Genome-wide and molecular evolution analysis of the subtilase gene family in Vitis vinifera

Background

Vitis vinifera (grape) is one of the most economically significant fruit crops in the world. The availability of the recently released grape genome sequence offers an opportunity to identify and analyze some important gene families in this species. Subtilases are a group of subtilisin-like serine proteases that are involved in many biological processes in plants. However, no comprehensive study incorporating phylogeny, chromosomal location and gene duplication, gene organization, functional divergence, selective pressure and expression profiling has been reported so far for the grape.

Results

In the present study, a comprehensive analysis of the subtilase gene family in V. vinifera was performed. Eighty subtilase genes were identified. Phylogenetic analyses indicated that these subtilase genes comprised eight groups. The gene organization is considerably conserved among the groups. Distribution of the subtilase genes is non-random across the chromosomes. A high proportion of these genes are preferentially clustered, indicating that tandem duplications may have contributed significantly to the expansion of the subtilase gene family. Analyses of divergence and adaptive evolution show that while purifying selection may have been the main force driving the evolution of grape subtilases, some of the critical sites responsible for the divergence may have been under positive selection. Further analyses of real-time PCR data suggested that many subtilase genes might be important in the stress response and functional development of plants.

Conclusions

Tandem duplications as well as purifying and positive selections have contributed to the functional divergence of subtilase genes in V. vinifera. The data may contribute to a better understanding of the grape subtilase gene family.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-1116) contains supplementary material, which is available to authorized users.     

Non-synonymous mutations mapped to chromosome X associated with andrological and growth traits in beef cattle

Background

Previous genome-wide association analyses identified QTL regions in the X chromosome for percentage of normal sperm and scrotal circumference in Brahman and Tropical Composite cattle. These traits are important to be studied because they are indicators of male fertility and are correlated with female sexual precocity and reproductive longevity. The aim was to investigate candidate genes in these regions and to identify putative causative mutations that influence these traits. In addition, we tested the identified mutations for female fertility and growth traits.

Results

Using a combination of bioinformatics and molecular assay technology, twelve non-synonymous SNPs in eleven genes were genotyped in a cattle population. Three and nine SNPs explained more than 1% of the additive genetic variance for percentage of normal sperm and scrotal circumference, respectively. The SNPs that had a major influence in percentage of normal sperm were mapped to LOC100138021 and TAF7L genes; and in TEX11 and AR genes for scrotal circumference. One SNP in TEX11 was explained ~13% of the additive genetic variance for scrotal circumference at 12 months. The tested SNP were also associated with weight measurements, but not with female fertility traits.

Conclusions

The strong association of SNPs located in X chromosome genes with male fertility traits validates the QTL. The implicated genes became good candidates to be used for genetic evaluation, without detrimentally influencing female fertility traits.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1595-0) contains supplementary material, which is available to authorized users.     

Dynamic thermal time model of cold hardiness for dormant grapevine buds

Background and Aims

Grapevine (Vitis spp.) cold hardiness varies dynamically throughout the dormant season, primarily in response to changes in temperature. The development and possible uses of a discrete-dynamic model of bud cold hardiness for three Vitis genotypes are described.

Methods

Iterative methods were used to optimize and evaluate model parameters by minimizing the root mean square error between observed and predicted bud hardiness, using up to 22 years of low-temperature exotherm data. Three grape cultivars were studied: Cabernet Sauvignon, Chardonnay (both V. vinifera) and Concord (V. labruscana). The model uses time steps of 1 d along with the measured daily mean air temperature to calculate the change in bud hardiness, which is then added to the hardiness from the previous day. Cultivar-dependent thermal time thresholds determine whether buds acclimate (gain hardiness) or deacclimate (lose hardiness).

Key Results

The parameterized model predicted bud hardiness for Cabernet Sauvignon and Chardonnay with an r² = 0·89 and for Concord with an r² = 0·82. Thermal time thresholds and (de-)acclimation rates changed between the early and late dormant season and were cultivar dependent but independent of each other. The timing of these changes was also unique for each cultivar. Concord achieved the greatest mid-winter hardiness but had the highest deacclimation rate, which resulted in rapid loss of hardiness in spring. Cabernet Sauvignon was least hardy, yet maintained its hardiness latest as a result of late transition to eco-dormancy, a high threshold temperature required to induce deacclimation and a low deacclimation rate.

Conclusions

A robust model of grapevine bud cold hardiness was developed that will aid in the anticipation of and response to potential injury from fluctuations in winter temperature and from extreme cold events. The model parameters that produce the best fit also permit insight into dynamic differences in hardiness among genotypes.     

Evaluation and optimisation of indel detection workflows for ion torrent sequencing of the BRCA1 and BRCA2 genes

Background

The Ion Torrent PGM is a popular benchtop sequencer that shows promise in replacing conventional Sanger sequencing as the gold standard for mutation detection. Despite the PGM’s reported high accuracy in calling single nucleotide variations, it tends to generate many false positive calls in detecting insertions and deletions (indels), which may hinder its utility for clinical genetic testing.

Results

Recently, the proprietary analytical workflow for the Ion Torrent sequencer, Torrent Suite (TS), underwent a series of upgrades. We evaluated three major upgrades of TS by calling indels in the BRCA1 and BRCA2 genes. Our analysis revealed that false negative indels could be generated by TS under both default calling parameters and parameters adjusted for maximum sensitivity. However, indel calling with the same data using the open source variant callers, GATK and SAMtools showed that false negatives could be minimised with the use of appropriate bioinformatics analysis. Furthermore, we identified two variant calling measures, Quality-by-Depth (QD) and VARiation of the Width of gaps and inserts (VARW), which substantially reduced false positive indels, including non-homopolymer associated errors without compromising sensitivity. In our best case scenario that involved the TMAP aligner and SAMtools, we achieved 100% sensitivity, 99.99% specificity and 29% False Discovery Rate (FDR) in indel calling from all 23 samples, which is a good performance for mutation screening using PGM.

Conclusions

New versions of TS, BWA and GATK have shown improvements in indel calling sensitivity and specificity over their older counterpart. However, the variant caller of TS exhibits a lower sensitivity than GATK and SAMtools. Our findings demonstrate that although indel calling from PGM sequences may appear to be noisy at first glance, proper computational indel calling analysis is able to maximize both the sensitivity and specificity at the single base level, paving the way for the usage of this technology for future clinical genetic testing.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-516) contains supplementary material, which is available to authorized users.     

A high quality draft consensus sequence of the genome of a heterozygous grapevine variety

Background

Worldwide, grapes and their derived products have a large market. The cultivated grape species Vitis vinifera has potential to become a model for fruit trees genetics. Like many plant species, it is highly heterozygous, which is an additional challenge to modern whole genome shotgun sequencing. In this paper a high quality draft genome sequence of a cultivated clone of V. vinifera Pinot Noir is presented.

Principal Findings

We estimate the genome size of V. vinifera to be 504.6 Mb. Genomic sequences corresponding to 477.1 Mb were assembled in 2,093 metacontigs and 435.1 Mb were anchored to the 19 linkage groups (LGs). The number of predicted genes is 29,585, of which 96.1% were assigned to LGs. This assembly of the grape genome provides candidate genes implicated in traits relevant to grapevine cultivation, such as those influencing wine quality, via secondary metabolites, and those connected with the extreme susceptibility of grape to pathogens. Single nucleotide polymorphism (SNP) distribution was consistent with a diffuse haplotype structure across the genome. Of around 2,000,000 SNPs, 1,751,176 were mapped to chromosomes and one or more of them were identified in 86.7% of anchored genes. The relative age of grape duplicated genes was estimated and this made possible to reveal a relatively recent Vitis-specific large scale duplication event concerning at least 10 chromosomes (duplication not reported before).

Conclusions

Sanger shotgun sequencing and highly efficient sequencing by synthesis (SBS), together with dedicated assembly programs, resolved a complex heterozygous genome. A consensus sequence of the genome and a set of mapped marker loci were generated. Homologous chromosomes of Pinot Noir differ by 11.2% of their DNA (hemizygous DNA plus chromosomal gaps). SNP markers are offered as a tool with the potential of introducing a new era in the molecular breeding of grape.     

Multiple origins of the determinate growth habit in domesticated common bean (Phaseolus vulgaris)

Background and Aims

The actual number of domestications of a crop is one of the key questions in domestication studies. Answers to this question have generally been based on relationships between wild progenitors and domesticated descendants determined with anonymous molecular markers. In this study, this question was investigated by determining the number of instances a domestication phenotype had been selected in a crop species. One of the traits that appeared during domestication of common bean (Phaseolus vulgaris) is determinacy, in which stems end with a terminal inflorescence. It has been shown earlier that a homologue of the arabidopsis TFL1 gene – PvTFL1y – controls determinacy in a naturally occurring variation of common bean.

Methods

Sequence variation was analysed for PvTFL1y in a sample of 46 wild and domesticated accessions that included determinate and indeterminate accessions.

Key Results

Indeterminate types – wild and domesticated – showed only synonymous nucleotide substitutions. Determinate types – observed only among domesticated accessions – showed, in addition to synonymous substitutions, non-synonymous substitutions, indels, a putative intron-splicing failure, a retrotransposon insertion and a deletion of the entire locus. The retrotransposon insertion was observed in 70 % of determinate cultivars, in the Americas and elsewhere. Other determinate mutants had a more restricted distribution in the Americas only, either in the Andean or in the Mesoamerican gene pool of common bean.

Conclusions

Although each of the determinacy haplotypes probably does not represent distinct domestication events, they are consistent with the multiple (seven) domestication pattern in the genus Phaseolus. The predominance of determinacy in the Andean gene pool may reflect domestication of common bean prior to maize introduction in the Andes.     

Association of IREB2 and CHRNA3 polymorphisms with airflow obstruction in severe alpha-1 antitrypsin deficiency

Background

The development of COPD in subjects with alpha-1 antitrypsin (AAT) deficiency is likely to be influenced by modifier genes. Genome-wide association studies and integrative genomics approaches in COPD have demonstrated significant associations with SNPs in the chromosome 15q region that includes CHRNA3 (cholinergic nicotine receptor alpha3) and IREB2 (iron regulatory binding protein 2).We investigated whether SNPs in the chromosome 15q region would be modifiers for lung function and COPD in AAT deficiency.

Methods

The current analysis included 378 PIZZ subjects in the AAT Genetic Modifiers Study and a replication cohort of 458 subjects from the UK AAT Deficiency National Registry. Nine SNPs in LOC123688, CHRNA3 and IREB2 were selected for genotyping. FEV₁ percent of predicted and FEV₁/FVC ratio were analyzed as quantitative phenotypes. Family-based association analysis was performed in the AAT Genetic Modifiers Study. In the replication set, general linear models were used for quantitative phenotypes and logistic regression models were used for the presence/absence of emphysema or COPD.

Results

Three SNPs (rs2568494 in IREB2, rs8034191 in LOC123688, and rs1051730 in CHRNA3) were associated with pre-bronchodilator FEV₁ percent of predicted in the AAT Genetic Modifiers Study. Two SNPs (rs2568494 and rs1051730) were associated with the post-bronchodilator FEV₁ percent of predicted and pre-bronchodilator FEV₁/FVC ratio; SNP-by-gender interactions were observed. In the UK National Registry dataset, rs2568494 was significantly associated with emphysema in the male subgroup; significant SNP-by-smoking interactions were observed.

Conclusions

IREB2 and CHRNA3 are potential genetic modifiers of COPD phenotypes in individuals with severe AAT deficiency and may be sex-specific in their impact.