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Sequence diversity in coding regions of candidate genes in the glycoalkaloid biosynthetic pathway of wild potato species.

Manrique-Carpintero NC, Tokuhisa JG, Ginzberg I, Holliday JA, Veilleux RE - G3 (Bethesda) (2013)

Bottom Line: More polymorphisms were found in introns than exons and in genes of secondary compared to primary metabolism.Although no significant deviation from neutrality was found, dN/dS ratios < 1 and negative values of Tajima's D test suggested purifying selection and genetic hitchhiking in the gene fragments.These results can be used to evaluate SGA accumulation in segregating or association mapping populations.

View Article: PubMed Central - PubMed

Affiliation: Department of Horticulture, Virginia Polytechnic Institute and State University, Blacksburg, Virginia 24061.

ABSTRACT
Natural variation in five candidate genes of the steroidal glycoalkaloid (SGA) metabolic pathway and whole-genome single nucleotide polymorphism (SNP) genotyping were studied in six wild [Solanum chacoense (chc 80-1), S. commersonii, S. demissum, S. sparsipilum, S. spegazzinii, S. stoloniferum] and cultivated S. tuberosum Group Phureja (phu DH) potato species with contrasting levels of SGAs. Amplicons were sequenced for five candidate genes: 3-hydroxy-3-methylglutaryl coenzyme A reductase 1 and 2 (HMG1, HMG2) and 2.3-squalene epoxidase (SQE) of primary metabolism, and solanidine galactosyltransferase (SGT1), and glucosyltransferase (SGT2) of secondary metabolism. SNPs (n = 337) producing 354 variations were detected within 3.7 kb of sequenced DNA. More polymorphisms were found in introns than exons and in genes of secondary compared to primary metabolism. Although no significant deviation from neutrality was found, dN/dS ratios < 1 and negative values of Tajima's D test suggested purifying selection and genetic hitchhiking in the gene fragments. In addition, patterns of dN/dS ratios across the SGA pathway suggested constraint by natural selection. Comparison of nucleotide diversity estimates and dN/dS ratios showed stronger selective constraints for genes of primary rather than secondary metabolism. SNPs (n = 24) with an exclusive genotype for either phu DH (low SGA) or chc 80-1 (high SGA) were identified for HMG2, SQE, SGT1 and SGT2. The SolCAP 8303 Illumina Potato SNP chip genotyping revealed eight informative SNPs on six pseudochromosomes, with homozygous and heterozygous genotypes that discriminated high, intermediate and low levels of SGA accumulation. These results can be used to evaluate SGA accumulation in segregating or association mapping populations.

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Comparison of hierarchical clusters based on 3841 polymorphic SNPs (A) and eight SGA informative SNPs (B) from whole genome analysis of germplasm panel. A neighbor-joining phylogenetic tree built in TASSEL classified species and accessions with similar patterns as A. Marks on the left of species name corresponded to defined clusters. Most of the accessions with high SGA levels cluster on big dots, intermediate levels on small dots, and low on diamonds. However, cmm 7 and sto 61 with low SGA levels were not in the right cluster.
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fig4: Comparison of hierarchical clusters based on 3841 polymorphic SNPs (A) and eight SGA informative SNPs (B) from whole genome analysis of germplasm panel. A neighbor-joining phylogenetic tree built in TASSEL classified species and accessions with similar patterns as A. Marks on the left of species name corresponded to defined clusters. Most of the accessions with high SGA levels cluster on big dots, intermediate levels on small dots, and low on diamonds. However, cmm 7 and sto 61 with low SGA levels were not in the right cluster.

Mentions: A whole genome SNP chip analysis was done on the twelve potato accessions to identify genomic regions putatively associated with SGA accumulation (Table S1). In this analysis, a monoploid line derived from chc 80 1−4 was used instead of chc 80−1 itself, and the ANOVA was used to identify the most likely SNPs associated with SGA accumulation. The stringency was increased to identify significant SNPs that clustered at least two accessions with low or high SGA levels, with mean difference between SNP clusters equal to or greater than 5 logarithm units (Table 8). Then the allelic structure of significant SNPs was analyzed by accessions to identify informative SNPs. Thirty-four significant SNPs located on 10 pseudochromosomes were initially associated with total SGA accumulation (Table 8). Cluster analysis identified eight informative SNPs on six pseudochromosomes with homozygous and heterozygous genotypes that discriminated high, intermediate and low levels of SGA accumulation (Figure 3). Two accessions, phu DH and spg 55, were mostly representative in the cluster of low SGA levels. Of four SNPs located on pseudochromosome 1, one (solcap_snp_c1_5656) at 63.6 Mb mainly clustered in two SNP genotypes (AA and AG) six accessions with the greatest accumulation of SGAs. Five SNPs were found on pseudochromosome 2, of which solcap_snp_c2_30160 at 19.1 Mb grouped in two SNP genotypes (CC and TC) eight accessions with the greatest levels of accumulation, with a gradual decrease from the four with CC to the four with TC. Pseudochromosomes 3, 4, and 5 with two, four and one SNPs did not group the accessions into any logical arrangement. Of five SNPs on pseudochromosome 6, solcap_snp_5775 at 45.3 Mb was informative in grouping eight accessions with the greatest levels of SGA accumulation in homozygous (CC) and heterozygous (TC) genotypes similar to the distribution by solcap_snp_c2_30160 on pseudochromosome 2. Solcap_snp_c2_18573 at 53.2 Mb of five SNPs on pseudochromosome 7 again grouped the 8 accessions with the greatest levels of SGAs under similar patterns. Solcap_snp_c1_1512 one of the two SNPs on pseudochromosome 9, at 28.8 Mb clustered nine accessions; the five with the greatest levels of SGAs had the CC allele whereas the four with lower levels had the TC genotype. The SNP on pseudochromosome 10 did not have any defined cluster associated with high or low SGA accessions. Finally, three informative SNPs were identified out of five on pseudochromosome 11 (solcap_snp_c1_2304, solcap_snp_c2_57429 and solcap_snp_c2_49311) between 4.5 and 8.9 Mb. These SNPs have at least three accessions with the greatest SGA level, three in intermediate levels and two with the lowest levels grouped in different SNP genotypes. The SNP on pseudochromosome 12 was not informative. The group of eight informative SNPs defined putative haplotypes for low, intermediate and high SGA accumulation (Figure 3). Comparison of hierarchical clustering trees, built in the statistical software JMP using 3841 SNPs with nonmissing data from the potato array and another with the eight informative SNPs, showed that the twelve accessions were grouped by species except for spl taxa in the first tree in contrast with the second where they were grouped by SGA levels (Figure 4). The neighbor-joining phylogenetic tree constructed in TASSEL 3.0 (Bradbury et al. 2007) (data not shown) using the 3841 SNPs followed the kinship between samples from the same species found in the analysis in JMP. Phu DH the only cultivated species in the germplasm panel was separated from all other samples. Three general clusters were generated based on informative SNPs that grouped by SGA levels for most of the accessions. Two accessions (cmm 7 and sto 61) with low levels of SGA were located in high and intermediate SGA cluster.


Sequence diversity in coding regions of candidate genes in the glycoalkaloid biosynthetic pathway of wild potato species.

Manrique-Carpintero NC, Tokuhisa JG, Ginzberg I, Holliday JA, Veilleux RE - G3 (Bethesda) (2013)

Comparison of hierarchical clusters based on 3841 polymorphic SNPs (A) and eight SGA informative SNPs (B) from whole genome analysis of germplasm panel. A neighbor-joining phylogenetic tree built in TASSEL classified species and accessions with similar patterns as A. Marks on the left of species name corresponded to defined clusters. Most of the accessions with high SGA levels cluster on big dots, intermediate levels on small dots, and low on diamonds. However, cmm 7 and sto 61 with low SGA levels were not in the right cluster.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3755908&req=5

fig4: Comparison of hierarchical clusters based on 3841 polymorphic SNPs (A) and eight SGA informative SNPs (B) from whole genome analysis of germplasm panel. A neighbor-joining phylogenetic tree built in TASSEL classified species and accessions with similar patterns as A. Marks on the left of species name corresponded to defined clusters. Most of the accessions with high SGA levels cluster on big dots, intermediate levels on small dots, and low on diamonds. However, cmm 7 and sto 61 with low SGA levels were not in the right cluster.
Mentions: A whole genome SNP chip analysis was done on the twelve potato accessions to identify genomic regions putatively associated with SGA accumulation (Table S1). In this analysis, a monoploid line derived from chc 80 1−4 was used instead of chc 80−1 itself, and the ANOVA was used to identify the most likely SNPs associated with SGA accumulation. The stringency was increased to identify significant SNPs that clustered at least two accessions with low or high SGA levels, with mean difference between SNP clusters equal to or greater than 5 logarithm units (Table 8). Then the allelic structure of significant SNPs was analyzed by accessions to identify informative SNPs. Thirty-four significant SNPs located on 10 pseudochromosomes were initially associated with total SGA accumulation (Table 8). Cluster analysis identified eight informative SNPs on six pseudochromosomes with homozygous and heterozygous genotypes that discriminated high, intermediate and low levels of SGA accumulation (Figure 3). Two accessions, phu DH and spg 55, were mostly representative in the cluster of low SGA levels. Of four SNPs located on pseudochromosome 1, one (solcap_snp_c1_5656) at 63.6 Mb mainly clustered in two SNP genotypes (AA and AG) six accessions with the greatest accumulation of SGAs. Five SNPs were found on pseudochromosome 2, of which solcap_snp_c2_30160 at 19.1 Mb grouped in two SNP genotypes (CC and TC) eight accessions with the greatest levels of accumulation, with a gradual decrease from the four with CC to the four with TC. Pseudochromosomes 3, 4, and 5 with two, four and one SNPs did not group the accessions into any logical arrangement. Of five SNPs on pseudochromosome 6, solcap_snp_5775 at 45.3 Mb was informative in grouping eight accessions with the greatest levels of SGA accumulation in homozygous (CC) and heterozygous (TC) genotypes similar to the distribution by solcap_snp_c2_30160 on pseudochromosome 2. Solcap_snp_c2_18573 at 53.2 Mb of five SNPs on pseudochromosome 7 again grouped the 8 accessions with the greatest levels of SGAs under similar patterns. Solcap_snp_c1_1512 one of the two SNPs on pseudochromosome 9, at 28.8 Mb clustered nine accessions; the five with the greatest levels of SGAs had the CC allele whereas the four with lower levels had the TC genotype. The SNP on pseudochromosome 10 did not have any defined cluster associated with high or low SGA accessions. Finally, three informative SNPs were identified out of five on pseudochromosome 11 (solcap_snp_c1_2304, solcap_snp_c2_57429 and solcap_snp_c2_49311) between 4.5 and 8.9 Mb. These SNPs have at least three accessions with the greatest SGA level, three in intermediate levels and two with the lowest levels grouped in different SNP genotypes. The SNP on pseudochromosome 12 was not informative. The group of eight informative SNPs defined putative haplotypes for low, intermediate and high SGA accumulation (Figure 3). Comparison of hierarchical clustering trees, built in the statistical software JMP using 3841 SNPs with nonmissing data from the potato array and another with the eight informative SNPs, showed that the twelve accessions were grouped by species except for spl taxa in the first tree in contrast with the second where they were grouped by SGA levels (Figure 4). The neighbor-joining phylogenetic tree constructed in TASSEL 3.0 (Bradbury et al. 2007) (data not shown) using the 3841 SNPs followed the kinship between samples from the same species found in the analysis in JMP. Phu DH the only cultivated species in the germplasm panel was separated from all other samples. Three general clusters were generated based on informative SNPs that grouped by SGA levels for most of the accessions. Two accessions (cmm 7 and sto 61) with low levels of SGA were located in high and intermediate SGA cluster.

Bottom Line: More polymorphisms were found in introns than exons and in genes of secondary compared to primary metabolism.Although no significant deviation from neutrality was found, dN/dS ratios < 1 and negative values of Tajima's D test suggested purifying selection and genetic hitchhiking in the gene fragments.These results can be used to evaluate SGA accumulation in segregating or association mapping populations.

View Article: PubMed Central - PubMed

Affiliation: Department of Horticulture, Virginia Polytechnic Institute and State University, Blacksburg, Virginia 24061.

ABSTRACT
Natural variation in five candidate genes of the steroidal glycoalkaloid (SGA) metabolic pathway and whole-genome single nucleotide polymorphism (SNP) genotyping were studied in six wild [Solanum chacoense (chc 80-1), S. commersonii, S. demissum, S. sparsipilum, S. spegazzinii, S. stoloniferum] and cultivated S. tuberosum Group Phureja (phu DH) potato species with contrasting levels of SGAs. Amplicons were sequenced for five candidate genes: 3-hydroxy-3-methylglutaryl coenzyme A reductase 1 and 2 (HMG1, HMG2) and 2.3-squalene epoxidase (SQE) of primary metabolism, and solanidine galactosyltransferase (SGT1), and glucosyltransferase (SGT2) of secondary metabolism. SNPs (n = 337) producing 354 variations were detected within 3.7 kb of sequenced DNA. More polymorphisms were found in introns than exons and in genes of secondary compared to primary metabolism. Although no significant deviation from neutrality was found, dN/dS ratios < 1 and negative values of Tajima's D test suggested purifying selection and genetic hitchhiking in the gene fragments. In addition, patterns of dN/dS ratios across the SGA pathway suggested constraint by natural selection. Comparison of nucleotide diversity estimates and dN/dS ratios showed stronger selective constraints for genes of primary rather than secondary metabolism. SNPs (n = 24) with an exclusive genotype for either phu DH (low SGA) or chc 80-1 (high SGA) were identified for HMG2, SQE, SGT1 and SGT2. The SolCAP 8303 Illumina Potato SNP chip genotyping revealed eight informative SNPs on six pseudochromosomes, with homozygous and heterozygous genotypes that discriminated high, intermediate and low levels of SGA accumulation. These results can be used to evaluate SGA accumulation in segregating or association mapping populations.

Show MeSH