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Garlic (A. sativum L.) alliinase gene family polymorphism reflects bolting types and cysteine sulphoxides content.

Ovesná J, Mitrová K, Kučera L - BMC Genet. (2015)

Bottom Line: We found additional sequence variants using partial amplicons.Detected polymorphism is thus also associated with cysteine-sulphoxide content in individual genotypes.Higher genetic variability found in bolting genotypes may indicates longer period of their sexual propagation in comparison with nonbolting genotypes.

View Article: PubMed Central - PubMed

Affiliation: Crop Research Institute, Drnovská 507/73, 161 06, Prague-Ruzyně, Czech Republic. ovesna@vurv.cz.

ABSTRACT

Background: Alliinase is an important enzyme occurring in Allium species that converts precursors of sulfuric compounds, cysteine sulfoxides into a biologically active substance termed allicin. Allicin facilitates garlic defense against pests and produces health-promoting compounds. Alliinase is encoded by members of a multigene family that has not yet been sufficiently characterized, namely with regard to the copy numbers occurring within the genome and the polymorphisms among the family members.

Results: We cloned 45 full-length alliinase amplicons of cultivar (cv.) Jovan. Sequence analyses revealed nine different sequence variants (SVs), confirming the multilocus nature of this gene family. Several mutations in exons, mainly occurring in the first exon coding for vacuolar signal peptide, were found. These results enabled us to identify sequences with putatively modified vacuole-targeting abilities. We found additional sequence variants using partial amplicons. We estimated that the minimum number of gene copies in the diploid genome of the investigated cultivar was fourteen. We obtained similar results for another three cultivars, which differed in bolting type and place of origin. The further identification of high degree of polymorphisms in the intron regions allowed us to develop a specific polymerase chain reaction assay capable to capture intron length polymorphism (ILP). This assay was used to screen 131 additional accessions. Polymorphic data were used for cluster analysis, which separated the bolting and non-bolting garlic types and those with high cysteine-sulfoxide contents in a similar way as AFLP analysis in previous study. These newly developed markers can be further applied for the selection of desirable garlic genotypes.

Conclusions: Detailed analysis of sequences confirmed multigenic nature of garlic alliinase. Intron and exon polymorphism analysis generated similar results as whole genome variability assessed previously by AFLP. Detected polymorphism is thus also associated with cysteine-sulphoxide content in individual genotypes. ILP markers capable to detect intron polymorphisms were newly developed. Developed markers could be applied in garlic breeding. Higher genetic variability found in bolting genotypes may indicates longer period of their sexual propagation in comparison with nonbolting genotypes.

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Related in: MedlinePlus

Protein sequence relationships of garlic alliinases from cv. Japo and two complete alliinase sequences. The phylogenetic tree was inferred using the neighbor-joining method [50]. The optimal tree with a branch length sum of 0.03415794 is shown. The percentage of replicate trees in which the associated sequence variants (SVs) clustered together in the bootstrap test (1000 replicates) is shown next to the branches [51]. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances. The evolutionary distances were computed using the Poisson correction method [52] and are represented as the number of amino acid substitutions per site. All ambiguous positions were removed for each sequence pair. There were a total of 487 positions in the final data set. Evolutionary analyses were conducted with MEGA6 [49]
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Fig2: Protein sequence relationships of garlic alliinases from cv. Japo and two complete alliinase sequences. The phylogenetic tree was inferred using the neighbor-joining method [50]. The optimal tree with a branch length sum of 0.03415794 is shown. The percentage of replicate trees in which the associated sequence variants (SVs) clustered together in the bootstrap test (1000 replicates) is shown next to the branches [51]. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances. The evolutionary distances were computed using the Poisson correction method [52] and are represented as the number of amino acid substitutions per site. All ambiguous positions were removed for each sequence pair. There were a total of 487 positions in the final data set. Evolutionary analyses were conducted with MEGA6 [49]

Mentions: The comparison of nine SVs from cv. Jovan with a reference sequence [GenBank: Z12622, GenBank: gi16108, protein Uni-Prot: Q01594] and alliin lyase 2 protein [Uni-Prot: Q41233] indicated that the obtained sequences were closer to each other than to the reference sequences, which was supported by the bootstrap values indicated in the dendrogram (Fig. 2).Fig. 2


Garlic (A. sativum L.) alliinase gene family polymorphism reflects bolting types and cysteine sulphoxides content.

Ovesná J, Mitrová K, Kučera L - BMC Genet. (2015)

Protein sequence relationships of garlic alliinases from cv. Japo and two complete alliinase sequences. The phylogenetic tree was inferred using the neighbor-joining method [50]. The optimal tree with a branch length sum of 0.03415794 is shown. The percentage of replicate trees in which the associated sequence variants (SVs) clustered together in the bootstrap test (1000 replicates) is shown next to the branches [51]. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances. The evolutionary distances were computed using the Poisson correction method [52] and are represented as the number of amino acid substitutions per site. All ambiguous positions were removed for each sequence pair. There were a total of 487 positions in the final data set. Evolutionary analyses were conducted with MEGA6 [49]
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4440563&req=5

Fig2: Protein sequence relationships of garlic alliinases from cv. Japo and two complete alliinase sequences. The phylogenetic tree was inferred using the neighbor-joining method [50]. The optimal tree with a branch length sum of 0.03415794 is shown. The percentage of replicate trees in which the associated sequence variants (SVs) clustered together in the bootstrap test (1000 replicates) is shown next to the branches [51]. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances. The evolutionary distances were computed using the Poisson correction method [52] and are represented as the number of amino acid substitutions per site. All ambiguous positions were removed for each sequence pair. There were a total of 487 positions in the final data set. Evolutionary analyses were conducted with MEGA6 [49]
Mentions: The comparison of nine SVs from cv. Jovan with a reference sequence [GenBank: Z12622, GenBank: gi16108, protein Uni-Prot: Q01594] and alliin lyase 2 protein [Uni-Prot: Q41233] indicated that the obtained sequences were closer to each other than to the reference sequences, which was supported by the bootstrap values indicated in the dendrogram (Fig. 2).Fig. 2

Bottom Line: We found additional sequence variants using partial amplicons.Detected polymorphism is thus also associated with cysteine-sulphoxide content in individual genotypes.Higher genetic variability found in bolting genotypes may indicates longer period of their sexual propagation in comparison with nonbolting genotypes.

View Article: PubMed Central - PubMed

Affiliation: Crop Research Institute, Drnovská 507/73, 161 06, Prague-Ruzyně, Czech Republic. ovesna@vurv.cz.

ABSTRACT

Background: Alliinase is an important enzyme occurring in Allium species that converts precursors of sulfuric compounds, cysteine sulfoxides into a biologically active substance termed allicin. Allicin facilitates garlic defense against pests and produces health-promoting compounds. Alliinase is encoded by members of a multigene family that has not yet been sufficiently characterized, namely with regard to the copy numbers occurring within the genome and the polymorphisms among the family members.

Results: We cloned 45 full-length alliinase amplicons of cultivar (cv.) Jovan. Sequence analyses revealed nine different sequence variants (SVs), confirming the multilocus nature of this gene family. Several mutations in exons, mainly occurring in the first exon coding for vacuolar signal peptide, were found. These results enabled us to identify sequences with putatively modified vacuole-targeting abilities. We found additional sequence variants using partial amplicons. We estimated that the minimum number of gene copies in the diploid genome of the investigated cultivar was fourteen. We obtained similar results for another three cultivars, which differed in bolting type and place of origin. The further identification of high degree of polymorphisms in the intron regions allowed us to develop a specific polymerase chain reaction assay capable to capture intron length polymorphism (ILP). This assay was used to screen 131 additional accessions. Polymorphic data were used for cluster analysis, which separated the bolting and non-bolting garlic types and those with high cysteine-sulfoxide contents in a similar way as AFLP analysis in previous study. These newly developed markers can be further applied for the selection of desirable garlic genotypes.

Conclusions: Detailed analysis of sequences confirmed multigenic nature of garlic alliinase. Intron and exon polymorphism analysis generated similar results as whole genome variability assessed previously by AFLP. Detected polymorphism is thus also associated with cysteine-sulphoxide content in individual genotypes. ILP markers capable to detect intron polymorphisms were newly developed. Developed markers could be applied in garlic breeding. Higher genetic variability found in bolting genotypes may indicates longer period of their sexual propagation in comparison with nonbolting genotypes.

Show MeSH
Related in: MedlinePlus