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Digital genotyping of sorghum – a diverse plant species with a large repeat-rich genome
BACKGROUND: Rapid acquisition of accurate genotyping information is essential for all genetic marker-based studies. For species with relatively small genomes, complete genome resequencing is a feasible approach for genotyping; however, for species with large and highly repetitive genomes, the acquis...
Autores principales: | , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
BioMed Central
2013
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3716661/ https://www.ncbi.nlm.nih.gov/pubmed/23829350 http://dx.doi.org/10.1186/1471-2164-14-448 |
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author | Morishige, Daryl T Klein, Patricia E Hilley, Josie L Sahraeian, Sayed Mohammad Ebrahim Sharma, Arun Mullet, John E |
author_facet | Morishige, Daryl T Klein, Patricia E Hilley, Josie L Sahraeian, Sayed Mohammad Ebrahim Sharma, Arun Mullet, John E |
author_sort | Morishige, Daryl T |
collection | PubMed |
description | BACKGROUND: Rapid acquisition of accurate genotyping information is essential for all genetic marker-based studies. For species with relatively small genomes, complete genome resequencing is a feasible approach for genotyping; however, for species with large and highly repetitive genomes, the acquisition of whole genome sequences for the purpose of genotyping is still relatively inefficient and too expensive to be carried out on a high-throughput basis. Sorghum bicolor is a C(4) grass with a sequenced genome size of ~730 Mb, of which ~80% is highly repetitive. We have developed a restriction enzyme targeted genome resequencing method for genetic analysis, termed Digital Genotyping (DG), to be applied to sorghum and other grass species with large repeat-rich genomes. RESULTS: DG templates are generated using one of three methylation sensitive restriction enzymes that recognize a nested set of 4, 6 or 8 bp GC-rich sequences, enabling varying depth of analysis and integration of results among assays. Variation in sequencing efficiency among DG markers was correlated with template GC-content and length. The expected DG allele sequence was obtained 97.3% of the time with a ratio of expected to alternative allele sequence acquisition of >20:1. A genetic map aligned to the sorghum genome sequence with an average resolution of 1.47 cM was constructed using 1,772 DG markers from 137 recombinant inbred lines. The DG map enhanced the detection of QTL for variation in plant height and precisely aligned QTL such as Dw3 to underlying genes/alleles. Higher-resolution NgoMIV-based DG haplotypes were used to trace the origin of DNA on SBI-06, spanning Ma1 and Dw2 from progenitors to BTx623 and IS3620C. DG marker analysis identified the correct location of two miss-assembled regions and located seven super contigs in the sorghum reference genome sequence. CONCLUSION: DG technology provides a cost-effective approach to rapidly generate accurate genotyping data in sorghum. Currently, data derived from DG are used for many marker-based analyses, including marker-assisted breeding, pedigree and QTL analysis, genetic map construction, map-based gene cloning and association studies. DG in combination with whole genome resequencing is dramatically accelerating all aspects of genetic analysis of sorghum, an important genetic reference for C(4) grass species. |
format | Online Article Text |
id | pubmed-3716661 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2013 |
publisher | BioMed Central |
record_format | MEDLINE/PubMed |
spelling | pubmed-37166612013-07-20 Digital genotyping of sorghum – a diverse plant species with a large repeat-rich genome Morishige, Daryl T Klein, Patricia E Hilley, Josie L Sahraeian, Sayed Mohammad Ebrahim Sharma, Arun Mullet, John E BMC Genomics Methodology Article BACKGROUND: Rapid acquisition of accurate genotyping information is essential for all genetic marker-based studies. For species with relatively small genomes, complete genome resequencing is a feasible approach for genotyping; however, for species with large and highly repetitive genomes, the acquisition of whole genome sequences for the purpose of genotyping is still relatively inefficient and too expensive to be carried out on a high-throughput basis. Sorghum bicolor is a C(4) grass with a sequenced genome size of ~730 Mb, of which ~80% is highly repetitive. We have developed a restriction enzyme targeted genome resequencing method for genetic analysis, termed Digital Genotyping (DG), to be applied to sorghum and other grass species with large repeat-rich genomes. RESULTS: DG templates are generated using one of three methylation sensitive restriction enzymes that recognize a nested set of 4, 6 or 8 bp GC-rich sequences, enabling varying depth of analysis and integration of results among assays. Variation in sequencing efficiency among DG markers was correlated with template GC-content and length. The expected DG allele sequence was obtained 97.3% of the time with a ratio of expected to alternative allele sequence acquisition of >20:1. A genetic map aligned to the sorghum genome sequence with an average resolution of 1.47 cM was constructed using 1,772 DG markers from 137 recombinant inbred lines. The DG map enhanced the detection of QTL for variation in plant height and precisely aligned QTL such as Dw3 to underlying genes/alleles. Higher-resolution NgoMIV-based DG haplotypes were used to trace the origin of DNA on SBI-06, spanning Ma1 and Dw2 from progenitors to BTx623 and IS3620C. DG marker analysis identified the correct location of two miss-assembled regions and located seven super contigs in the sorghum reference genome sequence. CONCLUSION: DG technology provides a cost-effective approach to rapidly generate accurate genotyping data in sorghum. Currently, data derived from DG are used for many marker-based analyses, including marker-assisted breeding, pedigree and QTL analysis, genetic map construction, map-based gene cloning and association studies. DG in combination with whole genome resequencing is dramatically accelerating all aspects of genetic analysis of sorghum, an important genetic reference for C(4) grass species. BioMed Central 2013-07-05 /pmc/articles/PMC3716661/ /pubmed/23829350 http://dx.doi.org/10.1186/1471-2164-14-448 Text en Copyright © 2013 Morishige et al.; licensee BioMed Central Ltd. http://creativecommons.org/licenses/by/2.0 This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. |
spellingShingle | Methodology Article Morishige, Daryl T Klein, Patricia E Hilley, Josie L Sahraeian, Sayed Mohammad Ebrahim Sharma, Arun Mullet, John E Digital genotyping of sorghum – a diverse plant species with a large repeat-rich genome |
title | Digital genotyping of sorghum – a diverse plant species with a large repeat-rich genome |
title_full | Digital genotyping of sorghum – a diverse plant species with a large repeat-rich genome |
title_fullStr | Digital genotyping of sorghum – a diverse plant species with a large repeat-rich genome |
title_full_unstemmed | Digital genotyping of sorghum – a diverse plant species with a large repeat-rich genome |
title_short | Digital genotyping of sorghum – a diverse plant species with a large repeat-rich genome |
title_sort | digital genotyping of sorghum – a diverse plant species with a large repeat-rich genome |
topic | Methodology Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3716661/ https://www.ncbi.nlm.nih.gov/pubmed/23829350 http://dx.doi.org/10.1186/1471-2164-14-448 |
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