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Published online 2 October 2006
Published in Crop Sci 46:2354-2360 (2006)
© 2006 Crop Science Society of America
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GENOMICS, MOLECULAR GENETICS & BIOTECHNOLOGY

Molecular Marker-Assisted Selection for Improved Cooking and Eating Quality of Two Elite Parents of Hybrid Rice

Qiao-Quan Liua,c,*, Qian-Feng Lia,c, Xiu-Ling Caib, Hong-Mei Wangc, Shu-Zhu Tanga, Heng-Xiu Yua, Zong-Yang Wangb and Ming-Hong Gua,**

a Jiangsu Key Lab. of Crop Genetics and Physiology, Key Lab. of Plant Functional Genomics of the Ministry of Education, Agric. College, Yangzhou Univ., Jiangsu 225009, P.R. China
b Shanghai Institute of Plant Physiology & Ecology, Chinese Academy of Science, Shanghai 200032, P.R. China
c College of Bioscience & Biotechnology, Yangzhou Univ., Jiangsu 225009, P.R. China

* Corresponding author (qqliu{at}yzu.edu.cn)

** Corresponding author (gumh{at}yzu.edu.cn)


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Long-te-fu (LTF) and Zhan-shan 97 (ZS) are two key female parents for the generation of indica hybrid rice, which have greatly contributed to the achievement of rice production in China. However, the high amylose content (AC) in the endosperm, controlled by the Waxy (Wx) gene encoded granule-bound starch synthase I, of both lines results in poor cooking and eating quality of the milled rice. Our previous studies have shown that AC was correlated with the ability to excise intron 1 from the leader sequence of the Wx transcript, and which is responsible by a single nucleotide polymorphism (G or T) located at the first nucleotide of the splice donor site of Wx intron 1. Thus, a CAPS marker was subsequently developed, and with this molecular marker-assisted selection (MAS) we successfully introgressed the Wx-TT locus of rice cultivars with good quality intermediate AC into LTF-B and ZS-B. These were subsequently introduced into their relevant male-sterile lines (LTF-A and ZS-A) to generate improved indica hybrids. In the selected lines LTF(tt)-B and ZS(tt)-B, the AC was reduced to a relatively low level (15%). Consequently, the hybrids crossed from the selected lines had dramatically reduced amylose levels. In the field trials, the agronomic performance in the improved lines and their hybrids was examined and compared to the originals. The other key factors involved in rice cooking and eating quality were also improved in the selected lines and their hybrids.

Abbreviations: AC, amylose content • ASV, alkali spreading value • CAPS, Cleaved Amplified Polymorphic Sequence • GBSS, granule-bound starch synthase • GC, gel consistency • GT, gelatinization temperature • MAS, marker-assisted selection • SNP, single nucleotide polymorphism • Wx, waxy gene


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
RICE IS a global food crop staple, particularly in China. Aside from increasing crop yields, improving the grain quality currently represents one of the major problems in rice production in most rice-producing regions. This issue is especially relevant for growers of the poor quality of indica hybrid rice in China. Hybrid rice was first developed in China in 1974 and has gained popularity since its release in 1976. Hybrid rice yields about 20% more rice than conventional rice varieties, and now accounts for more than half of the annual rice planting area in China. Therefore, rice researchers consider the improvement of the quality of hybrid rice in China a top priority.

In many rice-producing areas of the world, cooking and eating quality always represents a major criterion in evaluating rice grain. Cooking and eating quality in rice is mainly determined by the starch composition, especially by three physicochemical characteristics of the starch, i.e., AC, gel consistency (GC) and gelatinization temperature (GT) (Juliano, 1985; Mo et al., 1990). High AC in the endosperm is usually associated with dry, fluffy, and separated cooked rice grains, and represents the key determinant of poor cooking and eating quality (Juliano, 1985). In China, the cooking and eating quality of indica hybrid rice is usually poor; this is often because of a high AC in their parent lines. For example, the AC in LTF-B and ZS-B, the key Chinese maintainer lines to produce indica hybrid rice, reaches a dramatically high level (25–30%). As a result the derived hybrid rice quality has not been satisfactory. Therefore, AC downregulation in endosperm is a major objective for the improvement of rice grain quality, especially of indica hybrid rice.

In rice endosperm, amylose synthesis is initiated by GBSS, and encoded by the Wx gene on chromosome 6 (Okagaki and Wessler, 1988). We have previously cloned and sequenced the Wx gene from both the japonica and indica rice cultivars (Wang et al., 1990); and the regulation of its expression has been studied (Wang et al., 1995; Cai et al., 1998). The results demonstrated that AC, as well as GBSS content, was correlated with the excision of intron 1 from the leader sequence of the Wx transcript, and a SNP, the G to T mutation at the first nucleotide of the splice donor site of Wx intron 1, may be the key factor that produced the alteration in splicing efficiency (Fig. 1) (Cai et al., 1998; Isshiki et al., 1998). The cultivars with the Wx-G/Wx-G genotype contain a G base at the first nucleotide of the splice donor site of the Wx intron 1; so the Wx pre-transcripts were completely processed to the mature (2.3 kb) Wx mRNA, and therefore were abundant in GBSS, and had a high level of AC in rice endosperm. Also, the Wx pre-transcripts in the cultivars with the Wx-T/Wx-T genotype were alternatively spliced, and only a small mature Wx mRNA was produced. This resulted in relatively low or intermediate levels of GBSS and AC (Cai et al., 1998). Thus, a CAPS marker, referred to as PCR-AccI (Fig. 1), was subsequently developed to identify whether the first nucleotide of the splice donor site of Wx intron 1 was G or T, and to generate inferences regarding the AC level in the genetic materials. This is especially important for breeding programs that attempt to improve rice quality by MAS (Cai et al., 2002).


Figure 1
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  		Fig. 1. Comparison of the Wx exon 1-intron 1 junction sequences from rice cultivars with high (H) and intermediate (I) amylose content (AC). Exon and intron sequences are shown in capital and lowercase letters, respectively. The complete sequence is given only for cultivars with high AC, and only those that differ from the corresponding nucleotides are shown in boxes for cultivars with intermediate level of AC. The dashes (–) indicate nucleotide deletions in the intermediate AC cultivar, compared to the corresponding sequence of the high AC cultivar. The bolded sequences represent the recognition site by AccI in the high AC cultivar, where it is not due to the G to T mutation in the case of the intermediate AC cultivar. The primers used for the CAPS marker PCR-AccI determination are underlined.

 
Besides, it is known that the alkali disintegration property of rice grain is distinctly different among rice varieties, especially between japonica and indica types, which is highly correlated to the GT of the rice flour (Juliano, 1985). In general, the indica-type rice grain has higher alkali tolerance than that of the japonica-type. Genetic studies showed that the key gene controlling the varietal difference in alkali disintegration between japonica-type and indica-type, designated the ALK gene, had been mapped near the Wx locus on chromosome 6 (Umemoto et al., 2002; Gao et al., 2003). More recently, the ALK gene was proven to encode a starch-soluble synthase isoform, SSIIa, and a single base change in this gene might be responsible for the difference in GT between the two species (Gao et al., 2003; Nakamura et al., 2005).

The present study was conducted to improve the cooking and eating quality of LTF-B and ZS-B by downregulation of the amylose content. The Wx-T/Wx-T genotype, which was from good quality indica rice lines with intermediate AC, was introgressed into these two target lines using the developed PCR-AccI marker and its assisted selection. The results showed that substitution of this allele successfully downregulated AC. It greatly improved the cooking and eating quality of both lines, and their relevant hybrids, without significantly changing agronomic performance.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Plant Materials
Three types of indica rice parents for three-line hybrid rice production were used in this study, and included the following seven lines: (i) Two maintainer lines, LTF-B and ZS-B, were used as the target parents to be improved. (ii) Three restorer lines, ‘Minghui 63’ (MH63), ‘Yanhui 559’ (YH559), and ‘9311’, were used as Wx-T/Wx-T genotype donors and/or parents for the production of hybrids with selected lines. They were also well known as the key parents of elite hybrids, widely grown in China. These were of good quality; they possessed intermediate AC, soft GC, and a high GT. (iii) Two male-sterile lines, LTF-A and ZS-A, were the relevant parents of LTF-B and ZS-B, respectively.

Scheme for Crossing
Two crosses were first conducted to generate two F1 hybrids between LTF-B and 9311, and ZS-B and YH559, respectively. Following a recurrent backcrossing procedure combined with the PCR-AccI MAS, the Wx-T/Wx-T genotype in 9311 or YH559 was subsequently introgressed into LTF-B or ZS-B, respectively. Both introgressions were performed after four generations of backcrosses and two generations of self-fertilization occurred. In each generation, the Wx-G/Wx-T genotype individual was selected, after determination of the molecular marker, combined with the selection of agronomic traits, and used for the backcross with the original parent. The selected maintainer lines were designated LTF (tt)-B and ZS (tt)-B, and were derived from LTF-B and ZS-B, respectively. The Wx-T/Wx-T genotype was then introgressed into the male-sterile lines LTF-A and ZS-A by crossing and subsequently backcrossing with the selected LTF (tt)-B and ZS (tt)-B lines, respectively. The selected male-sterile lines containing the homozygous Wx-T allele were designated LTF (tt)-A and ZS (tt)-A, respectively.

Markers for Marker-Assisted Selection
The PCR-AccI CAPS marker (Fig. 1) was measured according to the method described previously by Cai et al. (2002). Briefly, a set of PCR primers, forward primer (5'-ACCATTCCTTCAGTTCTTTG-3') and reverse primer (5'-ATGA TTTAACGAGAGTTGAA-3'), located around the first nucleotide of the splice donor site of Wx intron 1 (Fig. 1), were used to amplify a DNA fragment with a size of 530 bp from the total genomic DNA of rice leaf. This PCR product was digested with the restriction enzyme Acc I. Once the PCR product from the Wx-G/Wx-G genotype was completely digested, and after electrophoresis, two DNA fragments (405 bp and 125 bp) were observed. Conversely, the PCR product from Wx-T/Wx-T genotype could not be digested. Additionally, if the PCR product, derived from the crossed materials between Wx-G/Wx-G and Wx-T/Wx-T genotype, was not completely digested, it would reveal three DNA fragments (530 bp, 405 bp, and 125 bp respectively) after Acc I digestion and subsequent electrophoresis. This pattern would be designated as a heterozygous Wx-G/Wx-T genotype. In this study, the two target parent lines LTF-B and ZS-B, as well as their relevant male-sterile lines LTF-A and ZS-A, contained the Wx-G/Wx-G genotype, while the three restorer lines were all Wx-T/Wx-T genotype.

Field Trials and Agronomic Trait Evaluation
Two field trials were conducted to evaluate the agronomic performance of the selected lines and their relevant hybrids. One trial was located on Hainan Island (Spring, 2003), and was designed to compare the selected maintainer and sterile lines with their original parents. Simultaneously, both the selected and original sterile lines were crossed with the restorer lines to produce the hybrids for another field trial. This trial was located in Yangzhou (Summer, 2003) and was designed to compare the agronomic traits between the new crosses and their original hybrids. This comparison included the following: (i) the original hybrid ‘Teyou 63’ (LTF-AxMH63) and the new hybrids LTF (tt)-AxMH63; (ii) the original hybrid ‘Teyou 559’ (LTF-AxYH559) and the new hybrids LTF (tt)-AxYH559; and (iii) the original hybrid ‘Shanyou 63’ (ZS-AxMH63) and the new hybrids ZS (tt)-AxMH63. In both trials, three replications were conducted for each test material, and in each replication the selected lines (or new crosses) and their original parents (or hybrids) were randomized into subplots. Each subplot consisted of four rows with 10 plants per row (a single plant per hill). To minimize the margin effect, only ten plants in the middle of each subplot were used to measure the agronomic traits. The traits measured included heading date, plant height, length of the main panicle, number of tillers per plant, number of grains per panicle, seed setting, and the weight of grains. Finally, the yield per single plant was theoretically obtained according to the main economic traits measured.

Grain Quality Measurement
The AC, GC, and GT in mature seeds were measured as described previously by Tan et al. (1999). Briefly, AC was determined by the colorimetric method with iodine-potassium iodide, as described previously (Juliano, 1971). GC was assessed by measuring gel length, such that the longer gel is considered to be softer than the shorter gel (Cagampang et al., 1973). The GT was calculated on the basis of individual grains. This measurement was expressed in terms of the alkali spreading value (ASV) as described by Little et al. (1958). For example, a high ASV represented more spreading in alkali, which indicated a low GT. Conversely, a low ASV indicated a high GT. Triplicate measurements were taken and the mean value was taken as the final result.

Starch pasting properties were determined using a Rapid-Visco Analyser, Model 3D (Newport Scientific, Warriewood, NSW, Australia) following the AACC Approved Method 61–02 (2000), described previously by Han et al. (2004). Three grams of rice flour were weighed in an aluminum RVA canister and 25 mL of distilled water was added. A paddle was placed in the canister and rotated at 960 rpm for 10 s to disperse the rice sample. The viscosity was evaluated using a constant paddle rotation of 160 rpm. The sequential temperature curve for a 12.5 min test was as follows: the sample was incubated at 50°C for 1 min, heated to 95°C in 3.75 min and then held at 95°C for 2.5 min, cooled to 50°C in 3.75 min and held at 50°C for 1.5 min. Viscosity values were recorded in rapid visco units (RVU) for peak viscosity (PV), holding or hot paste viscosity (HPV), final or cool paste viscosity (CPV), breakdown (BD = PV-HPV), setback (SB = CPV-PV), and consistency (CS = CPV-HPV). Each analysis was repeated at least twice.

Northern Blot Analysis
Total RNAs were isolated from developing rice seeds at 12 DAP by the cold-phenol method (Zheng et al., 1993). For each sample, a 15-µg sample of total RNA was subjected to electrophoresis on a 1.0% agarose gel containing formaldehyde, and blotted onto a Hybond N+ nylon membrane (Roche Applied Science, Penzberg, Germany). The Wx cDNA coding sequences were labeled with digoxigenin (DIG) using a DIG nucleic acid labeling kit (Roche), and were used as hybridizing probes. The experimental procedure was performed as described by Liu et al. (2003).

Electrophoresis and Detection of Wx Protein
The rice Wx protein was extracted from mature seeds as described by Sano (1984). Wx protein, extracted from 5 mg milled rice powder of each sample, was dissolved in 50 µL extraction buffer. Aliquots (10 µl) of the samples were loaded into lanes of a 12% polyacrylamide gel containing SDS. The proteins were subsequently visualized by staining with Coomassie brilliant blue.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Molecular Marker-Assisted Backcross Breeding
The Wx-genotype and grain quality in all original lines used in the present study were first confirmed (Fig. 2, Table 1). For the GT phenotype, it was relatively low, 7.0 and 6.4 grade of ASV, respectively, for the two target parents (Table 1). The genotype of the ALK locus was detected to be the indica-type in both the two target lines and one donor line, 9311, according to the method described by Gao et al. (2003) (data not shown), while another donor line, YH559, has the japonica-type ALK locus and shows very high GT (Table 1).


Figure 2
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  		Fig. 2. Determination of the PCR-AccI molecular marker in individuals of the BC4F2 generation from two target parents, LTF-B (A) and ZS-B (B). Panel (A) lane 1: 9311 (TT genotype); lane 2: LTF-B (GG genotype); lanes 3–12: individuals in BC4F2 generation; and lane 13: hybrid Teyou 559 (GT genotype). Panel (B) lane 1: YH559 (TT genotype); lane 2: ZS-B (GG genotype); lanes 3–12: individuals in BC4F2 generation; and lane 13: Shanyou 63 (GT genotype). Lane M: the 1 kb plus DNA molecular marker (Invitrogen).

 

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  		Table 1. Comparison of the AC, GC, and GT characteristics between the selected lines and their original parents.

 
According to the described MAS scheme, a total of four generations of backcrosses was conducted, and BC4F1 was finally generated. To evaluate the effect of MAS on AC, the Wx-G/Wx-T-type individual selected in each BCnF1 generation was allowed to be self-pollinated to produce BCnF2. In each generation of BCnF2, the co-segregation between the types of molecular marker and the AC was as predicted (detailed data not shown), which was consistent with our previous results in the F2 generations, derived from five crosses between indica rice cultivars with high and intermediate amylose levels (Cai et al., 2002).

Several individuals with Wx-G/Wx-T genotype in both BC4F1 cases were identified with comparable agronomic performance, as compared to their original parents, LTF-B and ZS-B, respectively. Therefore, the progenies with Wx-G/Wx-G, Wx-G/Wx-T, or Wx-T/Wx-T genotype in the generation of BC4F2 were identified. Most of the progenies were identical to the parents in appearance, and the AC in their mature seeds measured as predicted. For an example, among the BC4F2 progenies from LTF-Bx9311 the average AC was as follows: 27.3% (range = 25.0–29.9%), 21.7% (range = 19.0–23.8%), and 15.1% (range = 12.4–17.9%) in the mature seeds of the three genotype plants, respectively. Therefore, based on both the grain quality and agronomic appearance in the field, four individuals, homozygous for Wx-T allele, were selected as improved types (LTF (tt)-B or ZS (tt)-B) of LTF-B or ZS-B (Table 1). They were further used as the donor for the introgression of the Wx-T allele into the male-sterile lines LTF-A or ZS-A, and the eight relevant male-sterile lines, homozygous for the Wx-TT allele, were obtained and designated LTF (tt)-A or ZS (tt)-A.

Agronomic Performance of the Selected Lines and Hybrids in Field Trials
Table 2 shows the results of the major agronomic traits of the selected maintainer lines and their original parents LTF-B and ZS-B. Out of the measured agronomic traits, most presented no statistically significant differences in the selected lines, when compared to the originals. However, the heading date was significantly delayed by 1 to 3 d in several selected lines. Also, the yield in three selected lines, which was generated from the combination of major economic traits, was significantly higher than that of their originals. At the same time, a comparison of major agronomic traits, inculding the heading date, panicle number per plant, panicle length, and grain numbers per panicle, was also conducted between the improved sterile lines and their original lines. No significant differences were observed (data not shown). Subsequently, the field performance of the new hybrids produced with the improved restorer lines was compared with their original ones (Table 3). The results revealed that only the hybrids from LTF (tt)-AxMH63 exhibited significantly shorter plant height as compared to the original hybrid Teyou 63. All others were similar to the original hybrids.


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  		Table 2. Comparison of the major agronomic traits between the selected maintainer lines and their original parents (Hainan, 2003).

 

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  		Table 3. Comparison of the amylose content, major agronomic traits, and yielding between the hybrids from selected lines and their original parents (Jiangsu, 2003).

 
Amylose Content and Wx Gene Expression in the Selected Lines and Hybrids
Measurement of the AC in mature seeds from the field trials demonstrated that all eight selected maintainer lines contained an intermediate to low level of AC. This was similar to the donor parent 9311 or YH559, but was dramatically less than that of the original parent LTF-B or ZS-B (Table 1). Additionally, the amylose level in the new hybrids, derived from the improved sterile lines, was also less than those in the original hybrids (Table 3).

To explain the AC reduction, which resulted from the downregulation of Wx gene expression in selected lines, both the transcript and protein products of the Wx gene were compared with the selected lines and their original lines. In both original maintainer lines, LTF-B and ZS-B, only the mature Wx mRNA (2.3 kb) was detected, indicating that the Wx pre-mRNAs (3.4 kb) was completely processed, resulting in a very high level of Wx protein (Fig. 3). Among BC4F2, as expected, the expression patterns and levels of the Wx gene were also segregated and tightly correlated with the genotype, as revealed by both Northern blot and Wx protein analyses. In the case of LTF-B, for example, the individuals which still contained the Wx-G/Wx-G genotype showed the same expression pattern as the original line, while the Wx-G/Wx-T BC4F2 lines contained a relatively low level of Wx gene products. However, after substitution by the Wx-T allele in the selected lines, both the un-spliced pre-mRNAs and mature mRNAs of the Wx gene appeared, and the amount of mature mRNAs was dramatically decreased compared to the original LTF-B. This resulted in a very low level of Wx protein in the selected lines. This expression pattern was identical to that of the donor 9311. The Wx expression was also compared among the newly developed hybrids and their originals and the results showed that the levels of both mature Wx mRNAs and Wx proteins were greatly lower in the new hybrid from the selected line LTF (tt)-A and MH63, as compared to the original hybrid Teyou 63 (Fig. 3).


Figure 3
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  		Fig. 3. Comparison of Wx gene expression levels among the selected lines and hybrids, and their originals by both transcript (A and B) and protein levels (C). Northern blot analysis of hybridized signals with the Wx probe. The 3.4 kb Wx pre-mRNAs and 2.3 kb Wx mature mRNAs are indicated by arrows (panel A). Panel B shows the rRNAs by EB staining after electrophoresis; SDS-PAGE of Coomassei blue stained Wx proteins purified from the endosperm of mature seeds (panel C). The genotypes of PCR-AccI molecular marker of the analyzed samples is shown in panel D. [Lane 1: LTF-B original; lanes 2–6: five individual plants in BC4F2 generation; lanes 5–6 represent the two selected lines L-tt-1 B and L-tt-3 B; lane 7: 9311; lane 8: ZS-B original; lane 9–10: two selected lines Z-tt-1 B and Z-tt-2 B; lane 11: YH559; lane 12: original hybrid Teyou 63; and lane 13: one new hybrid LTF (tt)-A-1xMH63.]

 
Effects on Other Grain and Starch Properties
The other quality traits related to rice physicochemical properties were measured and compared among the selected versions and their originals. In response to a great decrease in AC, we observed a significant increase in GC and GT selected male lines (Table 1) and their relevant new hybrids (Table 4). This result was consistent with previous studies. The starch pasting property of original hybrids and their improved versions are shown in Table 4. This indicated that the selected hybrids changed their starch properties, and had lower hot viscosity, cool viscosity, setback, and consistence values. These hybrids had higher breakdown value than the original hybrids; however, they were similar in peak viscosity value, peak time, and pasting temperature (Table 4). The other rice grain qualities, including milling characteristics, appearance character of milled rice, and grain shape (width and length, data not shown), were also measured and compared among the original and improved lines (or hybrids). No significant differences were detected.


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  		Table 4. The AC, GC, GT, and starch pasting properties (starch viscosity characteristics) of the improved hybrid rice under regular field conditions (Jiangsu, China, 2003). Rapid viscosity units (RVU).

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Recently, it has become very important to generate new indica rice varieties or hybrid parent lines with an intermediate or low AC. However, AC is a character controlled by the triploid genotype of endosperm, and the AC between different seeds from one hybrid rice plant will segregate and have gene dosage effects (Mo et al., 1990). This differs from other characters that are controlled by a diploid genotype. It is thus an elaborate endeavor to improve the traits related to grain quality using conventional breeding programs. Modern molecular biotechnology makes it possible and convenient to improve the quality of rice, in particular the AC controlled by a single major locus, by using a genetic transformation or MAS procedure for downregulation of the expression of the endogenous Wx gene (Liu et al., 2005; Cai et al., 2002). In this study, using our previously developed MAS schedule, we introgressed the Wx-T allele of good quality rice cultivars with intermediate AC into the key maintainer lines, LTF-B and ZS-B. These were subsequently introduced into relevant male-sterile lines, LTF-A and ZS-A to generate improved indica hybrids. This MAS event greatly improved the cooking and eating quality in the selected versions of the maintainer lines, LTF (tt)-B and ZS (tt)-B, as well as their resulting hybrids. No significant alteration was observed in the essential agronomic characters of both the improved quality maintainer lines and relevant hybrids after a comparison with the originals.

Previous studies have demonstrated that a lower GT and a higher GC was present in rice with low AC. There was a linear correlation among the three factors in cooking and eating quality (Juliano, 1985). Among them, the AC is well documented to be controlled by the Wx locus as described above, and GC is known to be mainly controlled by the same Wx locus. However, the GT is not only controlled by Wx but also by the ALK locus (Umemoto et al., 2002; Gao et al., 2003). The present study showed that the GC and GT characteristics were improved in the grains of both AC improved maintainer lines and subsequently the new hybrids. As expected, the changed GC in the improved parents and their hybrids might be mainly due to the decrease of the AC after introgression of the Wx-T allele from the donors. For the improved GT phenotype, in the case of improved ZS(tt) lines, the increase of GT must be contributed by the substitution of the japonica-type ALK allele from the donor line YH559. But, in the case of the improved LTF(tt) versions, the change of GT might be due to some other reasons, as the ALK allele is the same between the target parent LTF and the donor line 9311. Additionally, several researchers have revealed a correlation between cooked rice texture and starch pasting properties (Champagne et al., 1999). The results reported in this paper indicate that starch pasting properties, measured by RVA, were also changed after the downregulation of AC and with GC and GT improvement in the selected LTF-B and ZS-B lines, and their relevant hybrids. Therefore, our results indicate that the poor cooking and eating quality in the key indica maintainer lines, and their hybrids, can be improved dramatically by molecular breeding assisted by the selection of the PCR-AccI marker at the Wx locus.

Additional improvements, other than cooking and eating quality, occurred after the transfer of the Wx-T allele. The uniformity of the quality among the grains from F1 hybrid was greatly increased due to the homozygosity of the Wx-T allele in the newly developed hybrid. Since AC is controlled by the triploid genotype of endosperm the genetic difference in endosperm quality between the two parents must segregate between individual seeds from their F1 hybrids (Mo et al., 1990). This produced the poor quality in hybrid rice generated from two parent lines of differing quality. For example, the original hybrid Teyou 559 in this study was derived from the LTF-A with very high AC and YH559 with intermediate AC. However, after the replacement of the Wx-G allele with the Wx-T allele from the male line, both the male and selected female parents carried the same Wx-T allele. Therefore, the expression of GBSS and AC was similar among individual kernels from the newly produced hybrids. Such AC uniformity, with respect to the other quality characteristics GC and GT, is highly desirable for improvement in hybrid rice production.

The present results from field tests revealed no or negligible change in the quality of the essential agronomic characteristics of both the selected maintainer lines and the relevant hybrids. This suggests that the improved versions of both of the male-sterile lines, after introgression of the Wx-T allele, maintained the wide adaptability and high combining ability with the tested restorer lines. Thus, these lines with improved quality could be immediately useful for high yield production of good quality rice hybrids.


   ACKNOWLEDGMENTS
 
This study was financially supported by the Ministry of Science and Technology of China (Grants 2004AA212092 and 2005CB120804), the National Natural and Science Foundation (Grants 30300226 and 30530470), and the Government of Jiangsu Province.

Received for publication March 21, 2006.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 




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K. K. Jena and D. J. Mackill
Molecular Markers and Their Use in Marker-Assisted Selection in Rice
Crop Sci., July 1, 2008; 48(4): 1266 - 1276.
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