (2000) reported that GC was predominately determined by the gene on chromosome 3. Besides this major gene, an epistastic genetic site was found to affect GC. The study of Tian et al. (2010) demonstrated that not only AC and GC were controlled by Wx gene, but also GT was influenced to some extent. Through positional cloning, it was found that GC was determined by Wx gene (Su et al., 2011). In this study, from the genetic rule of quality traits in different substitution lines, it was further proved that Wx gene had pleiotropies and there was negative correlation between AC and GC. Moreover, the substitution of Wx allele would not produce obvious effect on GT value, but a distinct negative correlation existing between AC and ASV value. As Wx was a major gene controlling both AC and GC but a minor gene affecting GT, while the SSIIa gene was a key determinant responsible for GT but a minor gene influencing AC and GC, all of which were tightly linked on the short arm of Chromosome 6 (Zhou et al., 2003; Tian et al., 2009). And this result was in agreement with the study of Tian et al. (2009), which indicated there was a positive relationship for the change of AC and GT. But surprisingly, the study of Sun et al. (2005) showed that there was no obvious relevance for AC and GT. Presumably, the difference was correlated with the genotype of the experiment materials, which might have the same haplotype combination for Wx and SSIIa gene.

Usually, rice grain quality is a comprehensive trait including the appearance, milling, cooking, and nutritional qualities. The high chalky traits influences the appearance, and the low HMRR decreases the milling quality, both of which results in poor rice quality. In this study, the Wx gene region associated with high or intermediate AC was successfully transferred into Y58S by selection of In1(G/T) site of Wx gene. The data showed that when AC was profoundly increased, two dramatic changes occurred with an accompanied reduction on GC and HMMR and an obvious increment of chalky traits. However, these genetic changes were not obvious for Wxb types in the BC3F2 population. It was possible that rice strains with the Wxb allele had low amylose, in contrast to rice strains of higher AC with the Wx* or Wxn allele, which had low genetic effects on the AC value (Teng et al., 2012). And Zhou et al. (2003) found that when Wx* allele was substituted by Wxb, it caused the decreasing of AC and chalky traits and the increasing of GC and HMRR, contributing the whole optimization of quality traits. Therefore, a complex correlation for Wx alleles was also defined among these quality traits. Given that the rice endosperm was mainly comprised with amylose and amylopectin, and the ratio between amylose and amylopectin was an important physiochemical indicator influencing rice quality. And it is well documented that the synthesis of amylose and amylopectin in endosperm was composed of a complex regulative network including ADP-glucose pyrophosphorylase, GBSS, soluble starch synthase, starch branching enzymes and starch de-branching enzymes. Each enzyme had rich variant isoforms, which was synthesized by different alleles. And these alleles functioned in different stages during starch synthesis, suggesting that the genetic control of starch synthesis was very complicated (Tian et al., 2010; Ardashir et al., 2012). Meanwhile, there were obvious fluctuations in physicochemical properties arising from selection of any single gene (Tian et al., 2009). In view of other regulative genes have been identified to produce minor effect on the fine structure and characteristics of starch, optimization of different haplotype combinations of minor genes might be an ideal choice for improving grain quality of rice.

CONCLUSION

In this study, Wx as a major gene influencing the quality traits, we found that the genetic effect value was in the order Wx* > Wxn > Wxb among these alleles, and the genetic effect caused by heterogeneous genotype was among homogenous dominate and recessive genotype. Meanwhile, during the course of physiochemical indexes changes for Wx* or Wx"1 alleles, accompanied with the rising of amylose content, gel consistency and the head milling rice recovery would be decreased, resulting in the increasing of the chalky traits, all of which caused noticeable fluctuations on quality traits. But for Wxb allele, the genetic effect was not influential on the comprehensive physiochemical properties.

ACKNOWLEDGEMENTS

This research was financially supported by the National High-tech Program ('863' program) of P.R. of China (Grant nr 2012AA101103).

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Received: 23 December 2014.
Accepted: 25 July 2015.



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