[Show abstract][Hide abstract] ABSTRACT: With the ever-increasing global demand for high quality rice in both local production regions and with Western consumers, we have a strong desire to understand better the importance of the different traits that make up the quality of the rice grain and obtain a full picture of rice quality demographics. Rice is by no means a 'one size fits all' crop. Regional preferences are not only striking, they drive the market and hence are of major economic importance in any rice breeding / improvement strategy. In this analysis, we have engaged local experts across the world to perform a full assessment of all the major rice quality trait characteristics and importantly, to determine how these are combined in the most preferred varieties for each of their regions. Physical as well as biochemical characteristics have been monitored and this has resulted in the identification of no less than 18 quality trait combinations. This complexity immediately reveals the extent of the specificity of consumer preference. Nevertheless, further assessment of these combinations at the variety level reveals that several groups still comprise varieties which consumers can readily identify as being different. This emphasises the shortcomings in the current tools we have available to assess rice quality and raises the issue of how we might correct for this in the future. Only with additional tools and research will we be able to define directed strategies for rice breeding which are able to combine important agronomic features with the demands of local consumers for specific quality attributes and hence, design new, improved crop varieties which will be awarded success in the global market.
PLoS ONE 01/2014; 9(1):e85106. DOI:10.1371/journal.pone.0085106 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Starch is a major component of human diets. The relative contribution of variation in the genes of starch biosynthesis to the nutritional and functional properties of the rice was evaluated in a rice breeding population. Sequencing 18 genes involved in starch synthesis in a population of 233 rice breeding lines discovered 66 functional SNPs in exonic regions. Five genes, AGPS2b, Isoamylase1, SPHOL, SSIIb and SSIVb showed no polymorphism. Association analysis found 31 of the SNP were associated with differences in pasting and cooking quality properties of the rice lines. Two genes appear to be the major loci controlling traits under human selection in rice, GBSSI (waxy gene) and SSIIa. GBSSI influenced amylose content and retrogradation. Other genes contributing to retrogradation were GPT1, SSI, BEI and SSIIIa. SSIIa explained much of the variation in cooking characteristics. Other genes had relatively small effects.
[Show abstract][Hide abstract] ABSTRACT: High-throughput sequencing of pooled DNA was applied to polymorphism discovery in candidate genes involved in starch synthesis. This approach employed semi- to long-range PCR (LR-PCR) followed by next-generation sequencing technology. A total of 17 rice starch synthesis genes encoding seven classes of enzymes, including ADP-glucose pyrophosphorylase (AGPase), granule starch synthase (GBSS), soluble starch synthase (SS), starch branching enzyme (BE), starch debranching enzyme (DBE) and starch phosphorylase (SPHOL) and phosphate translocator (GPT1) from 233 genotypes were PCR amplified using semi- to long-range PCR. The amplification products were equimolarly pooled and sequenced using massively parallel sequencing technology (MPS). By detecting single nucleotide polymorphism (SNP)/Indels in both coding and noncoding areas of the genes, we identified genetic differences and characterized the SNP/Indel variation and distribution patterns among individual starch candidate genes. Approximately, 60.9 million reads were generated, of which 54.8 million (90%) mapped to the reference sequences. The average coverage rate ranged from 12,708 to 38,300 times for SSIIa and SSIIIb, respectively. SNPs and single/multiple-base Indels were analysed in a total assembled length of 116,403 bp. In total, 501 SNPs and 113 Indels were detected across the 17 starch-related loci. The ratio of synonymous to nonsynonymous SNPs (Ka/Ks) test indicated GBSSI and isoamylase 1 (ISA1) as the least diversified (most purified) and conservative genes as the studied populations have been through cycles of selection. This report demonstrates a useful strategy for screening germplasm by MPS to discover variants in a specific target group of genes.
[Show abstract][Hide abstract] ABSTRACT: The barley genome sequence will be available soon, allowing barley to follow the rice model of genetic analysis, accelerating the rate of knowledge acquisition surrounding the genetic control of important traits. We have used the rice genome sequence to understand the genetic control of rice starch quality within the Australian rice breeding program. Illumina GAIIx sequencing of 17 rice starch synthesis genes from 233 genotypes within the Australian rice breeding program detected 501 SNPs and 113 Indels in both coding and non-coding regions. Five genes encoding the enzymes AGPL2a, Isoamylase1, SPHOL, SSIIb and SSIVb showed no polymorphism. Associations between 52 of the 110 functional SNP and the pasting and cooking quality of the rice were detected. GBSSI and SSIIa had a major influence on starch properties and the genes encoding other enzymes had minor associations. The ´G/T´ SNP at the boundary site of exon1/intron1 of GBSSI showed the strongest association with retrogradation and amylose content. The GC/TT SNP at exon 8 of SSIIa showed a very significant association with pasting temperature, gelatinisation temperature and peak time. A new ´C/T´ SNP was found at position 1188 in the GPT encoding gene which alters Leu24 to Phe. This SNP may be associated with retrogradation and amylose content. These associations provide new tools for deliberate selection of rice genotypes for specific functional and nutritional outcomes.
Acknowledgements: This project was funded by NSW DPI and the Australian Research Council. Thanks to Rachelle Ward NSW DPI for provision of rice starch phenotypic data and Stirling Bowen and Mark Edwards for technical assistance with Illumina GAIIx sequencing and MALDI-TOF MS (Sequenom MassARRAY) analysis.
[Show abstract][Hide abstract] ABSTRACT: In rice-consuming countries, specific varieties are recognized as premium, “gold standard” varieties, while others are recognized
as being superior but second best, despite being identical using the current suite of tools to evaluate quality. The objectives
of this study were to determine if there are distinguishable differences in sensory properties of premium and second best
varieties and whether these differences are common to premium varieties. Color, an important sensory property, was determined
on the raw and cooked rice using a colorimeter. As raw rice, some of the premium varieties were whiter than their second best
counterparts while others were not. However, when cooked, with two exceptions, the premium varieties were of the same or greater
whiteness than their counterparts. A trained sensory panel employed descriptive sensory analysis, an objective tool, to characterize
and analytically measure the flavor (aromatics, taste, mouthfeel) and texture of premium and second best varieties collected
from nine rice-consuming countries. Sweet taste, popcorn aroma/flavor, and water-like metallic mouthfeel showed significant
differences in intensity between the premium–second best variety pairs. Slickness, roughness, and springiness were the major
traits that distinguished the texture of varieties. Quality evaluation programs do not routinely measure these texture and
flavor traits, but the fact that they distinguished the varieties in most pairs indicates that their measurement should be
added to the suite of grain quality tests in the development of new higher-yielding, stress-tolerant varieties. The incorporation
of premium quality will ensure that quality is no impediment to widespread adoption leading to enhanced productivity and food
KeywordsRice flavor-Rice texture-Rice color-Rice quality-Descriptive sensory analysis-Amylose-Protein
[Show abstract][Hide abstract] ABSTRACT: Low temperature is a major abiotic stress for rice cultivation, causing serious yield loss in many countries. To identify QTL controlling low temperature induced spikelet sterility in rice, the progeny of F2, BC1F1 and BC2F1 populations derived from a Reiziq Lijiangheigu cross were exposed to 21/15C for 15 days at the booting stage, and spikelet sterility was assessed. For genotyping, 92 polymorphic markers from 373 SSR and 325 STS primer pairs were used. A major QTL was initially indentified on the short arm of chromosome 10 by selective genotyping using highly tolerant and susceptible progeny from F2 and BC1F1 populations. The QTL (qLTSPKST10. 1) was validated and mapped by genotyping the entire F2 (282 progeny) and BC1F1 (84 progeny) populations. The results from the F2 population showed that qLTSPKST 10.1 could explain 20.5% of the variation in spikelet sterility caused by low temperature treatment with additive (a = 14.4) and dominant effect (d = -7.5). From the analysis of 98 selected BC2F1 progeny, the QTL located in the 3.5 cM interval between S10010. 9 and S10014. 4 was further confirmed. Based on the studies of 3 generations in 2 years, it was clear that the QTL on chromosome 10 is a major determinant of the control of low temperature induced spikelet sterility at booting stage. 2010 Springer Science+Business Media B.V.
[Show abstract][Hide abstract] ABSTRACT: Next Generation allows rapid acquisition of genome wide sequence data for a range of key reference varieties which differ by important phenotypes. These data can be quickly converted to high throughput genotyping assays allowing rapid analysis of populations derived from the reference varieties. These data also can be used for variety identification and important quality controls. We have optimized a high-throughput multiplexed SNP assay for eight polymorphisms which explain two agronomic and three grain quality traits in rice. Gene fragments coding for the agronomic traits plant height (sd-1) and blast disease resistance (Pi-ta) and the quality traits amylose content (waxy), gelatinisation temperature (alk) and fragrance (fgr) were amplified in a multiplex PCR. A single base extension reaction carried out at the polymorphism responsible for each of these phenotypes within these genes generated extension products which were quantified by a MALDI–TOF Mass Spectrometry system, the Sequenom® MassARRAY®. The assay detects both SNPs and indels and is co-dominant, simultaneously in one 5 µL reaction, detecting both homozygous and heterozygous samples in a multiplex system. We are extending this approach to the analysis of rice starch biosynthesis genes and their association studies.
[Show abstract][Hide abstract] ABSTRACT: Gelatinisation temperature (GT) is one of the key traits measured in programs for breeding rice (Oryza sativa L.). It is commonly estimated by the alkali spreading value (ASV), and less commonly by differential scanning calorimetry (DSC). Using a diverse set of germplasm, it was determined that DSC values associate poorly with ASV, are not correlated with amylose content but correlate with cooking time. Rice varieties are traditionally grouped into three classes of GT based on ASV: high, intermediate and low. However, the distribution of DSC values of 4000 samples shows only two classes: high and low. Large differences in the distributions of chain lengths synthesised by starch synthase IIa (SSIIa) support the two classes as the major grouping, two haplotypes associating with each peak. Each peak of DSC values spanned 10C. The chain length distribution of the amylopectin molecules from varieties at the upper boundary of each peak showed significantly more chains that span both the crystalline and amorphous lamellae of a cluster than varieties at the other end of that distribution. Improved varieties, classified as intermediate GT by ASV, belong to both of the classes defined by DSC, implying that some enzyme, other than SSIIa is involved in intermediate GT.
[Show abstract][Hide abstract] ABSTRACT: Cereal Chem. 86(5):492–498 Amylose content is a parameter that correlates with the cooking behav-ior of rice. It is measured at the earliest possible stages of rice improve-ment programs to enable breeders to build the foundations of appropriate grain quality during cultivar development. Amylose is usually quantified by absorbance of the amylose-iodine complex. The International Network for Quality Rice (INQR) conducted a survey to determine ways that amy-lose is measured, reproducibility between laboratories, and sources of variation. Each laboratory measured the amylose content of a set of 17 cultivars of rice. The study shows that five different versions of the iodine
[Show abstract][Hide abstract] ABSTRACT: Automated DNA Extraction, Next Generation Sequencing and high throughput genotyping technologies used in combination allow more tightly targeted approaches to genetic analysis than ever before. It is now possible to quickly acquire genome wide data for a range of key reference varieties which differ by important phenotypes. These data can be quickly converted to high throughput genotyping assays allowing rapid analysis of populations derived from the reference varieties. The same data can be used for cereal variety identification, an important quality control tool. We have optimized a high-throughput multiplexed SNP assay for eight polymorphisms which explain two agronomic and three grain quality traits in rice. Gene fragments coding for the agronomic traits plant height (sd-1) and blast disease resistance (Pi-ta) and the quality traits amylose content (waxy), gelatinisation temperature (alk) and fragrance (fgr) were amplified in a multiplex PCR. A single base extension reaction carried out at the polymorphism responsible for each of these phenotypes within these genes generated extension products which were quantified by a MALDI –TOF system, the Sequenom® MassARRAY®. The assay detects both SNPs and indels and is co-dominant, simultaneously detecting both homozygous and heterozygous samples in a multiplex system. We are extending this approach to the analysis of rice starch biosynthesis genes.
[Show abstract][Hide abstract] ABSTRACT: Low temperature is a common production constraint in rice cultivation in temperate zones and high-elevation environments, with the potential to affect growth and development from germination to grain filling. There is a wide range of genotype-based differences in cold tolerance among rice varieties, these differences often reflecting growth conditions in the place of origin, as well as breeding history. However, improving low temperature tolerance of varieties has been difficult, due to a lack of clarity of the genetic basis to low temperature tolerance for different growth stages of the rice plant. Seeds or plants of 17 rice varieties of different origins were exposed to low temperature during germination (15°C), seedling, booting, and flowering stages (18.5°C), to assess their cold tolerance at different growth stages. Low temperature at the germination stage reduced both the percentage and speed of germination. Varieties from China (B55, Banjiemang, and Lijianghegu) and Hungary (HSC55) were more tolerant of low temperature than other varieties. Most of the varieties showed moderate levels of low temperature tolerance during the seedling stage, the exceptions being some varieties from Australia (Pelde, YRL39, and YRM64) and Africa (WAB160 and WAB38), which were susceptible to low temperature at the seedling stage. Low temperature at booting and flowering stages reduced plant growth and caused a significant decline in spikelet fertility. Some varieties from China (B55, Bangjiemang, Lijiangheigu), Japan (Jyoudeki), the USA (M103, M104), and Australia (Quest) were tolerant or moderately tolerant, while the remaining varieties were susceptible or moderately susceptible to low temperature at booting and flowering stages. Three varieties from China (B55, Lijianghegu, Banjiemang) and one from Hungary (HSC55) showed consistent tolerance to low temperature at all growth stages. These varieties are potentially important gene donors for breeding and genetic studies. The cold tolerance of the 17 rice varieties assessed at different growth stages was correlated. Screening for cold tolerance during early growth stages can therefore potentially be an effective way for assessing cold tolerance in breeding programs.
[Show abstract][Hide abstract] ABSTRACT: The cooking quality of rice is associated with the starch gelatinization temperature (GT). Rice genotypes with low GT have probably been selected for their cooking quality by humans during domestication. We now report polymorphisms in starch synthase IIa (SSIIa) that explain the variation in rice starch GT. Sequence analysis of the eight exons of SSIIa identified significant polymorphism in only exon 8. These single nucleotide polymorphisms (SNPs) were determined in 70 diverse genotypes of rice. Two SNPs could classify all 70 genotypes into either high GT or low GT types which differed in GT by 8 degrees C. 'A' rather than 'G' at base 2412 determined whether a methionine or valine was present at the corresponding amino acid residue in SSIIa, whilst two adjacent SNPs at bases 2543 and 2544 coded for either leucine (GC) or phenylalanine (TT). Rice varieties with high GT starch had a combination of valine and leucine at these residues. In contrast, rice varieties with low GT starch had a combination of either methionine and leucine or valine and phenylalanine at these same residues. At least two distinct polymorphisms have apparently been selected for their desirable cooking qualities in the domestication of rice.
[Show abstract][Hide abstract] ABSTRACT: We have previously determined that fragrance in rice, a recessive trait, is due to a large deletion (8bp) and 3 SNP’s in a gene on chromosome 8 which encodes a putative betaine aldehyde dehydrogenase 2 (BAD2). This mutation results in the formation of a truncated BAD2 enzyme because of the creation of an in-frame termination signal 800bp before that of the wild type. Because this truncated BAD2 is missing key binding domains, it is unlikely that it is capable of acting upon the target substrate and this leads to an accumulation of the principal fragrant molecule, 2-acetyl-1-pyrroline. Here we utilise single tube allele specific amplification (STASA) as a simple, low-cost, perfect assay to discriminate between fragrant and non-fragrant rice varieties in addition to homozygous fragrant, homozygous non-fragrant and heterozygous non-fragrant individuals in a population segregating for fragrance. Two external primers generate a 580bp fragment as a positive control for each sample. Internal primers in conjunction with their corresponding external primer pair produce a 355bp fragment from a non-fragrant allele and a 257bp fragment from a fragrant allele, allowing analysis on agarose gels.
[Show abstract][Hide abstract] ABSTRACT: Allele specific amplification (ASA) is a low-cost, robust technique that can be utilised to discriminate between alleles that
differ by SNP's, insertions or deletions, within a single PCR tube. Fragrance in rice, a recessive trait, has been shown to
be due to an eightbp deletion and three SNP's in a gene on chromosome 8 which encodes a putative betaine aldehyde dehydrogenase
2 (BAD2). Here we report a single tube ASA assay which allows discrimination between fragrant and non-fragrant rice varieties
and identifies homozygous fragrant, homozygous non-fragrant and heterozygous non-fragrant individuals in a population segregating
for fragrance. External primers generate a fragment of approximately 580bp as a positive control for each sample. Internal
and corresponding external primers produce a 355bp fragment from a non-fragrant allele and a 257bp fragment from a fragrant
allele, allowing simple analysis on agarose gels.
[Show abstract][Hide abstract] ABSTRACT: The whole rice genome sequence was used to assist in the identification of a single nucleotide polymorphism (SNP) marker linked to the fragrance gene (fgr) in rice. Genes flanked by restriction fragment length polymorphism and microsatellite markers known to be linked to the fragrance gene were identified by DNA sequence alignment of EST sequences against BAC clones covering this region of chromosome eight. Re-sequencing and comparison of parts of these genes derived from a fragrant and a non-fragrant cultivar revealed only one SNP (a C/T transition) in more than 6 kbp of sequence from 14 genes. Ten of eleven fragrant genotypes and six of 14 non-fragrant genotypes tested carried the C allele. This approach indicated a generally low level of SNP polymorphism in cultivated rice suggesting that association of SNP with phenotypes should be an efficient path to gene discovery in cultivated rice.