Rice is a major source of nutrition for most people in the developing world. Although tremendous achievements have been made for the improvement of many agronomic traits in rice in the last three decades, much less progress has been obtained for quality traits due to the lack of simple and efficient selection methods in rice breeding. With rapid advancement in crop molecular breeding, marker-aided selection has been successfully applied in many crop plants. Similarly, new methods for genetic engineering of better crop plants have been reported in the last decade by overexpressing or gene silencing of candidate genes. Although several eating quality QTLs have been identified in previous studies [18, 19], it is not clear whether these QTLs are useful for marker-aided selection or not because the genomic regions of these QTLs have not been further characterized. Recently Nelson et al.  identified six main-effect milling yield QTLs in the two RIL populations derived from crosses of common parent Cypress with RT0034, a low-milling yield japonica line and LaGrue, a low-milling yield japonica cultivar, respectively. In this study, we used two high throughput sequencing technologies to profile the transcriptome of five cultivars differing in milling yield and eating quality. Many genes specifically or commonly expressed in the high milling yield cultivar Cypress and the good eating quality cultivar Ilpumbyeo were identified from the MPSS and SBS libraries. These candidate genes are excellent starting materials for the development of molecular markers linked to milling quality in the US and eating quality in Korea for rice breeding. It is also possible that overexpression or silencing of some candidate genes will lead to the generation of transgenic rice plants with superior grain quality.
During the rice seed development, sugars, amino acids, and other important metabolites are transported from source (primarily leaves) to sink (seeds). Once in the seeds, these metabolites are allocated to different biosynthetic pathways (primarily starch metabolism and storage protein biosynthesis) to produce mainly starch and proteins in precise quantities and ratios. Achieving such a defined composition of starch and proteins require the regulation and coordination of various pathways so that, at each developmental stage, the participating enzymes are present in appropriate amounts and in the correct cellular compartments . AGPase and GBSS I play important roles during starch biosynthesis in rice . The genes encoding for AGPase and GBSS I enzymes are highly expressed 7 to 28 days after flowering during grain development, and their expression is highly correlated with the increases in both starch content and grain weight. The AGPase gene is also highly expressed in the high-yield cultivars of both glutinous and non-glutinous rice . In addition, AGPase (Os01g44220) undergoes alternative splicing similar to the AGPase small subunit gene in barley . Duan and Sun  showed that a mutation in the GBSS I gene leads to a lower level of functional GBSS I mRNA and correspondingly to a lower level of GBSS I enzyme for amylose synthesis, which causes a reduction in amylose accumulation. During rice seed formation, the genes encoding AGPases are active 3 days before flowering and maintain an intermediate although declining level of activity during seed maturation . Genetic variation survey showed that the polymorphism in the rice waxy gene encoding the GBSS enzyme explains much of the variation in apparent amylose content across 92 important long, medium and short grain US rice cultivars and 101 progeny of a cross between low-amylose and intermediate-amylose breeding lines [73, 74]. The amylose content and the level of waxy protein in 31 rice cultivars from China were correlated with the ability of the cultivar to excise intron I from the leader sequence of the Wx transcript . In this study, we found that the important starch biosynthesis related genes encoding AGPase (Os01g44220), 1,4-α-glucan branching enzyme (Os02g32660), limit dextrinase (Os04g08270), 1,4-α-glucan branching enzyme (Os06g51084), and α-amylase (Os09g29404) were up-regulated in Cypress compared to LaGrue and Nipponbare in six-days old developing seeds. Our time-course RT-PCR analysis also confirmed that expression of AGPase and GBSS I genes was higher in the high milling cultivar Cypress than in the low milling cultivar LaGrue early (6 and 9 DAF) in seed development. These results suggest that these two genes related to starch synthesis may greatly affect milling yield. Starch biosynthesis is also associated with complex genotypic-environmental interactions in maize endosperm . Since the plants in this study were grown in the controlled environmental conditions (growth chambers), the effect of environmental factors on the expression of the starch biosynthesis genes should be tested in the field conditions.
Cereal proteins are generally deficient in lysine, but lysine content might be increased with increased accumulation of the precursor molecules required for the enzymatic reactions involved in lysine metabolism. The key precursor molecules include lactate, acetyl CoA, malate, L-aspartate, L-asparagine, L-aspartate-semialdehyde, homoserine, homocysteine, 2-oxobutanoate, 2-aceto-1-hydroxybutyrate, and α-ketoglutarate, and the enzymes involved in their production are very important (Additional File 5). Enhancing the production of these precursor molecules will require the identification of the genes encoding these enzymes. In this study, we found that the genes encoding malate dehydrogenase (Os03g56280, Os01g46070) and aminotransferase (Os09g28050, Os03g18810) involved in the production of malate and aspartate in Cypress and Ilpumbyeo, respectively, were up-regulated compared to the controls. Genes encoding aspartate transaminase (Os01g55540) and enoyl-CoA hydratase (Os02g43720) enzymes, which are responsible for the production of acetyl CoA, were also up-regulated in Cypress compared to the controls. Similarly, the gene encoding lactoylglutathione lyase (Os05g07940), which is responsible for the production of lactate, was up-regulated in Cypress compared to the controls. As indicated, genetic manipulation of the expression levels of these precursors/enzymes may lead to an increased accumulation of lysine in the endosperm and thus an increased nutritional value of the rice seeds.
In the last decade, oligoarrays, SAGE, MPSS, and SBS have been widely used for transcriptome profiling. MPSS and SBS have been recently used for whole-genome transcription analysis and have generated abundant expression data for many organisms [42, 44, 45]. In this study, both MPSS and SBS technologies were used to analyze the transcriptomes of the 6-days-old developing seeds in five rice cultivars. The number of redundant and non-redundant signatures generated in this study were similar to those in previous reports in rice and Arabidopsis [43, 45, 77]. Although MPSS generates large volume of data, its complicated library-construction procedure and high sequencing cost limit its use in individual laboratories. As the cost of the next-generation sequencing methods has significantly decreased in the last few years, SBS sequencing has become a popular method for transcriptome analysis because it costs 90% less than MPSS and can generate at least three times more transcripts. Furthermore, in the current study, about 30% more transcripts were found in the SBS library than in the MPSS library. Many of these additional signatures are low-copy transcripts, indicating that SBS is a powerful method for identifying rare transcripts . The correlation coefficient is higher between MPSS and SBS than between RL-SAGE and microarray , or between RL-SAGE and MPSS or MPSS and microarrays as in previous studies . Therefore, SBS will undoubtedly become the preferred high throughput sequencing method for deep transcriptome analysis in plants.