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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.
Molecular Breeding 04/2012; 16(4):279-283. · 2.85 Impact Factor
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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.
Scientific Reports 01/2012; 2:557.
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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.
Plant Biotechnology Journal 06/2011; 9(9):1074-85. · 5.44 Impact Factor
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Melissa A Fitzgerald,
Christine J Bergman,
Adoracion P Resurreccion,
Jürgen Möller,
Rosario Jimenez, Russell F Reinke,
Margrit Martin,
Pedro Blanco,
Federico Molina,
Ming-Hsuan Chen, [......],
Siti Dewi Indrasari,
Asfaliza Ramli,
Rauf Ahmad,
Sharifa S Dipti,
Lihong Xie,
Nguyen Thi Lang,
Pratibha Singh,
Dámaso Castillo Toro,
Fatemeh Tavasoli,
Christian Mestres
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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
Cereal Chemistry 11/2009; 86(5):492-498. · 1.45 Impact Factor
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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.
Plant Biotechnology Journal 02/2006; 4(1):115-22. · 5.44 Impact Factor
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Centre for Plant Conservation Genetics Papers.
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Centre for Plant Conservation Genetics Papers.
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Centre for Plant Conservation Genetics Papers.
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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.
Centre for Plant Conservation Genetics Papers.
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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.
Centre for Plant Conservation Genetics Papers.
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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.
Centre for Plant Conservation Genetics Papers.
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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.
Centre for Plant Conservation Genetics Papers.
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ABSTRACT: Traditionally, identification of genes which control important traits has been labour intensive and time consuming. We have demonstrated the annotated rice genome sequence used in combination with re-sequencing by PCR greatly facilitates the discovery of both genes and polymorphisms within genes, which control commercially important traits. Identification of the gene which controls fragrance was achieved using a relatively small mapping population of 168 F2 individuals. Analysis of the recombination data and the relatively large tract (385 kbp) of annotated genome sequence between the flanking markers revealed a candidate gene in this region which plausibly explained the known biochemistry of fragrance. Re-sequencing the gene in a fragrant variety found a mutation which was in accord with the known genetics of fragrance. In the absence of a genome sequence, a much larger mapping population, a genome library and more sequencing would have been necessary. Likewise, availability of the rice genome sequence greatly simplified the task of re-sequencing the SSIIa encoding gene which allowed us to identify single nucleotide polymorphisms (SNP) in soluble starch synthase IIa which explain gelatinisation temperature (GT). This important quantitative trait seems to be determined by two SNP in the 3’ end of the coding sequence. Because starch bio-synthetic genes display high levels of interspecific conservation, it is likely this knowledge will have utility in other species.
Centre for Plant Conservation Genetics Papers.
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Centre for Plant Conservation Genetics Papers.
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Centre for Plant Conservation Genetics Papers.
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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.
Centre for Plant Conservation Genetics Papers.
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Centre for Plant Conservation Genetics Papers.
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ABSTRACT: An important quality trait in rice breeding is the production of basmati- and jasmine-style fragrance in cooked rice. This trait is controlled by a number of chemical constituents, however, it has been established that in most varieties a single recessive gene controls the most important chemical compound 2-acetyl-l-pyrroline (AP). Traditional methods of fragrance detection are through sensory or chemical means, however, each of these have their disadvantages. Microsatellite or simple sequence repeats (SSRs) have the ability to overcome the disadvantages. Using both the publicly available and restricted access sequence information of the Monsanto rice sequence databases, we have identified an (AT)40 repeat that easily distinguishes fragrant and non-fragrant alleles of the fgr gene. Using a mapping population of 50 F2 individuals, the polymorphic marker (PIC = 0.9) was genotyped and two recombinants identified, placing the marker at a maximum of 4 cM from the fgr gene. This study demonstrates that analysis of the rice genome sequence is an effective option for identification of markers for use in rice improvement.
Centre for Plant Conservation Genetics Papers.
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ABSTRACT: PCR based molecular markers will enhance the efficiency and effectiveness of the Australian rice improvement program. PCR based molecular markers have been developed for the fragrance (fgr) and semi-dwarf (sd-1) genes in rice. A strategy is described for determining further target characters for development of molecular markers. Breeding objectives were assessed with regard to importance, mode of inheritance, environmental influence, difficulty and accuracy of assessment, and stage of program at which the trait is currently assessed. The traits selected as candidates for development of molecular markers are: More markers for fragrance (not all desirable parent combinations can be distinguished with existing markers); disease resistance, blast (Magnaporthe grisea), bacterial blight (Xanthamonas oryzae), and sheath blight (Rhizoctonia solani); grain extension on cooking; predisposition to chalkiness; gelatinisation temperature; minor fragrance components; and seedling vigour and early establishment. Marker development for this suite of traits includes short-, medium- and long-term goals. For some of these traits PCR based markers exist, but require testing on Australian populations. Some traits require conversion of molecular markers to PCR based systems for practical application to selection programs. Other traits are mapped but require development of markers, yet others require more fundamental genetic studies. Molecular markers will be particularly important for incorporation of disease resistance into Australian breeding populations, due to the absence of these pathogens from Australia. The aim is to have marker-assisted selection operating as an integral part of the Australian rice improvement program in the short to medium term.
Centre for Plant Conservation Genetics Papers.
