Russell F Reinke

Identification of microsatellite markers for fragrance in rice by analysis of the rice genome sequence

Several chemical constituents are important to the fragrance of cooked rice. However, the chemical compound 2-acetyl-1-pyrroline (AP) is regarded as the most important component of fragrance in the basmati- and jasmine-style fragrant rices. AP is found in all parts of the plant except the roots. It is believed that a single recessive gene is responsible for the production of fragrance in most rice plants. The detection of fragrance can be carried out via sensory or chemical methods, although each has their disadvantages. To overcome these difficulties, we have identified an (AT)40 repeat microsatellite or simple sequence repeat (SSR) marker for fragrant and non-fragrant alleles of the fgr gene. Identification of this marker was facilitated through use of both the publicly available and restricted access sequence information of the Monsanto rice sequence databases. Fifty F2 individuals from a mapping population were genotyped for the polymorphic marker. This marker has a high polymorphism information content (PIC = 0.9). Other SSR markers linked to fragrance could be identified in the same way of use in other populations. This study demonstrates that analysis of the rice genome sequence is an effective option for identification of markers for use in rice improvement.

Diversity of Global Rice Markets and the Science Required for Consumer-Targeted Rice Breeding

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.

A single nucleotide polymorphism (SNP) marker linked to the fragrance gene in rice (Oryza sativa L.)

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.

Addressing the dilemmas of measuring amylose in rice.

ABSTRACT Amylose content is a parameter that correlates with the cooking behavior of rice. It is measured at the earliest possible stages of rice improvement 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 amylose 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 binding method are in use. The data show that repeatability was high within laboratories but reproducibility between laboratories was low. The major sources of variability were the way the standard curve was constructed and the iodine binding capacity of the potato amylose used to produce the standard. Reproducibility is much lower between laboratories using ...

SNP in starch biosynthesis genes associated with nutritional and functional properties of rice

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.

PCR-based molecular markers for the fragrance gene in rice (Oryza sativa L.).

Abstract The genomic DNA clone RG28, linked to the major fragrance gene of rice (fgr), was assessed for polymorphism in order to produce a PCR-based marker for fragrance. A small mono-nucleotide repeat, that was polymorphic between a pair of fragrant and non-fragrant cultivars, was identified and developed into a co-dominant PCR-based marker. The polymorphism-information-content determinations for three microsatellite markers, that have been genetically mapped near RG28, are also presented. These PCR-based markers will be highly useful in distinguishing fragrance-producing alleles from non-fragrance-producing alleles at the fgr locus.

Melting the secrets of gelatinisation temperature in rice

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 10°C. 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.

Important Sensory Properties Differentiating Premium Rice Varieties

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 security.

Discovery of polymorphisms in starch‐related genes in rice germplasm by amplification of pooled DNA and deeply parallel sequencing

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.

Lower-than-expected floret sterility of rice under extremely hot conditions in a flood-irrigated field in New South Wales, Australia.

Abstract Rice florets are susceptible to high-temperature damage at anthesis, but rice production remains stable in the Riverina region of Australia even when the air temperature during flowering exceeds 40°C. To identify the mechanism that supports rice production under these conditions, we examined sterility and pollination in relation to microclimate and panicle temperature in an extremely hot paddy field in the Riverina region of New South Wales. In windy > 40°C weather, the panicle temperature was > 38°C at the windward edge of the crop but around 35°C inside the crop, probably because of strong transpirational cooling due to the extremely dry wind (15% RH). Pollen from the windward edge of the crop showed extremely poor germination, yet that from inside the crop showed sufficient germination for fertilization. Moreover, sterility inside the crop was significantly lower than that at windward edge. We concluded that the wind with large vapor pressure deficit enabled stable rice production under the extreme heat during flowering.