Performance and Inheritance of Rice Starch RVA Profile Characteristics

Transcription

1 Rice Science, 5, 12(1): Performance and Inheritance of Rice Starch RVA Profile Characteristics YAN Chang-jie, LI Xin, ZHANG Rong, SUI Jiong-ming, LIANG Guo-hua, SHEN Xin-ping, GU Shi-liang, GU Ming-hong (The Key Laboratory of Education Ministry for Plant Functional Genomics, Yangzhou University, Yangzhou 2259, China) Abstract: The classification and inheritance of rice starch RVA profiles were studied by testing RVA profiles of F 2 clonally propagated population and some varieties, including japonica, indica, and glutinous types. All the RVA profiles of accessions could be divided into six kinds based on the cluster analysis, that is A, B, C, D, E and F. Feature profile of sticky variety was type A. The profiles of indica varieties could be presented as B, C, D, E and F types and that of japonica varieties fell into D, E and F types. RVA profiles of the rice were mainly affected by apparent amylose content (AAC), but the difference of RVA profiles could also be detected in the varieties with similar AAC. The close relationship between RVA profile indices and sensory of low or middle AAC varieties suggested that the RVA profiles can be used as indirect index to evaluate eating and cooking quality. In four hybrid combinations, the component indices of RVA profiles of F 1 were between those of its parents except peak viscosity and pasting temperature. Also, in F 2 clonally propagated population, all the components of the RVA profiles showed continuous segregation, suggesting that all of them were controlled by a series of minor genes, although Wx gene may play an important role in determining the RVA profiles. These results may provide some new information useful for rice quality breeding program. Key words: rice; RVA profiles; apparent amylose content; eating quality; F 2 clonally propagated population Rice is the main staple food for over half of world s population. In China, rice breeders presently have focused on rice quality improvement, especially eating and cooking attributes. Because starch contributes about 9% to milled rice, rice eating and cooking quality is mainly influenced by the starch properties. So understanding the genetic behavior of starch properties and finding an index to evaluate the eating and cooking characteristics, will contribute to the successful identification and development of lines with good quality in rice breeding. It has been widely documented that rice eating and cooking quality were mainly affected by two components, apparent amylose content (AAC) and fine structure of amylopectin. In previous research the genetic control of AAC, gelatinization temperature (GT), gel consistency [1, 2] [3, 4] and paste viscosity were also studied. After marketing of Rapid Visco-Analyser (RVA) developed by New Scientific Instruments Pty Ltd, it was widely used to evaluate Received: 8 November 4; Accepted: 5 January 5 the starch properties of wheat, maize, and rice due to its many merits. It was reported that some characteristics of RVA, such as breakdown and setback, were closely related to eating and cooking quality of rice [5, 6], and RVA profile might be used as an indirect index to evaluate the rice grain quality, which would be helpful for selection of lines with good quality in early generations in rice breeding program. Although the paste viscosity of rice flour was studied by many scientists [4, 7, 8], yet the number of varieties involved was small, and comprehensive information of genetic behavior of paste viscosity of rice was too limited yet. In this study, we focused on the investigation and classification of rice paste viscosity of more than hundred varieties, including japonica, indica and glutinous types, and try to find the relationship between the RVA profile and sensory taste. The genetic behavior of RVA profiles was also studied based on an F 2 clonally propagated population and 4 indica-type hybrid combinations.

2 Rice Science, Vol. 12, No. 1, 5 Plant materials MATERIALS AND METHODS Three sets of rice plant materials were applied in this experiment. Set-one included 46 indica varieties, 42 japonica varieties, 13 glutinous ones, and 4 indica-type hybrid combinations. Set-two, Wx antisense RNA transgenic lines, including 7 lines derived from japonica variety Wuyunjing 7, coded as B21 27, and 5 lines from Wuyunjing 8, as B The AAC of each line was showed in Table 2. Set-three was an F 2 clonally propagated population, which was derived from Balilla (japonica) and Dular (indica) hybrid by tissue culture method [9]. A total of 188 fully fertile lines were chosen for further quality study. All the materials were raised during the growing season 2, at the farm of College of Agriculture, Yangzhou University. The seeds of many varieties, F 1, and segregation population were harvested at maturity stage from 5 plants in the middle part of plot, then dried and stored at room temperature ( to 26 ) for about 3 months. Rice samples were firstly milled to white rice using a Satake Rice Machine (Satake Corporation, Japan), and then ground to flour with a Cyclone Sample Mill (UDY Corporation, Fort Collins, Colo., USA) and sieved through a 1-mesh. Rice quality assays AAC and sensory tests were conducted according to the national standard of People s Republic of China (GB ) [1]. The eating quality was presented by synthetic evaluation, including odor, 15% (peculiar smell, 6%; perfume, 9%); appearance, 15% (gloss, 5%; hue, 5%; integrality degree, 5%); palatability, 6%, (hardness, %; adhesiveness, %; flavor, %); and texture of cool rice, 1%. Rice viscosity (RVA profile) was measured by Rapid Visco-Analyzer model 3D developed by Newport Scientific Corporation, and analyzed with Thermal Cycle for Windows software according to American Association of Cereal Chemists Standard Method AACC 61-2 [11]. Three-gram flour of each sample was weighed into aluminum canister when the samples were at 14% moisture, to which 25 ml distilled water was added. A paddle was placed in the canister, and its blade was vigorously jogged up and down through the sample for ten times. The RVA disperses the samples by rotating the paddle at 96 r/min for the first 1 s of the test, after which the viscosity is sensed using a constant paddle rotation speed of 16 r/min. The idle temperature is set to 5, and the following 12.5-min test profiles were run: 1) 5 held for 1. min, 2) the temperature is linearly ramped up to 95 until 4.8 min, 3) the temperature is held at 95 until 7.5 min, 4) the temperature is linearly ramped down to 5 at 11 min, and 5) held at 5 until 12.5 min. Heating and cooling were linearly ramped between the profile set points. Before each assay, calibration was carried out by using the standards. RVA curve was described by six parameters, i.e. peak viscosity (PKV), hot paste viscosity (HPV) or trough viscosity, cool paste viscosity (CPV) or final viscosity, breakdown (defined as subtracting the HPV from PKV), setback (defined as subtracting PKV from CPV) and consistence (defined as subtracting HPV from CPV). All the viscosity parameters were expressed in Rapid Visco Units (RVU). Statistic analysis Non-hierarchical cluster analysis, with min (SSw) algorithm was used to partition the materials into different groups, as described [12], and χ 2 test was carried out using SAS software. RESULTS Performance of the RVA profiles in rice flour Types of RVA based on 11 varieties In general, remarkable difference existed among all varieties assayed for six parameters, and the profile type could not be distinguished simply according to the japonica or indica properties, suggesting that there were multiple alleles existing in indica and japonica subspecies, which were involved in starch synthesis pathway. Nevertheless for glutinous varieties, significant difference was detected for indica and japonica ones. The pasting times of glutinous varieties

3 YAN Chang-jie, et al. Performance and Inheritance of Rice Starch RVA Profile Characteristics 41 were shorter than those of typical indica and japonica varieties, and retro-gradation phenomenon was not found in the process of cooling too. Due to the low amylose content or no amylose in glutinous rice endosperms, the interaction of starch granule was very little. Even at relatively low temperature, the interaction among starch granules could be easily disrupted. So the pasting curves of glutinous varieties exhibited that the temperature at peak viscosity was lower than those of indica and japonica ones, and almost no retro-gradations were found, resulting in very low final viscosity. In order to evaluate the RVA profiles of japonica, indica and glutinous varieties further, cluster analysis was carried out based on the min (SSw) method. One hundred and one varieties could be divided into six groups (Table 1), i.e. A, B, C, D, E, and F. Table 1 showed that all glutinous varieties were included in A group, while the indica varieties presented in 5 groups except A, and japonica varieties fell into D, E and F groups, suggesting that variation for starch property was widely existed among rice germplasm, either in japonica or indica varieties. The cluster analysis results also demonstrated that the degree of RVA profiles variations in indica varieties was much larger than those of japonica. These results were helpful for identifying favorable resources in high quality rice breeding. On the other hand, B and C groups consisted of indica varieties only, rather than japonica varieties. When compared the B and C groups to D, E and F groups, we found that the differences existed in BDV, CSV and SBV, the BDVs of B and C groups were smaller than those of D, E and F, while SBVs and CSVs were larger than those of the latter. But for Fig. 1. RVA profiles of varieties only different in AAC. other parameters, no obvious trend could be observed. The result suggested that BDV, CSV and SBV might be the main factors related to the difference in eating and cooking quality between indica and japonica varieties. RVA profiles of varieties differing in AAC In order to understand the effect of AAC on RVA profiles, several transgenic lines and their parents were chosen to assay their RVA profiles; these transgenic lines had been transformed with antisense Wx RNA to decrease their AAC. Previous study proved that the AAC was decreased to some degree, and no difference was found between parents and their transgenic lines as respect to gelatinization temperature and gel consistence even their AAC was different [13]. Table 2 listed the RVA profile parameters of Wuyunjing 7 and Wuyunjing 8 and their transgenic lines with anti-wx RNA gene. Fig. 1 showed the pasting curves of Wuyunjing 7 and its four transgenic lines. AAC of Wuyunjing 7 was 16.7%, and those of the transgenic lines were.1%, 2.1 %, 3.3 % and 13.5 %, respectively. As the AAC decreased, peak viscosity, trough viscosity, final Table 1. Comparison of rice RVA profile characteristics of six types resulting from cluster analysis. RVA group PKV HPV BDV CPV SBV CSV No. of varieties indica japonica Glutinous rice A B C D E F PKV, Peak viscosity; HPV, Hot paste viscosity; BDV, Breakdown; CPV, Cool paste viscosity; SBV, Setback viscosity; CSV, Consistence viscosity. The same as in the tables below.

4 42 Rice Science, Vol. 12, No. 1, 5 Fig. 2. RVA profiles of varieties with similar AAC. viscosity, breakdown viscosity, setback viscosity and consistence viscosity also decreased simultaneously, and the starch pasting time brought forward. Moreover, 11 transgenic lines changed their RVA profile characteristics from E to A group. The results indicated that AAC played an essential role in determining the starch properties. RVA profiles of varieties with similar AAC It is widely accepted fact that the AAC is an important determinant for rice eating and processing quality. The recent studies indicated that the fine structure of amylopectin plays an important role in this regard. In addition, differences were detected for eating quality by tasting among varieties with similar AAC. But the relationships between RVA profiles parameters and the fine structure of amylopection were unclear yet. In this experiment, many differences were realized in eating quality and RVA profiles among varieties with similar AAC (Fig. 2, Table 3). In glutinous varieties (very low AAC), the RVA curves Table 2. AAC and RVA profiles parameters of Wuyunjing 7, Wuyunjing 8 and their corresponding transgenic lines with antisense Wx. Variety (line) AAC (%) PKV HPV BDV CPV SBV CSV PeT (min) PaT ( ) RVA type Wuyunjing E B E B A B A B A B A B A B A Wuyunjing F B A B A B A B A B A AAC, Apparent amylose content; PeT, Peak time; PaT, Pasting temperature.

5 YAN Chang-jie, et al. Performance and Inheritance of Rice Starch RVA Profile Characteristics 43 were entirely different, especially in peak viscosity, trough viscosity and others although AAC being similar. In low, middle and high AAC types, the RVA curves also changed regularly in despite of similar AAC. For example, for low AAC type, the eating quality of Wuxiangjing 3 (AAC, 14.4%), Nongken 57 (AAC, 15.2%), 337 (15.5%) was better than Yizhila (AAC, 15.3%), and the breakdown viscosity of the former three samples was higher than that of Yizhila, while peak viscosity, trough viscosity, final viscosity, setback viscosity, consistence viscosity and peak time were smaller than those of Yizhila. For middle AAC type, the eating quality of Zhongzao 1 and Peidi was significantly different although their AAC being similar, their AAC were 25. and 23.8%, respectively. Their RVA profiles were also discrete obviously; peak viscosity, trough viscosity, final viscosity, setback viscosity, consistence viscosity, peak time and pasting temperature of inferior quality variety Zhongzao 1 were markedly higher than those of Peidi, while the breakdown viscosity of the former was lower than that of the latter. Similarly, this phenomenon was also found in high AAC type. Compared Kuidao with M214-1, with AAC being 28.8% and 28.4%, respectively, the eating quality of Kuidao in terms of synthetic evaluation was better than that of M214-1, and the breakdown viscosity of Kuidao was higher than that of M Therefore, the eating quality of varieties with similar AAC might be differentiated by RVA profile. It suggested that the good quality varieties usually have higher breakdown viscosity, lower setback and final viscosity; in contrast, the inferior eating quality varieties have lower breakdown viscosity, and higher final and setback viscosity. These results suggested that RVA profile have the potential to be applied in evaluating the eating quality of rice. Inheritance of RVA profile parameters Performance of RVA profile of hybrid rice and their parents In order to understand the inheritance pattern of RVA profiles, two elite sterile lines Zhenshan 97A and Xieqingzao A, their maintainer lines Zhenshan 97B and Xieqingzao B, respectively, and two restorer lines Minghui 63 and 559 were used to develop 4 hybrid rice combinations, and their RVA profiles were assayed. Previous studies showed that the cytoplasm Table 3. RVA profile characteristics and eating quality scores of varieties with similar AAC. AAC type Variety AAC PKV HPV BDV CPV SBV CSV PeT PaT SE RVA type (line) (%) (RVU) (RVU) (RVU) (RVU) (RVU) (RVU) (min) ( ) VLC Jinlingnuo A B A Xiangjingnuo A B A LC Wuxiangjing D Nongken E Yizhila C F MC Peidi E IR C Zengchengbai C Zhongzao C HC IR B M C Luhuangzhan C Kuidao F VLC, Very low content; LC, Low content; MC, Middle content; HC, High content.

6 44 Rice Science, Vol. 12, No. 1, 5 had no significant effect on RVA profile and apparent amylose content [8]. So in this study, we assumed that RVA profiles of the maintainer lines were similar to those of the sterile lines, and they were used to represent the corresponding RVA profiles of the sterile lines. Fig. 3 showed the RVA profile curves of 4 hybrid rice combinations and their parents. It is obvious that the F 1 RVA profiles were between their parents, including pasting temperature, breakdown, final viscosity, setback, consistence and peak time, while the performance of peak viscosity varied with the different combinations. From the RVA profile curves, it was clear that the curves of the restorer lines always occupied the lowest position compared with F 1 s and sterile lines after the viscosity reached peak point (peak viscosity). These results implied that both of parents had some influence on F 1 in terms of RVA profiles, and the RVA profiles may be governed by several minor genes, with AAC being a key determinant. Because the final viscosity indirectly reflected the cool rice texture, the low final viscosity was required for good quality. According to the results of F 1 RVA profiles in this study, we should pay more attention to sterile lines improvement due to their contribution of viscosity to corresponding hybrid rice. It should be noted that hybrid rice quality was affected by two parents, and it was important to improve both parents simultaneously. Inheritance of RVA profile parameters in F 2 clonally propagated population It is well established that F 2 population is the best material for genetic analysis due to its plenty of genetic information. However F 2 population cannot be maintained from generation to generation because of heterozygous genotypes at many loci segregated in next generation, and it does not fit for quantitative trait analysis as one genotype can not be replicated at different environments and environment error is difficult to be estimated in statistic analysis. Wang et al [9] developed a special population, F 2 clonally propagated population (F 2 CP), which was maintained by tissue culture, had the same genetic composition as normal F 2 population. Each genotype could be propagated by tissue culture, and many individuals (clones) with same genotype could be obtained and replicated in different plots in order to estimate the experiment error in further statistic analysis. Fig. 3. RVA profiles of four hybrid rice combinations.

7 YAN Chang-jie, et al. Performance and Inheritance of Rice Starch RVA Profile Characteristics 45 PKV HPV SBV Number of plants CPV BDV CSV Number of plants RVA Viscosity( RVU) RVA Viscosity ( RVU) RVA RVAviscosity Viscosity ( RVU) / RVU Fig. 4. Distribution of RVA profile characteristics of F 2CP population. Table 4. RVA profiles of parents, F 1 and their segregation population F 2CP. Characteristic Balilla Dular Mean of parents F 1 Range of distribution in F 2CP PKV HPV BDV CPV SBV CSV RVA profiles of 188 lines, their parents and F 1 of F 2 CP were assayed. Table 4 and Fig. 4 summarized the performances of RVA profiles of F 2 CP. The two parents showed significant discrepancy in RVA profiles for all six parameters, and all parameters of F 1 were between their parents, near the mid-parent value except for BDV (Table 4). We found all the components of RVA profiles in F 2 CP segregated widely, and transgressive inheritance occurred. The bell-shaped distributions were observed, suggesting that all 6 indices were controlled by a series of minor genes. It should be noted that, previous studies and the result from RVA profile of different AAC lines in this study showed the RVA profiles that were mainly determined by AAC. Theoretically, if two parents showed difference in ACC, it would segregate and the population, and RVA profile would be similar to AAC. Actually, although the AAC of two parents, Dular and Balilla, was significantly different, the distribution patterns did not show two peaks and, one peak distribution was observed. It should be noted that in this experiments, the seeds from F 2 individuals, were harvested to assay their quality traits and they represented the F 2 phenotype. Continuous distribution with one peak may be resulted from the mixed genotypes of different seeds, which ultimately lead to the minor difference among different genotypes with different Wx dosage. Another reason is that the RVA profiles were also controlled by minor genes besides major gene, Wx. This emphasized that attention also should be paid for the minor genes in good quality rice breeding. distribution with two peaks was expected in F 2

8 46 Rice Science, Vol. 12, No. 1, 5 DISCUSSION Inheritance of RVA profile Due to the little amount of starch of single rice grain, RVA analysis was restricted when applied in estimating the rice quality, and the genetic mechanism of RVA profiles had not been understood thoroughly. Gravois et al [3] found that paste viscosity, peak viscosity, trough viscosity and final viscosity each was governed by a single allele with additive effect. This result was supported by both F 2 :F 3 segregation analysis and diallel analysis. Bao et al [4] found RVA profile was mainly controlled by Wx gene, and modified by several minor genes, but Wx gene had no effect on peak viscosity. Bao et al [14] used a doubled haploid population derived from an indica/japonica cross to accomplish QTL mapping of paste viscosity characteristics in rice. Twenty QTLs were identified for six parameters of RVA profile at least one location. They proposed that Wx gene was the main locus affecting the RVA profile because it explained 19.5% to 63.7% of total variation in population, while the difference in AAC between two parents (ZYQ8 and JX17) was about 6% [2]. In this study, two sets of transgenic rice lines with different AAC were used to study the effect of AAC on RVA. The results indicated that RVA was mainly influenced by AAC, suggesting the Wx gene played an essential role in determining the RVA pattern. A F 2 CP population derived from Balilla/Dular was also applied to study the RVA genetic mechanism. In this population, six parameters segregated widely, and some transgressive individuals appeared, and its frequency distribution was bell-like shape, suggesting that RVA profiles were controlled by some minor genes except for Wx gene, a major gene. The results were further confirmed in four rice hybrid combinations for RVA profile study in this case. Based on RVA profile characteristics, all the tested varieties could be divided into six groups with indica varieties into five and japonica ones into three groups. This indirectly suggested the variation was more pronounced in indica type than japonica one, which was similar to previous studies based on other traits measured. This result also indicated that there was abundance of variation in terms of RVA in both indica and japonica types. This provided some new information on rice quality. Based on the relationship between RVA profiles, and eating and cooking quality, the RVA profile could be a useful indicator in selecting a desirable donor parent in rice good quality breeding. Application of RVA profile in evaluating eating quality Although paste viscosity has long been used in rice breeding program to select the desirable eating, cooking properties of new rice varieties, RVA was not extensively used due to large sample and long period required. Currently, a new instrument is available, the Rapid Vsico Analyzer, which is made in Australia (Newport Scientific Pty Ltd). The RVA has become increasingly popular for investigating the viscosity properties of rice starch and other cereal crops, as it requires only a small sample size and is easy to manipulate. RVA test simulated the rice cooking process; it has been proved to be potential to represent the eating and cooking qualities. Amylose content is widely recognized as the most important determinant of the cooking, the processing and edibility of rice. However, it is also known that there are differences existed among varieties with similar AAC. These differences include RVA profile, gelatinization temperature, etc. Present study showed that there were differences in eating quality among varieties with similar AAC (Table 3), and these differences could be reflected by RVA profile, suggesting that in good quality rice breeding program, combining AAC with RVA profile as indices will be helpful for evaluating the rice eating quality, especially for varieties with similar AAC. In other words, we can predict eating, cooking and processing quality based on the AAC and RVA profiles with RVA-assisted selection. Wu et al [15] had successfully obtained a good quality indica line from the progeny of early indica, further confirming the RVA profile can be used as a secondary index in good rice quality breeding.

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