REDHORSE-REcombination and Double crossover detection in Haploid Organisms using next-geneRation SEquencing data

Background

Next-generation sequencing technology provides a means to study genetic exchange at a higher resolution than was possible using earlier technologies. However, this improvement presents challenges as the alignments of next generation sequence data to a reference genome cannot be directly used as input to existing detection algorithms, which instead typically use multiple sequence alignments as input. We therefore designed a software suite called REDHORSE that uses genomic alignments, extracts genetic markers, and generates multiple sequence alignments that can be used as input to existing recombination detection algorithms. In addition, REDHORSE implements a custom recombination detection algorithm that makes use of sequence information and genomic positions to accurately detect crossovers. REDHORSE is a portable and platform independent suite that provides efficient analysis of genetic crosses based on Next-generation sequencing data.

Results

We demonstrated the utility of REDHORSE using simulated data and real Next-generation sequencing data. The simulated dataset mimicked recombination between two known haploid parental strains and allowed comparison of detected break points against known true break points to assess performance of recombination detection algorithms. A newly generated NGS dataset from a genetic cross of Toxoplasma gondii allowed us to demonstrate our pipeline. REDHORSE successfully extracted the relevant genetic markers and was able to transform the read alignments from NGS to the genome to generate multiple sequence alignments. Recombination detection algorithm in REDHORSE was able to detect conventional crossovers and double crossovers typically associated with gene conversions whilst filtering out artifacts that might have been introduced during sequencing or alignment. REDHORSE outperformed other commonly used recombination detection algorithms in finding conventional crossovers. In addition, REDHORSE was the only algorithm that was able to detect double crossovers.

Conclusion

REDHORSE is an efficient analytical pipeline that serves as a bridge between genomic alignments and existing recombination detection algorithms. Moreover, REDHORSE is equipped with a recombination detection algorithm specifically designed for Next-generation sequencing data. REDHORSE is portable, platform independent Java based utility that provides efficient analysis of genetic crosses based on Next-generation sequencing data. REDHORSE is available at http://redhorse.sourceforge.net/.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1309-7) contains supplementary material, which is available to authorized users.     

Comparative linkage mapping of the Grey coat colour gene in horses

Grey horses are born coloured, turn progressively grey and often develop melanomas late in life. Grey shows an autosomal dominant inheritance and the locus has previously been mapped to horse chromosome 25 (ECA25), around the TXN gene. We have now developed eight new single nucleotide polymorphisms (SNPs) associated with genes on ECA25 using information on the linear order of genes on human chromosome 9q, as well as the human and mouse coding sequences. These SNPs were mapped in relation to the Grey locus using more than 300 progeny from matings between two Swedish Warmblood grey stallions and non-grey mares. Grey was firmly assigned to an interval with flanking markers NANS and ABCA1. This corresponds to a region of approximately 6.9 Mb on human chromosome 9q. Furthermore, no recombination was observed between Grey, TGFBR1 and TMEFF1, the last two being 1.4 Mb apart in human. There are no obvious candidate genes in this region and none of the genes has been associated with pigmentation disorders or melanoma development, suggesting that the grey phenotype is caused by a mutation in a novel gene.     

Pushing, pulling and trapping--modes of motor protein supported protein translocation

Protein translocation across the cellular membranes is an ubiquitous and crucial activity of cells. This process is mediated by translocases that consist of a protein conducting channel and an associated motor protein. Motor proteins interact with protein substrates and utilize the free energy of ATP binding and hydrolysis for protein unfolding, translocation and unbinding. Since motor proteins are found either at the cis- or trans-side of the membrane, different mechanisms for translocation have been proposed. In the Power stroke model, cis-acting motors are thought to push, while trans-motors pull on the substrate protein during translocation. In the Brownian ratchet model, translocation occurs by diffusion of the unfolded polypeptide through the translocation pore while directionality is achieved by trapping and refolding. Recent insights in the structure and function of the molecular motors suggest that different mechanisms can be employed simultaneously.     

Genome scans of variation and adaptive change: extended analysis of a candidate locus close to the phantom gene region in Drosophila melanogaster

Nucleotide variation in populations originating from the recent range expansion of a species should reflect their adaptation to new habitats as well as their demographic history. A survey of nucleotide variation at 109 noncoding X-chromosome fragments in a European population of Drosophila melanogaster allowed identifying some candidates to have been recently affected by positive selection. Adaptive changes leave a spatial differential footprint that can be used to discriminate among candidates by extending their study to neighboring regions. Here, we surveyed variation at an approximately 190-kb region spanning a locus exhibiting a significantly skewed frequency spectrum. A stretch of approximately 12 kb with reduced variation was detected within a continuously sequenced region that included the focal fragment. Moreover, the regions flanking this stretch exhibited an excess of high-frequency derived variants. Application of maximum likelihood ratio and goodness-of-fit tests suggested that the pattern of variation detected at the studied region (at cytological bands 17C-17D) might have been shaped by a recent selective change, most probably at or around the phantom gene that encodes CYP306A1, a cytochrome P450 enzyme in the ecdysteroidogenic pathway.     

不同性别类型黄瓜ACC合酶基因的单核苷酸多态性标记和酶切扩增长度多态性标记

黄瓜的性别分化与乙烯密切相关,1-氨基环丙烷-1-羧酸(ACC)合酶是乙烯生物合成过程中的关键酶.根据ACC合酶基因家族的保守序列设计PCR引物,从8个不同性别类型(雌雄同株、强雌性和全雌性)黄瓜品种中克隆了长度为1188bp的ACC合酶基因(CS-ACS2)片段(GenBank登记号为:DQ115884～DQ115886和DQ115875～DQ115879).经测序分析,3个雌雄同株性别类型品种的序列完全相同.与之相比,5个强雌性和全雌性品种中存在8个单核苷酸多态性(SNPs)标记,SNPs标记为4个A←→G和4个T←→C之间的转换.在8个SNPs中,有1个SNP位于内含子区域,其余7个SNPs都位于外显子区域.在7个位于外显子区域的SNPs中,有3个为非编码区的SNPs,4个为cSNPs.而在4个cSNPs中,有3个导致了编码的氨基酸序列改变.研究结果表明,与雌雄同株性别类型相比,雌性系中均存在单核苷酸的变异,这提示ACC合酶基因CS-ACS2的单核苷酸变异可能与黄瓜雌性系的发生形成有关.另一方面,根据SNP多态性还发展了一个酶切扩增长度多态性(CAPS)标记C-MT700.利用CAPS标记C-MT700能将强雌性优良品种MT-705与其他黄瓜品种相区别,该标记在黄瓜育种生产上具有一定的应用价值.此外,研究获得的SNPs标记和CAPS标记丰富了黄瓜的分子标记种类.     

Genetic association between bovine NOD2 polymorphisms and infection by Mycobacterium avium subsp. paratuberculosis in Holstein‐Friesian cattle

Nucleotide‐Binding Oligomerization Domain 2 (NOD2) has been reported to be a candidate gene for Mycobacterium avium subsp. paratuberculosis (MAP) infection in a Bos taurus × Bos indicus mixed breed based on a genetic association with the c.2197T>C single nucleotide polymorphism (SNP). Nevertheless, this SNP has also been reported to be monomorphic in the B. taurus species. In the present work, 18 SNPs spanning the bovine NOD2 gene have been analysed in a genetic association study of two independent populations of Holstein‐Friesian cattle. We found that the C allele of SNP c.*1908C>T, located in the 3′‐UTR region of the gene, is significantly more frequent in infected animals than in healthy ones, which supports the idea that the bovine NOD2 gene plays a role in susceptibility to MAP infection. However, in silico analyses of the NOD2 nucleotide sequence did not yield definitive data about a possible direct effect of SNP c.*1908C>T on susceptibility to infection and led us to consider its linkage disequilibrium with the causative variant. A more exhaustive genetic association study including all putative, functional SNPs from this gene and subsequent functional analyses needs to be conducted to achieve a more complete understanding of how different variants of NOD2 may affect susceptibility to MAP infection in cattle.     

慢性乙型肝炎抗病毒治疗研究进展

乙肝病毒的慢性感染是肝纤维化、肝细胞癌的重要病因,积极有效地抗病毒治疗,可显著改善HBV感染者的生活质量。干扰素、核苷（酸）类药物的抗乙肝病毒疗效已得到全球公认,本文就上述两类药物的研究进展进行综述。     

Parallel habitat-driven differences in the phylogeographical structure of two independent lineages of Mediterranean saline water beetles

It has been hypothesized that species living in small lentic water bodies, because of the short-term geological persistence of their habitat, should show higher dispersal ability, with increased gene flow among populations and a less pronounced phylogeographical structure. Conversely, lotic species, living in more geologically stable habitats, should show reduced dispersal and an increased phylogeographical structure at the same geographical scales. In this work we tested the influence of habitat type in two groups of aquatic Coleoptera ( Nebrioporus ceresyi and Ochthebius notabilis groups, families Dytiscidae and Hydraenidae respectively), each of them with closely related species typical of lotic and lentic saline Western Mediterranean water bodies. We used mitochondrial cox1 sequence data of 453 specimens of 77 populations through the range of nine species to compare a lotic vs. a lentic lineage in each of the two groups. Despite the differences in biology (predators vs. detritivorous) and evolutionary history, in both lotic lineages there was a higher proportion of nucleotide diversity among than within groups of populations, and a faster rate of accumulation of haplotype diversity (as measured by rarefaction curves) than in the lentic lineages. Similarly, lotic lineages had a higher absolute phylogenetic diversity, more remarkable considering their smaller absolute geographical ranges. By comparing closely related species, we were able to show the effect of contrasting habitat preferences in two different groups, in agreement with predictions derived from habitat stability.     

Molecular basis of guanine nucleotide dissociation inhibitor activity of human neuroglobin by chemical cross-linking and mass spectrometry

Oxidized human neuroglobin (Ngb), a heme protein expressed in the brain, has been proposed to act as a guanine nucleotide dissociation inhibitor (GDI) for the GDP-bound form of the heterotrimeric G protein alpha-subunit (Galpha(i)). Here, to elucidate the molecular mechanism underlying the GDI activity of Ngb, we used an glutathione-S-transferase pull-down assay to confirm that Ngb competes with G-protein betagamma-subunits (Gbetagamma) for binding to Galpha(i), and identified the Galpha(i)-binding site in Ngb by chemical cross-linking with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and sulfo-N-hydroxysuccinimide, coupled with mass spectrometry (MS). Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS analysis for tryptic peptides derived from the cross-linked Ngb-Galpha(i) complex revealed several binding regions in Ngb. Furthermore, MALDI-TOF/TOF MS analysis of the cross-linked Ngb and Galpha(i) peptides, together with the MS/MS scoring method, predicted cross-linking between Glu60 (Ngb) and Ser206 (Galpha(i)), and between Glu53 (Ngb) and Ser44 (Galpha(i)). Because Ser206 of Galpha(i) is located in the region that contacts Gbetagamma, binding of Ngb could facilitate the release of Gbetagamma from Galpha(i). Binding of Ngb to Galpha(i) would also inhibit the exchange of GDP for GTP, because Ser44 (Galpha(i)) is adjacent to the GDP-binding site and Glu53 (Ngb), which is cross-linked to Ser44 (Galpha(i)), could be located close to GDP. Thus, we have identified, for the first time, the sites of interaction between Ngb and Galpha(i), enabling us to discuss the functional significance of this binding on the GDI activity of Ngb.     

Potential role of subunit c of F0F1-ATPase and subunit c of storage body in the mitochondrial permeability transition. Effect of the phosphorylation status of subunit c on pore opening

Phosphorylated and non-phosphorylated forms of the F₀F₁-ATPase subunit c from rat liver mitochondria (RLM) were purified and their effect on the opening of the permeability transition pore (mPTP) was investigated. Addition of dephosphorylated subunit c to RLM induced mitochondrial swelling, decreased the membrane potential and reduced the Ca²⁺ uptake capacity, which was prevented by cyclosporin A. The same effect was observed in the presence of storage subunit c purified from livers of sheep affected with ceroid lipofuscinosis. In black-lipid bilayer membranes subunit c increased the conductance due to formation of single channels with fast and slow kinetics. The dephosphorylated subunit c formed channels with slow kinetics, i.e. the open state being of significantly longer duration than in the case of channels formed by the phosphorylated form that had short life spans and fast kinetics. The channels formed were cation-selective more so with the phosphorylated form. Subunit c of rat liver mitochondria was able to bind Ca²⁺. Collectively, the data allowed us to suppose that subunit c F₀F₁-ATPase might be a structural/regulatory component of mPTP exerting its role in dependence on phosphorylation status.