Gelatinization temperature of rice explained by polymorphisms in starch synthase

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 °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.     

Functional analysis of starch-synthesis genes in determining rice eating and cooking qualities

Apparent amylose content (AAC), gel consistency (GC), and gelatinization temperature (GT) are recognized as the most important determinants of rice eating and cooking qualities. The contributions of major starch-synthesis genes to these three traits have been investigated in the three consecutive experiments. In an initial QTL mapping with 130 doubled haploid (DH) lines, derived from an inter-subspecific cross of ‘Nanjing11’/‘Balilla’, the major QTLs responsible for AAC, GC, and GT coincided with the Wx (granule-bound starch synthase gene), Wx, and Sss IIa (soluble starch synthase gene) loci, respectively. In the second experiment, contributions of the major starch-synthesis genes to AAC, GC, and GT variations were estimated by using a multiple linear regression analysis. As shown, the Wx locus was a principal determinant for both AAC and GC, and could account for 58.5% and 38.9% of the phenotypic variations, respectively; while the Sss IIa locus was associated with GT, and could explain 25.5% of the observed variation. Eventually, a F₂ population consisting of 501 individuals, derived from an inter-subspecific cross of the two sticky rice varieties ‘Suyunuo’ and ‘Yangfunuo 4’, was examined with gene-tagged markers. In the absence of the Wx gene, none of the starch-synthesis genes investigated could dominate the GC variation, however, the Sss IIa locus could also explain 25.1% of the GT variation. In summary, the Wx locus dominates the AAC variation, and meanwhile plays a major role in the GC variation. The Sss IIa locus is a major factor in explaining the GT variation. Apart from the major genes, other genetic factors may also contribute to the GC/GT variations.     

Sequence diversity, haplotype analysis, association mapping and functional marker development in the waxy and starch synthase IIa genes for grain-yield-related traits in hexaploid wheat (Triticum aestivum L.)

Development of high-yielding cereal crops could meet increasing global demands for food, feed and bio-fuels. Wheat is one of the world??s most important cereal crops. The biosynthesis of starch is the major determinant of yield in wheat. Two starch biosynthesis genes, the waxy (Wx) genes and the starch synthase IIa (SSIIa) genes, were amplified and sequenced in 92 diverse wheat genotypes using genome-specific primers. Nucleotide diversity, haplotype analysis and association mapping were performed. The first exon (5??-UTR) and the first intron of the three homoeologous Wx genes were isolated using expressed sequence tag sequences. The Wx genes contained 12 exons separated by 11 introns. SNP (single nucleotide polymorphism) frequency ranged from 1 SNP/3,648?bp for Wx-D1 to 1 SNP/135?bp for SSIIa-A1, with an average of 1 SNP/230?bp. The average SNP frequencies in exon and intron regions were 1 SNP/322?bp and 1 SNP/228?bp, respectively. Thirty, 23 and 5 SNPs were identified and formed five, six and five haplotypes for SSIIa-A1, SSIIa-B1 and SSIIa-D1, respectively. However, no association was found between these SNPs and seven yield-related traits. Twenty-two, 15 and 1 SNPs were detected and formed nine, five and two haplotypes for Wx-A1, Wx-B1 and Wx-D1, respectively. Three unique nucleotides C+A+T at SNP5, SNP6 and SNP12 formed Wx-B1-H3, which was significantly associated with increased grain weight, thousand kernel weight, and total starch content in three spring wheat genotypes and five winter wheat genotypes. Cost-effective and co-dominant SNP markers were developed using temperature-switch (TS)-PCR and are being used for marker-assisted selection of doubled haploid lines with enhanced grain yield and starch content in winter wheat breeding programs.     

Nucleotide Diversity and Molecular Evolution of the <Emphasis Type="Italic">ALK</Emphasis> Gene in Cultivated Rice and its Wild Relatives

Gelatinization temperature (GT), an important parameter for rice cooking quality, is mainly regulated by the ALK gene encoding starch synthase IIa. Here, we reported the nucleotide diversity of the ALK gene in 122 cultivated accessions and 199 wild rice accessions that were collected around the Pearl River Basin in China. A total of 93 single nucleotide ploymorphisms (SNPs) were identified, with an average of one SNP per 40 bp. Tajima D statistics revealed that the DNA sequences covering the last exon have probably evolved under balancing selection. Based on two functional SNPs (an A to G substitution at 4198 bp and a GC to TT dinucleotide substitution at 4330/4331 bp), three haplotypes, G/GC, G/TT, and A/GC, were identified in both wild and cultivated accessions, with the G/GC haplotype being predominant. Interestingly, the A/GC haplotype was exclusively found in the wild accessions from Guangdong province, while the G/TT haplotype was only present in the wild accessions from Jiangxi province and Hainan Island. This suggests that the G/TT and A/GC variants may have arisen independently and undergone balancing selection on separate haplotypes in multiple populations. Our result supports earlier hypothesis that cultivated rice was independently domesticated from multiple domestication events in China. Our study aids in the understanding of the domestication process that led to the improvement of rice grain quality.     

Microsatellites, single nucleotide polymorphisms and a sequence tagged site in starch-synthesizing genes in relation to starch physicochemical properties in nonwaxy rice (Oryza sativa L.)

Starch characteristics determine the quality of various products of rice, e.g., eating, cooking and processing qualities. Our previous study indicated that molecular markers inside or close to starch synthesizing genes can differentiate the starch properties of 56 waxy rices. Here we report microsatellite (or simple sequence repeat, SSR) polymorphism in the Waxy (Wx) gene, soluble starch synthase I gene (SS1) and starch branching enzyme 1 gene (SBE1), single nucleotide polymorphism (SNP) in Wx and starch branching enzyme 3 gene (SBE3), and a sequence tagged site (STS) in starch branching enzyme 1 gene (SBE1) among 499 nonwaxy rice samples and their relationships with starch physicochemical properties. The nonwaxy rice samples consist of landraces (n = 172) obtained from germplasm centers and cultivars and breeding lines (n = 327) obtained from various breeding programs. Ten (CT) _n microsatellite alleles, (CT)₈, (CT)₁₀, (CT)₁₁, (CT)_12, (CT)₁₇, (CT)₁₈, (CT)₁₉, (CT)₂₀, (CT)₂₁, and (CT)₂₂, were found at the Wx locus, of which (CT)₁₁ was the most frequent, and (CT)₁₂, (CT)₂₁ and (CT)₂₂ were identified for the first time. Four (CT) _n microsatellite alleles were found at the SBE1 locus, (CT)₈, (CT)₉, and (CT)₁₀ together with an insertion sequence of CTCTCGGGCGA, and (CT)₈ alone without the insertion, of which (CT)₉ and the insertion was a new allele identified in only one rice, IR1552. Multiple microsatellites clustered at the SS1 locus, and in addition to the three alleles previously detected (SSS-A = (AC)₂...TCC(TC)₁₁...(TC)₅C(ACC)₁₁, SSS-B = (AC)₃...TCT(TC)₆...(TC)₄C(ACC)₉, and SSS-C = (AC)₃...TCT(TC)₆...(TC)₄C(ACC)₈), one new allele (SSS-D = (AC)₂...TCC(TC)₁₀...(TC)₄C(ACC)₉) was found. Analysis of the starch physicochemical properties of the samples with different microsatellites, SNPs and STS groups indicated that these molecular markers can differentiate almost all the physicochemical properties examined, e.g., apparent amylose content (AAC), pasting viscosity characteristics, and gel textural properties. Wx SSR and Wx SNP alone explained more variations for all physicochemical properties than the other molecular markers. The total six markers could explain 92.2, 81 and 86% of total variation of AAC, gel hardness (HD), and gel cohesiveness (COH), respectively, and they could explain more than 40% of the total variation of hot paste viscosity (HPV), cool paste viscosity (CPV), breakdown viscosity (BD), setback viscosity (SB) and gel adhesiveness (ADH). However, only 29% of the total variation of peak viscosity (PV) and 37% of pasting temperature (PT) could be explained by all the molecular markers. Some of these markers can differentiate the starch physicochemical properties among the rice samples with the same Wx allele, indicating that the variation within Wx allele classes can be explained by other starch synthesizing genes. These SSRs, SNPs and STS are useful in marker-assisted breeding for the improvement of starch quality of rice.Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.     

Pyramiding transgenes for multiple resistance in rice against bacterial blight,yellow stem borer and sheath blight

The present investigation revealed that the alk and gel(t) genes, which cause the differences between a japonica rice variety Nipponbare and an indica rice variety Kasalath in terms of the disintegration of endosperm starch granules in alkali solution and their gelatinisation in a 4 M urea solution, respectively, cosegregated in backcross inbred lines derived from a cross between the two varieties. The segregation pattern of the profile for amylopectin chain-length, which was distinguished by enrichment in short chains of DP≦11 and depletion in intermediate-size chains of 12≦DP≦24 in japonica as compared with indica, was exactly the same as those of the above physico-chemical properties of starch granules, and the gene was designated as acl(t). Gene-mapping analysis showed that the starch synthase IIa (SSIIa) gene is located at the alk locus on chromosome 6 in the rice genome. These results lead us to the possibility that different alleles of the SSIIa gene are responsible for differences in amylopectin structure between the two varieties, in that SSIIa plays a distinct role in the elongation of short chains within clusters (A+B₁ chains) of amylopectin. It is proposed that the activity of SSIIa in japonica rice is reduced in amount or functional capacity relative to the activity of this enzyme in indica rice. This, in turn, would explain why starch from japonica rice has a lower gelatinisation temperature than starch from indica rice and is more susceptible to disintegration in alkali or urea. The evidence for this hypothesis is that the alk(t), gel(t), acl(t) and SSIIa genes all map to the same locus. Received: 29 January 2001 / Accepted: 12 April 2001     

Development of SNP-based CAPS and dCAPS markers in eight different genes involved in starch biosynthesis in rice

Single nucleotide polymorphisms (SNPs), which are inexhaustible, highly stable, and simply detectable sequence polymorphisms, can lead to phenotypic variations by affecting protein composition changes. Here, we report development of 25 new cleaved amplified polymorphic sequence or derived cleaved amplified polymorphic sequence markers that have discrete band sizes in relation to the SNP genotypes in eight putative gene regions. The average frequency of DNA polymorphisms was 1 per 175 bp (SNPs, 1 per 217 bp; In/dels, 1 per 906 bp). In primary statistical analysis of each marker on 55 diverse rice accessions, including different ecotypes, the mean value of the major allele frequency was 0.658 (0.509–0.927). The average polymorphism information content was 0.326 (0.126–0.375). The mean value of the inbreeding coefficient (f) was 0.950 and was positive (heterozygote deficiency) at all loci, corresponding to the inbreeding system in rice. In cluster analysis, all rice accessions clustered mainly into three groups according to the ecotypes. The association analysis showed that the SNP of Granule-bound starch synthase I and ADP-glucose pyrophosphorylase small subunit (ADPase-S) genes were highly associated with apparent amylose content variation than the others. These new SNP markers may be useful in genotyping rice germplasm, in marker-assisted selection for improving starch quality and content, and in linkage as well as association studies. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Development of formulae for estimating amylose content and resistant starch content based on the pasting properties measured by RVA of Japonica polished rice and starch

We searched for the easy and simple method to measure the novel indicators which reflect not only AAC, but also (RS) based on pasting properties using RVA. Novel indexes such as SB/Con and Max/Fin (Maximum viscosity/Minimum viscosity) ratios had a very high correlation with proportion of intermediate and long chains of amylopectin; Fb₁₊₂₊₃ (DP ≧ 13). In Japonica polished rice, estimation formulae for AAC and RS content were developed using novel indexes based on pasting properties by RVA, and these equations showed determination coefficients of 0.89 and 0.80 for calibration and 0.71 and 0.75 for validation test. We developed the estimation formulae for AAC and RS content for Japonica starch samples. These equations showed determination coefficients of 0.86 and 1.00 for calibration and 0.76 and 0.83 for validation test, which showed that these equations can be applied to the unknown rice samples.     

Essential amino acids of starch synthase IIa differentiate amylopectin structure and starch quality between <Emphasis Type="Italic">japonica</Emphasis> and <Emphasis Type="Italic">indica</Emphasis> rice varieties

Four amino acids were variable between the ‘active’ indica-type and ‘inactive’ japonica-type soluble starch synthase IIa (SSIIa) of rice plants; Glu-88 and Gly-604 in SSIIa of indica-cultivars IR36 and Kasalath were replaced by Asp-88 and Ser-604, respectively, in both japonica cultivars Nipponbare and Kinmaze SSIIa, whereas Val-737 and Leu-781 in indica SSIIa were replaced by Met-737 in cv. Nipponbare and Phe-781 in cv. Kinmaze SSIIa, respectively. The SSIIa gene fragments shuffling experiments revealed that Val-737 and Leu-781 are essential not only for the optimal SSIIa activity, but also for the capacity to synthesize indica-type amylopectin. Surprisingly, however, a combination of Phe-781 and Gly-604 could restore about 44% of the SSIIa activity provided that Val-737 was conserved. The introduction of the ‘active’ indica-type SSIIa gene enabled the japonica-type cv. Kinmaze to synthesize indica-type amylopectin. The starch in the transformed japonica rice plants exhibited gelatinization-resistant properties that are characteristic of indica-rice starch. Transformed lines expressing different levels of the IR36 SSIIa protein produced a variety of starches with amylopectin chain-length distribution patterns that correlated well with their onset temperatures of gelatinization. The present study confirmed that the SSIIa activity determines the type of amylopectin structure of rice starch to be either the typical indica-type or japonica-type, by playing a specific role in the synthesis of the long B₁ chains by elongating short A and B₁ chains, notwithstanding the presence of functional two additional SSII genes, a single SSI gene, two SSIII genes, and two SSIV genes in rice plants.     

A whole‐genome SNP array (RICE6K) for genomic breeding in rice

The advances in genotyping technology provide an opportunity to use genomic tools in crop breeding. As compared to field selections performed in conventional breeding programmes, genomics‐based genotype screen can potentially reduce number of breeding cycles and more precisely integrate target genes for particular traits into an ideal genetic background. We developed a whole‐genome single nucleotide polymorphism (SNP) array, RICE6K, based on Infinium technology, using representative SNPs selected from more than four million SNPs identified from resequencing data of more than 500 rice landraces. RICE6K contains 5102 SNP and insertion–deletion (InDel) markers, about 4500 of which were of high quality in the tested rice lines producing highly repeatable results. Forty‐five functional markers that are located inside 28 characterized genes of important traits can be detected using RICE6K. The SNP markers are evenly distributed on the 12 chromosomes of rice with the average density of 12 SNPs per 1 Mb and can provide information for polymorphisms between indica and japonica subspecies as well as varieties within indica and japonica groups. Application tests of RICE6K showed that the array is suitable for rice germplasm fingerprinting, genotyping bulked segregating pools, seed authenticity check and genetic background selection. These results suggest that RICE6K provides an efficient and reliable genotyping tool for rice genomic breeding.     

Genome-specific primer sets for starch biosynthesis genes in wheat

Common wheat (Triticum aestivum L., 2n=6x=42) is an allohexaploid composed of three closely related genomes, designated A, B, and D. Genetic analysis in wheat is complicated, as most genes are present in triplicated sets located in the same chromosomal regions of homoeologous chromosomes. The goal of this report was to use genomic information gathered from wheat–rice sequence comparison to develop genome-specific primer sets for five genes involved in starch biosynthesis. Intron locations in wheat were inferred through the alignment of wheat cDNA sequences with rice genomic sequence. Exon-anchored primers, which amplify across introns, allowed the sequencing of introns from the three genomes for each gene. Sequence variation within introns among the three wheat genomes provided the basis for genome-specific primer design. For three genes, ADP-glucose pyrophosphorylase (Agp-L), sucrose transporter (SUT), and waxy (Wx), genome-specific primer sets were developed for all three genomes. Genome-specific primers were developed for two of the three genomes for Agp-S and starch synthase I (SsI). Thus, 13 of 15 possible genome-specific primer sets were developed using this strategy. Seven genome-specific primer combinations were used to amplify alleles in hexaploid wheat lines for sequence comparison. Three single nucleotide polymorphisms (SNPs) were identified in a comparison of 5,093 bp among a minimum of ten wheat accessions. Two of these SNPs could be converted into cleaved amplified polymorphism sequence (CAPS) markers. Our results indicated that the design of genome-specific primer sets using intron-based sequence differences has a high probability of success, while the identification of polymorphism among alleles within a genome may be a challenge.     

Endosperm enrichment and physicochemical properties of superior and inferior grain starch in super hybrid rice

• A significant asynchronous phenomenon exists in super hybrid rice because of the differences in spike and spikelet positions, which affect the accumulation and properties of starch. However, little is known about the endosperm enrichment and physicochemical properties of starch in superior and inferior grains in super hybrid rice.
• Rice YY2640 was selected as study material to investigate the enrichment and physicochemical properties of starch in superior and inferior grains in super rice using semi‐thin sections, X‐ray diffraction and related technologies.
• Superior grain filling was a continuous process, whereas inferior grain only started 8–10 days after anthesis. The order of starch accumulation starts in the central endosperm, then in the endosperm of the proximal vascular bundle and finally in the aleurone layer. Compared with the inferior grains, the superior grains have a higher 1000‐grain weight, apparent amylose content, total starch content, average starch granule size, relative crystallinity, solubility and a resonance peak ratio at 1022/995 cm^?1, whereas the swelling power and ratio of the resonance peak at 1045/1022 cm^?1 were lower. The final degree of hydrolysis of HCl, AAG and PPA of the superior grains were significantly lower than those of the inferior grains.
• The findings indicate that the different physicochemical properties of starch were mainly related to the development order of superior and inferior grains and the spatial enrichment of starch.

     

The three important traits for cooking and eating quality of rice grains are controlled by a single locus in an elite rice hybrid, Shanyou 63

The cooking and eating quality of the rice grain is one of the most serious problems in many rice-producing areas of the world. In this study, we conducted a molecular marker-based genetic analysis of three traits, amylose content (AC), gel consistency (GC) and gelatinization temperature (GT), that are the most important constituents of the cooking and eating quality of rice grains. The materials used in the analysis included F₂ seeds, an F_2:3 population, and an F₉ recombinant inbred-line population from a cross between the parents of ’Shanyou 63’, the most widely grown hybrid in rice production in China. Segregation analyses of these three generations showed that each of the three traits was controlled by a single Mendelian locus. Molecular marker-based QTL (quantitative trait locus) analyses, both by one-way analysis of variance using single marker genotypes and by whole-genome scanning with MAPMAKER/QTL, revealed a single locus that controls the expression of all three traits. This locus coincided with the Wx region on the short arm of chromosome 6, indicating that all three traits were either controlled by the Wx locus or by a genomic region tightly linked to this locus. This finding has provided clues to resolving the molecular bases of GC and GT in future studies. The results also have direct implications for the quality improvement of rice varieties. Received: 5 January 1999 / Accepted 30 January 1999     

High-throughput single nucleotide polymorphism genotyping for breeding applications in rice using the BeadXpress platform

Multiplexed single nucleotide polymorphism (SNP) markers have the potential to increase the speed and cost-effectiveness of genotyping, provided that an optimal SNP density is used for each application. To test the efficiency of multiplexed SNP genotyping for diversity, mapping and breeding applications in rice (Oryza sativa L.), we designed seven GoldenGate VeraCode oligo pool assay (OPA) sets for the Illumina BeadXpress Reader. Validated markers from existing 1536 Illumina SNPs and 44?K Affymetrix SNP chips developed at Cornell University were used to select subsets of informative SNPs for different germplasm groups with even distribution across the genome. A 96-plex OPA was developed for quality control purposes and for assigning a sample into one of the five O. sativa population subgroups. Six 384-plex OPAs were designed for genetic diversity analysis, DNA fingerprinting, and to have evenly-spaced polymorphic markers for quantitative trait locus (QTL) mapping and background selection for crosses between different germplasm pools in rice: Indica/Indica, Indica/Japonica, Japonica/Japonica, Indica/O. rufipogon, and Japonica/O. rufipogon. After testing on a diverse set of rice varieties, two of the SNP sets were re-designed by replacing poor-performing SNPs. Pilot studies were successfully performed for diversity analysis, QTL mapping, marker-assisted backcrossing, and developing specialized genetic stocks, demonstrating that 384-plex SNP genotyping on the BeadXpress platform is a robust and efficient method for marker genotyping in rice.     

Development and evaluation of single-nucleotide polymorphism markers in allotetraploid rapeseed (<Emphasis Type="Italic">Brassica napus</Emphasis> L.)

Single-nucleotide polymorphisms (SNPs) and insertion–deletions (INDELs) are currently the important classes of genetic markers for major crop species. In this study, methods for developing SNP markers in rapeseed (Brassica napus L.) and their in silico mapping and use for genotyping are demonstrated. For the development of SNP and INDEL markers, 181 fragments from 121 different gene sequences spanning 86 kb were examined. A combination of different selection methods (genome-specific amplification, hetero-duplex analysis and sequence analysis) allowed the detection of 18 singular fragments that showed a total of 87 SNPs and 6 INDELs between 6 different rapeseed varieties. The average frequency of sequence polymorphism was estimated to be one SNP every 247 bp and one INDEL every 3,583 bp. Most SNPs and INDELs were found in non-coding regions. Polymorphism information content values for SNP markers ranged between 0.02 and 0.50 in a set of 86 varieties. Using comparative genetics data for B. napus and Arabidopsis thaliana, an allocation of SNP markers to linkage groups in rapeseed was achieved: a unique location was determined for seven gene sequences; two and three possible locations were found for six and four sequences, respectively. The results demonstrate the usefulness of existing genomic resources for SNP discovery in rapeseed.     

Genetic diversity and population structure of a diverse set of rice germplasm for association mapping

Germplasm diversity is the mainstay for crop improvement and genetic dissection of complex traits. Understanding genetic diversity, population structure, and the level and distribution of linkage disequilibrium (LD) in target populations is of great importance and a prerequisite for association mapping. In this study, 100 genome-wide simple sequence repeat (SSR) markers were used to assess genetic diversity, population structure, and LD of 416 rice accessions including landraces, cultivars and breeding lines collected mostly in China. A model-based population structure analysis divided the rice materials into seven subpopulations. 63% of the SSR pairs in these accessions were in LD, which was mostly due to an overall population structure, since the number of locus pairs in LD was reduced sharply within each subpopulation, with the SSR pairs in LD ranging from 5.9 to 22.9%. Among those SSR pairs showing significant LD, the intrachromosomal LD had an average of 25–50 cM in different subpopulations. Analysis of the phenotypic diversity of 25 traits showed that the population structure accounted for an average of 22.4% of phenotypic variation. An example association mapping for starch quality traits using both the candidate gene mapping and genome-wide mapping strategies based on the estimated population structure was conducted. Candidate gene mapping confirmed that the Wx and starch synthase IIa (SSIIa) genes could be identified as strongly associated with apparent amylose content (AAC) and pasting temperature (PT), respectively. More importantly, we revealed that the Wx gene was also strongly associated with PT. In addition to the major genes, we found five and seven SSRs were associated with AAC and PT, respectively, some of which have not been detected in previous linkage mapping studies. The results suggested that the population may be useful for the genome-wide marker–trait association mapping. This new association population has the potential to identify quantitative trait loci (QTL) with small effects, which will aid in dissecting complex traits and in exploiting the rich diversity present in rice germplasm.     

Association between nonsynonymous mutations of starch synthase IIa and starch quality in rice (Oryza sativa)

Starch quality is one of the most important agronomic traits in Asian rice, Oryza sativa. Starch synthase IIa (SsIIa) is a major candidate gene for starch quality variation. Within SsIIa, three nonsynonymous mutations in exon 8 have been shown to affect enzyme activity when expressed in Escherichia coli. To search for the variation in SsIIa that is responsible for starch quality variation in rice, we sequenced the SsIIa exon 8 region and measured starch quality as starch disintegration in alkali for 289 accessions of cultivated rice and 57 accessions of its wild ancestor, Oryza rufipogon. A general linear model and nested clade analysis were used to identify the associations between the three nonsynonymous single nucleotide polymorphisms (SNPs) and starch quality. Among the three nonsynonymous SNPs, we found strong evidence of association at one nucleotide site ('SNP 3'), corresponding to a Leu/Phe replacement at codon 781. A second SNP, corresponding to a Val/Met replacement at codon 737, could potentially show an association with increased sample sizes. Variation in SsIIa enzyme activity is associated with the cohesiveness of rice grains when cooked, and our findings are consistent with selection for more cohesive grains during the domestication of tropical japonica rice.     

SNP haplotypes of the <Emphasis Type="Italic">BADH1</Emphasis> gene and their association with aroma in rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.)

Betaine aldehyde dehydrogenase (BADH) is a key enzyme involved in the synthesis of glycinebetaine—a powerful osmoprotectant against salt and drought stress in a large number of species. Rice is not known to accumulate glycinebetaine but it has two functional genes coding for the BADH enzyme. A non-functional allele of the BADH2 gene located on chromosome 8 is a major factor associated with rice aroma. However, similar information is not available regarding the BADH1 gene located on chromosome 4 despite the similar biochemical function of the two genes. Here we report on the discovery and validation of SNPs in the BADH1 gene by re-sequencing of diverse rice varieties differing in aroma and salt tolerance. There were 17 SNPs in introns with an average density of one per 171 bp, but only three SNPs in exons at a density of one per 505 bp. Each of the three exonic SNPs led to changes in amino acids with functional significance. Multiplex SNP assays were used for genotyping of 127 diverse rice varieties and landraces. In total 15 SNP haplotypes were identified but only four of these, corresponding to two protein haplotypes, were common, representing more than 85% of the cultivars. Determination of population structure using 54 random SNPs classified the varieties into two groups broadly corresponding to indica and japonica cultivar groups, aromatic varieties clustering with the japonica group. There was no association between salt tolerance and the common BADH1 haplotypes, but aromatic varieties showed specific association with a BADH1 protein haplotype (PH2) having lysine₁₄₄ to asparagine₁₄₄ and lysine₃₄₅ to glutamine₃₄₅ substitutions. Protein modeling and ligand docking studies show that these two substitutions lead to reduction in the substrate binding capacity of the BADH1 enzyme towards gamma-aminobutyraldehyde (GABald), which is a precursor of the major aroma compound 2-acetyl-1-pyrroline (2-AP). This association requires further validation in segregating populations for potential utilization in the rice breeding programs.