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Discovery of polymorphisms in starch-related genes in rice germplasm by amplification of pooled DNA and deeply parallel sequencing.
Plant Biotechnol J. 2011 Dec; 9(9):1074-85.PB

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.

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Publisher Full Text (DOI)

Authors+Show Affiliations

Kharabian-Masouleh A
Southern Cross Plant Science, Centre for Plant Conservation Genetics, Southern Cross University, Lismore, NSW 2480, Australia. akhara10@scu.edu.au
Waters DL
No affiliation info available
Reinke RF
No affiliation info available
Henry RJ
No affiliation info available

MeSH

1,4-alpha-Glucan Branching EnzymeDNA, PlantGenes, PlantGenetic LociGenotypeGlucose-1-Phosphate AdenylyltransferaseGlycoside HydrolasesINDEL MutationIsoamylaseOryzaPlant ProteinsPolymerase Chain ReactionPolymorphism, Single NucleotideSequence Analysis, DNAStarchStarch Synthase

Pub Type(s)

Journal Article

Language

eng

PubMed ID

21645201

Citation

Kharabian-Masouleh, Ardashir, et al. "Discovery of Polymorphisms in Starch-related Genes in Rice Germplasm By Amplification of Pooled DNA and Deeply Parallel Sequencing." Plant Biotechnology Journal, vol. 9, no. 9, 2011, pp. 1074-85.
Kharabian-Masouleh A, Waters DL, Reinke RF, et al. Discovery of polymorphisms in starch-related genes in rice germplasm by amplification of pooled DNA and deeply parallel sequencing. Plant Biotechnol J. 2011;9(9):1074-85.
Kharabian-Masouleh, A., Waters, D. L., Reinke, R. F., & Henry, R. J. (2011). Discovery of polymorphisms in starch-related genes in rice germplasm by amplification of pooled DNA and deeply parallel sequencing. Plant Biotechnology Journal, 9(9), 1074-85. https://doi.org/10.1111/j.1467-7652.2011.00629.x
Kharabian-Masouleh A, et al. Discovery of Polymorphisms in Starch-related Genes in Rice Germplasm By Amplification of Pooled DNA and Deeply Parallel Sequencing. Plant Biotechnol J. 2011;9(9):1074-85. PubMed PMID: 21645201.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Discovery of polymorphisms in starch-related genes in rice germplasm by amplification of pooled DNA and deeply parallel sequencing. AU - Kharabian-Masouleh,Ardashir, AU - Waters,Daniel L E, AU - Reinke,Russell F, AU - Henry,Robert J, Y1 - 2011/06/07/ PY - 2011/6/8/entrez PY - 2011/6/8/pubmed PY - 2012/3/1/medline SP - 1074 EP - 85 JF - Plant biotechnology journal JO - Plant Biotechnol J VL - 9 IS - 9 N2 - 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. SN - 1467-7652 UR - https://www.unboundmedicine.com/medline/citation/21645201/Discovery_of_polymorphisms_in_starch_related_genes_in_rice_germplasm_by_amplification_of_pooled_DNA_and_deeply_parallel_sequencing_ L2 - https://doi.org/10.1111/j.1467-7652.2011.00629.x DB - PRIME DP - Unbound Medicine ER -