Sox genes in grass carp (Ctenopharyngodon idella) with their implications for genome duplication and evolution

The Sox gene family is found in a broad range of animal taxa and encodes important gene regulatory proteins involved in a variety of developmental processes. We have obtained clones representing the HMG boxes of twelve Sox genes from grass carp (Ctenopharyngodon idella), one of the four major domestic carps in China. The cloned Sox genes belong to group B1, B2 and C. Our analyses show that whereas the human genome contains a single copy of Sox4, Sox11 and Sox14, each of these genes has two co-orthologs in grass carp, and the duplication of Sox4 and Sox11 occurred before the divergence of grass carp and zebrafish, which support the "fish-specific whole-genome duplication" theory. An estimation for the origin of grass carp based on the molecular clock using Sox1, Sox3 and Sox11 genes as markers indicates that grass carp (subfamily Leuciscinae) and zebrafish (subfamily Danioninae) diverged approximately 60 million years ago. The potential uses of Sox genes as markers in revealing the evolutionary history of grass carp are discussed.     

Prenatal diagnosis of foetuses with congenital abnormalities and duplication of the MECP2 region

MECP2 duplication results in a well-recognised syndrome in 100% of affected male children; this syndrome is characterised by severe neurodevelopmental disabilities and recurrent infections. However, no sonographic findings have been reported for affected foetuses, and prenatal molecular diagnosis has not been possible for this disease due to lack of prenatal clinical presentation. In this study, we identified a small duplication comprising the MECP2 and L1CAM genes in the Xq28 region in a patient from a family with severe X-linked mental retardation and in a prenatal foetus with brain structural abnormalities. Using high-resolution chromosome microarray analysis (CMA) to screen 108 foetuses with congenital structural abnormalities, we identified additional three foetuses with the MECP2 duplication. Our study indicates that ventriculomegaly, hydrocephalus, agenesis of the corpus callosum, choroid plexus cysts, foetal growth restriction and hydronephrosis might be common ultrasound findings in prenatal foetuses with the MECP2 duplication and provides the first set of prenatal cases with MECP2 duplication, the ultrasonographic phenotype described in these patients will help to recognise the foetuses with possible MECP2 duplication and prompt the appropriate molecular testing.     

Identifying new sex-linked genes through BAC sequencing in the dioecious plant Silene latifolia

Background

Silene latifolia represents one of the best-studied plant sex chromosome systems. A new approach using RNA-seq data has recently identified hundreds of new sex-linked genes in this species. However, this approach is expected to miss genes that are either not expressed or are expressed at low levels in the tissue(s) used for RNA-seq. Therefore other independent approaches are needed to discover such sex-linked genes.

Results

Here we used 10 well-characterized S. latifolia sex-linked genes and their homologs in Silene vulgaris, a species without sex chromosomes, to screen BAC libraries of both species. We isolated and sequenced 4 Mb of BAC clones of S. latifolia X and Y and S. vulgaris genomic regions, which yielded 59 new sex-linked genes (with S. vulgaris homologs for some of them). We assembled sequences that we believe represent the tip of the Xq arm. These sequences are clearly not pseudoautosomal, so we infer that the S. latifolia X has a single pseudoautosomal region (PAR) on the Xp arm. The estimated mean gene density in X BACs is 2.2 times lower than that in S. vulgaris BACs, agreeing with the genome size difference between these species. Gene density was estimated to be extremely low in the Y BAC clones. We compared our BAC-located genes with the sex-linked genes identified in previous RNA-seq studies, and found that about half of them (those with low expression in flower buds) were not identified as sex-linked in previous RNA-seq studies. We compiled a set of ~70 validated X/Y genes and X-hemizygous genes (without Y copies) from the literature, and used these genes to show that X-hemizygous genes have a higher probability of being undetected by the RNA-seq approach, compared with X/Y genes; we used this to estimate that about 30 % of our BAC-located genes must be X-hemizygous. The estimate is similar when we use BAC-located genes that have S. vulgaris homologs, which excludes genes that were gained by the X chromosome.

Conclusions

Our BAC sequencing identified 59 new sex-linked genes, and our analysis of these BAC-located genes, in combination with RNA-seq data suggests that gene losses from the S. latifolia Y chromosome could be as high as 30 %, higher than previous estimates of 10-20 %.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1698-7) contains supplementary material, which is available to authorized users.     

Genetic diversity and population structure inferred from the partially duplicated genome of domesticated carp,Cyprinus carpio L.

Genetic relationships among eight populations of domesticated carp (Cyprinus carpio L.), a species with a partially duplicated genome, were studied using 12 microsatellites and 505 AFLP bands. The populations included three aquacultured carp strains and five ornamental carp (koi) variants. Grass carp (Ctenopharyngodon idella) was used as an outgroup. AFLP-based gene diversity varied from 5% (grass carp) to 32% (koi) and reflected the reasonably well understood histories and breeding practices of the populations. A large fraction of the molecular variance was due to differences between aquacultured and ornamental carps. Further analyses based on microsatellite data, including cluster analysis and neighbor-joining trees, supported the genetic distinctiveness of aquacultured and ornamental carps, despite the recent divergence of the two groups. In contrast to what was observed for AFLP-based diversity, the frequency of heterozygotes based on microsatellites was comparable among all populations. This discrepancy can potentially be explained by duplication of some loci in Cyprinus carpio L., and a model that shows how duplication can increase heterozygosity estimates for microsatellites but not for AFLP loci is discussed. Our analyses in carp can help in understanding the consequences of genotyping duplicated loci and in interpreting discrepancies between dominant and co-dominant markers in species with recent genome duplication.     

The dormancy-breaking stimuli “chilling,hypoxia and cyanamide exposure” up-regulate the expression of α-amylase genes in grapevine buds

It has been suggested that respiratory stress is involved in the mechanism underlying the dormancy-breaking effect of hydrogen cyanamide (H₂CN₂) and sodium azide in grapevine buds; indeed, reductions in oxygen levels (hypoxia) and inhibitors of respiration promote bud-break in grapevines. In this study, we showed that, hypoxia increased starch hydrolysis soluble sugar consumption and up-regulated the expression of α-amylase genes (Vvα-AMYs) in grapevine buds, suggesting that these biochemical changes induced by hypoxia, may play a relevant role in the release of buds from endodormancy (ED). Three of the four Vvα-AMY genes that are expressed in grapevine buds were up-regulated by hypoxia and a correlation between changes in sugar content and level of Vvα-AMY gene expression during the hypoxia treatment was found, suggesting that soluble sugars mediate the effect of hypoxia on Vvα-AMY gene expression. Exogenous applications of soluble sugars and sugar analogs confirmed this finding and revealed that osmotic stress induces the expression of Vvα-AMY1 and Vvα-AMY3 and that soluble sugars induces Vvα-AMY2 and Vvα-AMY4 gene expression. Interestingly, the plant hormone gibberellic acid (GA₃) induced the expression of Vvα-AMY3 and Vvα-AMY4 genes, while dormancy breaking stimuli, chilling and cyanamide exposure, mainly induced the expression of Vvα-AMY1 and Vvα-AMY2 genes, suggesting that these two α-amylase genes might be involved in the release of grapevine buds from the ED.     

The repetitive component of the A genome of peanut (Arachis hypogaea) and its role in remodelling intergenic sequence space since its evolutionary divergence from the B genome

Background and Aims

Peanut (Arachis hypogaea) is an allotetraploid (AABB-type genome) of recent origin, with a genome of about 2·8 Gb and a high repetitive content. This study reports an analysis of the repetitive component of the peanut A genome using bacterial artificial chromosome (BAC) clones from A. duranensis, the most probable A genome donor, and the probable consequences of the activity of these elements since the divergence of the peanut A and B genomes.

Methods

The repetitive content of the A genome was analysed by using A. duranensis BAC clones as probes for fluorescence in situ hybridization (BAC-FISH), and by sequencing and characterization of 12 genomic regions. For the analysis of the evolutionary dynamics, two A genome regions are compared with their B genome homeologues.

Key Results

BAC-FISH using 27 A. duranensis BAC clones as probes gave dispersed and repetitive DNA characteristic signals, predominantly in interstitial regions of the peanut A chromosomes. The sequences of 14 BAC clones showed complete and truncated copies of ten abundant long terminal repeat (LTR) retrotransposons, characterized here. Almost all dateable transposition events occurred <3·5 million years ago, the estimated date of the divergence of A and B genomes. The most abundant retrotransposon is Feral, apparently parasitic on the retrotransposon FIDEL, followed by Pipa, also non-autonomous and probably parasitic on a retrotransposon we named Pipoka. The comparison of the A and B genome homeologous regions showed conserved segments of high sequence identity, punctuated by predominantly indel regions without significant similarity.

Conclusions

A substantial proportion of the highly repetitive component of the peanut A genome appears to be accounted for by relatively few LTR retrotransposons and their truncated copies or solo LTRs. The most abundant of the retrotransposons are non-autonomous. The activity of these retrotransposons has been a very significant driver of genome evolution since the evolutionary divergence of the A and B genomes.     

A triallelic genetic male sterility locus in Brassica napus: an integrative strategy for its physical mapping and possible local chromosome evolution around it

Background and Aims

Spontaneous male sterility is an advantageous trait for both constructing efficient pollination control systems and for understanding the developmental process of the male reproductive unit in many crops. A triallelic genetic male-sterile locus (BnMs5) has been identified in Brassica napus; however, its complicated genome structure has greatly hampered the isolation of this locus. The aim of this study was to physically map BnMs5 through an integrated map-based cloning strategy and analyse the local chromosomal evolution around BnMs5.

Methods

A large F₂ population was used to integrate the existing genetic maps around BnMs5. A map-based cloning strategy in combination with comparative mapping among B. napus, Arabidopsis, Brassica rapa and Brassica oleracea was employed to facilitate the identification of a target bacterial artificial chromosome (BAC) clone covering the BnMs5 locus. The genomic sequences from the Brassica species were analysed to reveal the regional chromosomal evolution around BnMs5.

Key Results

BnMs5 was finally delimited to a 0·3-cM genetic fragment from an integrated local genetic map, and was anchored on the B. napus A8 chromosome. Screening of a B. napus BAC clone library and identification of the positive clones validated that JBnB034L06 was the target BAC clone. The closest flanking markers restrict BnMs5 to a 21-kb region on JBnB034L06 containing six predicted functional genes. Good collinearity relationship around BnMs5 between several Brassica species was observed, while violent chromosomal evolutionary events including insertions/deletions, duplications and single nucleotide mutations were also found to have extensively occurred during their divergence.

Conclusions

This work represents major progress towards the molecular cloning of BnMs5, as well as presenting a powerful, integrative method to mapping loci in plants with complex genomic architecture, such as the amphidiploid B. napus.     

Putative Virulence Genes and Biofilm Production Among Typical Enteroaggregative Escherichia coli Isolates from Diarrhoeic Children in Kashmir and Andhra Pradesh

Fifty-eight typical EAEC isolates from children with diarrhoea were examined for HEp-2 cell adherence assay, presence of dispersin (aap), yersiniabactin (irp2), plasmid encoded toxins (pet), Shigella enterotoxin1 (set1A) and cryptic open reading frame (shf) putative virulence genes by polymerase chain reaction as well as for biofilm production. All the isolates showed aggregative adherence pattern on HEp-2 cells. All but five isolates (91.3 %) carried aap gene. While irp2, pet, set1A and shf genes were detected in 68.9, 5.1, 39.6, and 60.3 % isolates, respectively. Thirty-three (64.7 %) isolates out of 51 tested were found to produce biofilm which was found to be significantly associated only with set1A virulence gene (P = 0.025). Highest amount of biofilm was produced by a strain that possessed all the genes studied. Out of 14 isolates in which the most frequent gene combination (aap, irp2 and shf) was observed, only six produced biofilm. It is concluded that there is significant heterogeneity in putative virulence genes of EAEC isolates from diarrhoeic children and biofilm formation is associated with multiple genes.     

Hypoxia-inducible factor 1-mediated regulation of PPP1R3C promotes glycogen accumulation in human MCF-7 cells under hypoxia

Hundreds of genes can be regulated by hypoxia-inducible factor 1 (HIF1) under hypoxia. Here we demonstrated a HIF1-mediated induction of protein phosphatase 1, regulatory subunit 3C gene (PPP1R3C) in human MCF7 cells under hypoxia. By mutation analysis we confirmed the presence of a functional hypoxia response element that is located 229 bp upstream from the PPP1R3C gene. PPP1R3C induction correlates with a significant glycogen accumulation in MCF7 cells under hypoxia. Knockdown of either HIF1α or PPP1R3C attenuated hypoxia-induced glycogen accumulation significantly. Knockdown of HIF2α reduced hypoxia-induced glycogen accumulation slightly (but not significantly). Our results demonstrated that HIF1 promotes glycogen accumulation through regulating PPP1R3C expression under hypoxia, which revealed a novel metabolic adaptation of cells to hypoxia.     

Molecular evolution of glycinin and β-conglycinin gene families in soybean (Glycine max L. Merr.)

There are two main classes of multi-subunit seed storage proteins, glycinin (11S) and β-conglycinin (7S), which account for approximately 70% of the total protein in a typical soybean seed. The subunits of these two protein classes are encoded by a number of genes. The genomic organization of these genes follows a complex evolutionary history. This research was designed to describe the origin and maintenance of genes in each of these gene families by analyzing the synteny, phylogenies, selection pressure and duplications of the genes in each gene family. The ancestral glycinin gene initially experienced a tandem duplication event; then, the genome underwent two subsequent rounds of whole-genome duplication, thereby resulting in duplication of the glycinin genes, and finally a tandem duplication likely gave rise to the Gy1 and Gy2 genes. The β-conglycinin genes primarily originated through the more recent whole-genome duplication and several tandem duplications. Purifying selection has had a key role in the maintenance of genes in both gene families. In addition, positive selection in the glycinin genes and a large deletion in a β-conglycinin exon contribute to the diversity of the duplicate genes. In summary, our results suggest that the duplicated genes in both gene families prefer to retain similar function throughout evolution and therefore may contribute to phenotypic robustness.     

Phylogenetic utility of the nuclear genes AGAMOUS 1 and PHYTOCHROME B in palms (Arecaceae): an example within Bactridinae

Background and Aims

Molecular phylogenetic studies of palms (Arecaceae) have not yet provided a fully resolved phylogeny of the family. There is a need to increase the current set of markers to resolve difficult groups such as the Neotropical subtribe Bactridinae (Arecoideae: Cocoseae). We propose the use of two single-copy nuclear genes as valuable tools for palm phylogenetics.

Methods

New primers were developed for the amplification of the AGAMOUS 1 (AG1) and PHYTOCHROME B (PHYB) genes. For the AGAMOUS gene, the paralogue 1 of Elaeis guineensis (EgAG1) was targeted. The region amplified contained coding sequences between the MIKC K and C MADS-box domains. For the PHYB gene, exon 1 (partial sequence) was first amplified in palm species using published degenerate primers for Poaceae, and then specific palm primers were designed. The two gene portions were sequenced in 22 species of palms representing all genera of Bactridinae, with emphasis on Astrocaryum and Hexopetion, the status of the latter genus still being debated.

Key Results

The new primers designed allow consistent amplification and high-quality sequencing within the palm family. The two loci studied produced more variability than chloroplast loci and equally or less variability than PRK, RPBII and ITS nuclear markers. The phylogenetic structure obtained with AG1 and PHYB genes provides new insights into intergeneric relationships within the Bactridinae and the intrageneric structure of Astrocaryum. The Hexopetion clade was recovered as monophyletic with both markers and was weakly supported as sister to Astrocaryum sensu stricto in the combined analysis. The rare Astrocaryum minus formed a species complex with Astrocaryum gynacanthum. Moreover, both AG1 and PHYB contain a microsatellite that could have further uses in species delimitation and population genetics.

Conclusions

AG1 and PHYB provide additional phylogenetic information within the palm family, and should prove useful in combination with other genes to improve the resolution of palm phylogenies.