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High-resolution melting analysis for SNP genotyping and mapping in tetraploid alfalfa (Medicago sativa L.).

Han Y, Khu DM, Monteros MJ - Mol. Breed. (2011)

Bottom Line: HRM using a 384-well format is a fast, consistent, and efficient approach for SNP discovery and genotyping, useful in polyploid species with uncharacterized genomes.Possible applications of this method include variation discovery, analysis of candidate genes, genotyping for comparative and association mapping, and integration of genome-wide selection in breeding programs.ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s11032-011-9566-x) contains supplementary material, which is available to authorized users.

View Article: PubMed Central - PubMed

Affiliation: Forage Improvement Division, The Samuel Roberts Noble Foundation, 2510 Sam Noble Parkway, Ardmore, OK 73401 USA.

ABSTRACT
Single nucleotide polymorphisms (SNPs) represent the most abundant type of genetic polymorphism in plant genomes. SNP markers are valuable tools for genetic analysis of complex traits of agronomic importance, linkage and association mapping, genome-wide selection, map-based cloning, and marker-assisted selection. Current challenges for SNP genotyping in polyploid outcrossing species include multiple alleles per loci and lack of high-throughput methods suitable for variant detection. In this study, we report on a high-resolution melting (HRM) analysis system for SNP genotyping and mapping in outcrossing tetraploid genotypes. The sensitivity and utility of this technology is demonstrated by identification of the parental genotypes and segregating progeny in six alfalfa populations based on unique melting curve profiles due to differences in allelic composition at one or multiple loci. HRM using a 384-well format is a fast, consistent, and efficient approach for SNP discovery and genotyping, useful in polyploid species with uncharacterized genomes. Possible applications of this method include variation discovery, analysis of candidate genes, genotyping for comparative and association mapping, and integration of genome-wide selection in breeding programs. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s11032-011-9566-x) contains supplementary material, which is available to authorized users.

No MeSH data available.


a and b Small amplicon analysis of ten parents of six alfalfa mapping populations using primer MTTF063 with three replicates. One SNP is present at position 56 in the 111-bp amplicon. Each color represents a different SNP genotype: (CCCC = blue, TT (2 ×) and TTTT (4 ×) = green, CTTT = gray, CT (2 ×) and CCTT (4 ×) = orange, CCCT = red). a Normalized melting peaks of ten mapping parents: P1-4 (orange), P6-1(green), MS-13 (blue), MS-186 (blue), Chilean (gray), Wisfal (blue), F1-192 (red), Altet-4 (gray), 95–608 (orange), and NECS-141 (gray). b Normalized melting curves of ten mapping parents. c and d Small amplicon analysis of ten parents of six alfalfa mapping populations using primer MTTF509 with three replicates. One SNP is present at position 123 in the 149-bp amplicon. A second SNP at 79 bp is only present in P6-1. Each color represents a different SNP genotype. At MTTF509 SNP 123, AA (2 ×) and AAAA (4 ×) = gray, AAAG = green, AG (2 ×) and AAGG (4 ×) = red, AGGG = orange, GGGG = blue. At SNP 79, CCCC = blue and TTTT = all other colors. c Normalized melting peaks of ten mapping parents: P1-4, MS-13, Chilean and Altet-4 (gray), P6-1 (blue), MS-186, F1-192 and NECS-141(red), Wisfal (orange), and 95–608 (green). d Normalized melting curves of ten mapping parents. e and f Small amplicon analysis of ten parents of six alfalfa mapping populations using primer MTTF562 with three replicates. A total of nine SNPs are present in the parental genotypes. Each color represents a different SNP genotype (Table 2 and Supplementary Table 3). e Normalized melting peaks of ten mapping parents: P1-4 (blue), P6-1 (green), MS-13 (gray), MS-186 (red), Chilean (orange), Wisfal (aqua), F1-192 (light blue), Altet-4 (pink), 95–608 (purple), NECS-141 (light green). f Normalized melting curves of ten mapping parents
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Fig1: a and b Small amplicon analysis of ten parents of six alfalfa mapping populations using primer MTTF063 with three replicates. One SNP is present at position 56 in the 111-bp amplicon. Each color represents a different SNP genotype: (CCCC = blue, TT (2 ×) and TTTT (4 ×) = green, CTTT = gray, CT (2 ×) and CCTT (4 ×) = orange, CCCT = red). a Normalized melting peaks of ten mapping parents: P1-4 (orange), P6-1(green), MS-13 (blue), MS-186 (blue), Chilean (gray), Wisfal (blue), F1-192 (red), Altet-4 (gray), 95–608 (orange), and NECS-141 (gray). b Normalized melting curves of ten mapping parents. c and d Small amplicon analysis of ten parents of six alfalfa mapping populations using primer MTTF509 with three replicates. One SNP is present at position 123 in the 149-bp amplicon. A second SNP at 79 bp is only present in P6-1. Each color represents a different SNP genotype. At MTTF509 SNP 123, AA (2 ×) and AAAA (4 ×) = gray, AAAG = green, AG (2 ×) and AAGG (4 ×) = red, AGGG = orange, GGGG = blue. At SNP 79, CCCC = blue and TTTT = all other colors. c Normalized melting peaks of ten mapping parents: P1-4, MS-13, Chilean and Altet-4 (gray), P6-1 (blue), MS-186, F1-192 and NECS-141(red), Wisfal (orange), and 95–608 (green). d Normalized melting curves of ten mapping parents. e and f Small amplicon analysis of ten parents of six alfalfa mapping populations using primer MTTF562 with three replicates. A total of nine SNPs are present in the parental genotypes. Each color represents a different SNP genotype (Table 2 and Supplementary Table 3). e Normalized melting peaks of ten mapping parents: P1-4 (blue), P6-1 (green), MS-13 (gray), MS-186 (red), Chilean (orange), Wisfal (aqua), F1-192 (light blue), Altet-4 (pink), 95–608 (purple), NECS-141 (light green). f Normalized melting curves of ten mapping parents

Mentions: Distinct HRM peaks were observed for each SNP genotype in the alfalfa mapping population parents, each of which is represented by a different color (Fig. 1). In all samples evaluated, the three replicates always resulted in similar curves and peaks and were assigned to the same group, indicating the consistency and reproducibility of the HRM assay. Primer MTTF063 was designed using the sequence of a putative M. truncatula transcription factor encoding a transducin family protein similar to the Arabidopsis At5G49430 gene to produce a PCR amplicon 111 bp long (Supplementary Table 2). Five groups of melting curves were obtained from the diploid and tetraploid parental genotypes (Fig. 1a, b). Two groups (green and blue) exhibited the high and narrow melting curve and peaks which are typical for homozygotes. The remaining melting curve and peaks shown in gray, red, and orange are broader and shorter compared to the homozygous peaks, and correspond to the heterozygous genotypes.Fig. 1


High-resolution melting analysis for SNP genotyping and mapping in tetraploid alfalfa (Medicago sativa L.).

Han Y, Khu DM, Monteros MJ - Mol. Breed. (2011)

a and b Small amplicon analysis of ten parents of six alfalfa mapping populations using primer MTTF063 with three replicates. One SNP is present at position 56 in the 111-bp amplicon. Each color represents a different SNP genotype: (CCCC = blue, TT (2 ×) and TTTT (4 ×) = green, CTTT = gray, CT (2 ×) and CCTT (4 ×) = orange, CCCT = red). a Normalized melting peaks of ten mapping parents: P1-4 (orange), P6-1(green), MS-13 (blue), MS-186 (blue), Chilean (gray), Wisfal (blue), F1-192 (red), Altet-4 (gray), 95–608 (orange), and NECS-141 (gray). b Normalized melting curves of ten mapping parents. c and d Small amplicon analysis of ten parents of six alfalfa mapping populations using primer MTTF509 with three replicates. One SNP is present at position 123 in the 149-bp amplicon. A second SNP at 79 bp is only present in P6-1. Each color represents a different SNP genotype. At MTTF509 SNP 123, AA (2 ×) and AAAA (4 ×) = gray, AAAG = green, AG (2 ×) and AAGG (4 ×) = red, AGGG = orange, GGGG = blue. At SNP 79, CCCC = blue and TTTT = all other colors. c Normalized melting peaks of ten mapping parents: P1-4, MS-13, Chilean and Altet-4 (gray), P6-1 (blue), MS-186, F1-192 and NECS-141(red), Wisfal (orange), and 95–608 (green). d Normalized melting curves of ten mapping parents. e and f Small amplicon analysis of ten parents of six alfalfa mapping populations using primer MTTF562 with three replicates. A total of nine SNPs are present in the parental genotypes. Each color represents a different SNP genotype (Table 2 and Supplementary Table 3). e Normalized melting peaks of ten mapping parents: P1-4 (blue), P6-1 (green), MS-13 (gray), MS-186 (red), Chilean (orange), Wisfal (aqua), F1-192 (light blue), Altet-4 (pink), 95–608 (purple), NECS-141 (light green). f Normalized melting curves of ten mapping parents
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Related In: Results  -  Collection

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Fig1: a and b Small amplicon analysis of ten parents of six alfalfa mapping populations using primer MTTF063 with three replicates. One SNP is present at position 56 in the 111-bp amplicon. Each color represents a different SNP genotype: (CCCC = blue, TT (2 ×) and TTTT (4 ×) = green, CTTT = gray, CT (2 ×) and CCTT (4 ×) = orange, CCCT = red). a Normalized melting peaks of ten mapping parents: P1-4 (orange), P6-1(green), MS-13 (blue), MS-186 (blue), Chilean (gray), Wisfal (blue), F1-192 (red), Altet-4 (gray), 95–608 (orange), and NECS-141 (gray). b Normalized melting curves of ten mapping parents. c and d Small amplicon analysis of ten parents of six alfalfa mapping populations using primer MTTF509 with three replicates. One SNP is present at position 123 in the 149-bp amplicon. A second SNP at 79 bp is only present in P6-1. Each color represents a different SNP genotype. At MTTF509 SNP 123, AA (2 ×) and AAAA (4 ×) = gray, AAAG = green, AG (2 ×) and AAGG (4 ×) = red, AGGG = orange, GGGG = blue. At SNP 79, CCCC = blue and TTTT = all other colors. c Normalized melting peaks of ten mapping parents: P1-4, MS-13, Chilean and Altet-4 (gray), P6-1 (blue), MS-186, F1-192 and NECS-141(red), Wisfal (orange), and 95–608 (green). d Normalized melting curves of ten mapping parents. e and f Small amplicon analysis of ten parents of six alfalfa mapping populations using primer MTTF562 with three replicates. A total of nine SNPs are present in the parental genotypes. Each color represents a different SNP genotype (Table 2 and Supplementary Table 3). e Normalized melting peaks of ten mapping parents: P1-4 (blue), P6-1 (green), MS-13 (gray), MS-186 (red), Chilean (orange), Wisfal (aqua), F1-192 (light blue), Altet-4 (pink), 95–608 (purple), NECS-141 (light green). f Normalized melting curves of ten mapping parents
Mentions: Distinct HRM peaks were observed for each SNP genotype in the alfalfa mapping population parents, each of which is represented by a different color (Fig. 1). In all samples evaluated, the three replicates always resulted in similar curves and peaks and were assigned to the same group, indicating the consistency and reproducibility of the HRM assay. Primer MTTF063 was designed using the sequence of a putative M. truncatula transcription factor encoding a transducin family protein similar to the Arabidopsis At5G49430 gene to produce a PCR amplicon 111 bp long (Supplementary Table 2). Five groups of melting curves were obtained from the diploid and tetraploid parental genotypes (Fig. 1a, b). Two groups (green and blue) exhibited the high and narrow melting curve and peaks which are typical for homozygotes. The remaining melting curve and peaks shown in gray, red, and orange are broader and shorter compared to the homozygous peaks, and correspond to the heterozygous genotypes.Fig. 1

Bottom Line: HRM using a 384-well format is a fast, consistent, and efficient approach for SNP discovery and genotyping, useful in polyploid species with uncharacterized genomes.Possible applications of this method include variation discovery, analysis of candidate genes, genotyping for comparative and association mapping, and integration of genome-wide selection in breeding programs.ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s11032-011-9566-x) contains supplementary material, which is available to authorized users.

View Article: PubMed Central - PubMed

Affiliation: Forage Improvement Division, The Samuel Roberts Noble Foundation, 2510 Sam Noble Parkway, Ardmore, OK 73401 USA.

ABSTRACT
Single nucleotide polymorphisms (SNPs) represent the most abundant type of genetic polymorphism in plant genomes. SNP markers are valuable tools for genetic analysis of complex traits of agronomic importance, linkage and association mapping, genome-wide selection, map-based cloning, and marker-assisted selection. Current challenges for SNP genotyping in polyploid outcrossing species include multiple alleles per loci and lack of high-throughput methods suitable for variant detection. In this study, we report on a high-resolution melting (HRM) analysis system for SNP genotyping and mapping in outcrossing tetraploid genotypes. The sensitivity and utility of this technology is demonstrated by identification of the parental genotypes and segregating progeny in six alfalfa populations based on unique melting curve profiles due to differences in allelic composition at one or multiple loci. HRM using a 384-well format is a fast, consistent, and efficient approach for SNP discovery and genotyping, useful in polyploid species with uncharacterized genomes. Possible applications of this method include variation discovery, analysis of candidate genes, genotyping for comparative and association mapping, and integration of genome-wide selection in breeding programs. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s11032-011-9566-x) contains supplementary material, which is available to authorized users.

No MeSH data available.