Deletion variant near ZNF389 is associated with control of ovine lentivirus in multiple sheep flocks

Ovine lentivirus (OvLV) is a macrophage‐tropic lentivirus found in many countries that causes interstitial pneumonia, mastitis, arthritis and cachexia in sheep. There is no preventive vaccine and no cure, but breed differences suggest marker‐assisted selective breeding might improve odds of infection and control of OvLV post‐infection. Although variants in TMEM154 have consistent association with odds of infection, no variant in any gene has been associated with host control of OvLV post‐infection in multiple animal sets. Proviral concentration is a live‐animal diagnostic measure of OvLV control post‐infection related to severity of OvLV‐induced lesions. A recent genome‐wide association study identified a region including four zinc finger genes associated with proviral concentration in one Rambouillet flock. To refine this region, we tested additional variants and identified a small insertion/deletion variant near ZNF389 that showed consistent association with proviral concentration in three animal sets (P < 0.05). These animal sets contained Rambouillet, Polypay and crossbred sheep from multiple locations and management conditions. Strikingly, one flock had exceptionally high prevalence (>87%, including yearlings) and mean proviral concentration (>950 copies/μg), possibly due to needle sharing. The best estimate of proviral concentration by genotype, obtained from all 1310 OvLV‐positive animals tested, showed insertion homozygotes had less than half the proviral concentration of other genotypes (P < 0.0001). Future work will test additional breeds, management conditions and viral subtypes, and identify functional properties of the haplotype this deletion variant tracks. To our knowledge, this is the first genetic variant consistently associated with host control of OvLV post‐infection in multiple sheep flocks.     

Reduced lentivirus susceptibility in sheep with TMEM154 mutations

Visna/Maedi, or ovine progressive pneumonia (OPP) as it is known in the United States, is an incurable slow-acting disease of sheep caused by persistent lentivirus infection. This disease affects multiple tissues, including those of the respiratory and central nervous systems. Our aim was to identify ovine genetic risk factors for lentivirus infection. Sixty-nine matched pairs of infected cases and uninfected controls were identified among 736 naturally exposed sheep older than five years of age. These pairs were used in a genome-wide association study with 50,614 markers. A single SNP was identified in the ovine transmembrane protein (TMEM154) that exceeded genome-wide significance (unadjusted p-value 3×10(-9)). Sanger sequencing of the ovine TMEM154 coding region identified six missense and two frameshift deletion mutations in the predicted signal peptide and extracellular domain. Two TMEM154 haplotypes encoding glutamate (E) at position 35 were associated with infection while a third haplotype with lysine (K) at position 35 was not. Haplotypes encoding full-length E35 isoforms were analyzed together as genetic risk factors in a multi-breed, matched case-control design, with 61 pairs of 4-year-old ewes. The odds of infection for ewes with one copy of a full-length TMEM154 E35 allele were 28 times greater than the odds for those without (p-value<0.0001, 95% CI 5-1,100). In a combined analysis of nine cohorts with 2,705 sheep from Nebraska, Idaho, and Iowa, the relative risk of infection was 2.85 times greater for sheep with a full-length TMEM154 E35 allele (p-value<0.0001, 95% CI 2.36-3.43). Although rare, some sheep were homozygous for TMEM154 deletion mutations and remained uninfected despite a lifetime of significant exposure. Together, these findings indicate that TMEM154 may play a central role in ovine lentivirus infection and removing sheep with the most susceptible genotypes may help eradicate OPP and protect flocks from reinfection.     

羊慢病毒及其抗性基因研究进展

羊慢病毒也称小反刍动物慢病毒, 主要包括绵羊梅迪–维斯纳病毒和山羊关节炎–脑炎病毒, 二者主要感染绵羊和山羊, 目前该病在世界范围内流行并给养羊业带来很大的经济损失。研究表明, 不同绵羊品种对慢病毒易感性存在差异, 这种差异表明易感性不同的绵羊可能存在遗传多样性的差异。全基因组关联分析发现绵羊跨膜蛋白TMEM154 (Transmembrane protein 154)基因中的一个点突变E35K与抗病力高度相关, 可以作为绵羊抗病选育的分子标记。文章详述了绵羊TMEM154基因E35K突变对抗病力的影响和当前慢病毒抗病基因研究概况, 包括锌指家族、趋化因子受体CCR5、三重基序蛋白TRIM5α、载脂蛋白B mRNA剪辑酶催化多肽样蛋白3、多能发育相关基因2和4, 并简要介绍了羊慢病毒特征和我国羊慢病毒病的流行状况, 以期为我国绵羊养殖业和抗病选育提供参考。     

Beta‐globin gene evolution in the ruminants: evidence for an ancient origin of sheep haplotype B

Domestic sheep (Ovis aries) can be divided into two groups with significantly different responses to hypoxic environments, determined by two allelic beta‐globin haplotypes. Haplotype A is very similar to the goat beta‐globin locus, whereas haplotype B has a deletion spanning four globin genes, including beta‐C globin, which encodes a globin with high oxygen affinity. We surveyed the beta‐globin locus using resequencing data from 70 domestic sheep from 42 worldwide breeds and three Ovis canadensis and two Ovis dalli individuals. Haplotype B has an allele frequency of 71.4% in O. aries and was homozygous (BB) in all five wild sheep. This shared ancestry indicates haplotype B is at least 2–3 million years old. Approximately 40 kb of the sequence flanking the ~37‐kb haplotype B deletion had unexpectedly low identity between haplotypes A and B. Phylogenetic analysis showed that the divergent region of sheep haplotype B is remarkably distinct from the beta‐globin loci in goat and cattle but still groups with the Ruminantia. We hypothesize that this divergent ~40‐kb region in haplotype B may be from an unknown ancestral ruminant and was maintained in the lineage to O. aries, but not other Bovidae, evolving independently of haplotype A. Alternatively, the ~40‐kb sequence in haplotype B was more recently acquired by an ancestor of sheep from an unknown non‐Bovidae ruminant, replacing part of haplotype A. Haplotype B has a lower nucleotide diversity than does haplotype A, suggesting a recent bottleneck, whereas the higher frequency of haplotype B suggests a subsequent spread through the global population of O. aries.     

Mitochondrial DNA and Y‐chromosome diversity in East Adriatic sheep

Variation in mitochondrial DNA (mtDNA) and Y‐chromosome haplotypes was analysed in nine domestic sheep breeds (159 rams) and 21 mouflon ( Ovis musimon) sampled in the East Adriatic. Mitochondrial DNA analyses revealed a high frequency of type B haplotypes, predominantly in European breeds, and a very low frequency of type A haplotypes, which are more frequent in some Asian breeds. Mitochondrial haplotype Hmt‐3 was the most frequent (26.4%), and 37.1%, 20.8% and 7.6% of rams had haplotypes one, two and three mutations remote from Hmt‐3 respectively. In contrast, Y‐chromosome analyses revealed extraordinary paternal allelic richness: HY‐6, 89.3%; HY‐8, 5.0%; HY‐18, 3.1%; HY‐7, 1.3%; and HY‐5, 1.3%. In fact, the number of haplotypes observed is comparable to the number found in Turkish breeds and greater than the number found in European breeds so far. Haplotype HY‐18 (A‐oY1/135‐SRYM18), identified here for the first time, provides a link between the haplotype HY‐12 (A‐oY1/139‐SRYM18) found in a few rams in Turkey and haplotype HY‐9 (A‐oY1/131‐SRYM18) found in one ram in Ethiopia. All mouflons had type B mtDNA haplotypes, including the private haplotype (Hmt‐55), and all were paternally monomorphic for haplotype HY‐6. Our data support a quite homogeneous maternal origin of East Adriatic sheep, which is a characteristic of European breeds. At the same time, the high number of haplotypes found was surprising and intriguing, and it begs for further analysis. Simultaneous analysis of mtDNA and Y‐chromosome information allowed us to detect a large discrepancy between maternal and paternal lineages in some populations. This is most likely the result of breeder efforts to ‘upgrade’ local populations using rams with different paternal origins.     

Y chromosome haplotype diversity of domestic sheep (Ovis aries) in northern Eurasia

Variation in two SNPs and one microsatellite on the Y chromosome was analyzed in a total of 663 rams representing 59 breeds from a large geographic range in northern Eurasia. SNPA‐oY1 showed the highest allele frequency (91.55%) across the breeds, whereas SNPG‐oY1 was present in only 56 samples. Combined genotypes established seven haplotypes (H4, H5, H6, H7, H8, H12 and H19). H6 dominated in northern Eurasia, and H8 showed the second‐highest frequency. H4, which had been earlier reported to be absent in European breeds, was detected in one European breed (Swiniarka), whereas H7, which had been previously identified to be unique to European breeds, was present in two Chinese breeds (Ninglang Black and Large‐tailed Han), one Buryatian (Transbaikal Finewool) and two Russian breeds (North Caucasus Mutton‐Wool and Kuibyshev). H12, which had been detected only in Turkish breeds, was also found in Chinese breeds in this work. An overall low level of haplotype diversity (median h = 0.1288) was observed across the breeds with relatively higher median values in breeds from the regions neighboring the Near Eastern domestication center of sheep. H6 is the dominant haplotype in northwestern and eastern China, in which the haplotype distribution could be explained by the historical translocations of the H4 and H8 Y chromosomes to China via the Mongol invasions followed by expansions to northwestern and eastern China. Our findings extend previous results of sheep Y chromosomal genetic variability and indicate probably recent paternal gene flows between sheep breeds from distinct major geographic regions.     

A genome‐wide significant association on chromosome 2 for footrot resistance/susceptibility in Swiss White Alpine sheep

Footrot is one of the most important causes of lameness in global sheep populations and is characterized by a bacterial infection of the interdigital skin. As a multifactorial disease, its clinical representation depends not only on pathogen factors and environmental components but also on the individual resistance/susceptibility of the host. A genetic component has been shown in previous studies; however, so far no causative genetic variant influencing the risk of developing footrot has been identified. In this study, we genotyped 373 Swiss White Alpine sheep, using the ovine high‐density 600k SNP chip, in order to run a DNA‐based comparison of individuals with known clinical footrot status. We performed a case–control genome‐wide association study, which revealed a genome‐wide significant association for SNP rs418747104 on ovine chromosome 2 at 81.2 Mb. The three best associated SNP markers were located at the MPDZ gene, which codes for the multiple PDZ domain crumbs cell polarity complex component protein, also known as multi‐PDZ domain protein 1 (MUPP1). This protein is possibly involved in maintaining the barrier function and integrity of tight junctions. Therefore, we speculate that individuals carrying MPDZ variants may differ in their footrot resistance/susceptibility due to modified horn and interdigital skin integrity. In conclusion, our study reveals that MPDZ might represent a functional candidate gene, and further research is needed to explore its role in footrot affected sheep.     

Genetic control of α‐farnesene production in apple fruit and its role in fungal pathogenesis

Terpenes are important compounds in plant trophic interactions. A meta‐analysis of GC‐MS data from a diverse range of apple (Malus × domestica) genotypes revealed that apple fruit produces a range of terpene volatiles, with the predominant terpene being the acyclic branched sesquiterpene (E,E)‐α‐farnesene. Four quantitative trait loci (QTLs) for α‐farnesene production in ripe fruit were identified in a segregating ‘Royal Gala’ (RG) × ‘Granny Smith’ (GS) population with one major QTL on linkage group 10 co‐locating with the MdAFS1 (α‐farnesene synthase‐1) gene. Three of the four QTLs were derived from the GS parent, which was consistent with GC‐MS analysis of headspace and solvent‐extracted terpenes showing that cold‐treated GS apples produced higher levels of (E,E)‐α‐farnesene than RG. Transgenic RG fruit downregulated for MdAFS1 expression produced significantly lower levels of (E,E)‐α‐farnesene. To evaluate the role of (E,E)‐α‐farnesene in fungal pathogenesis, MdAFS1 RNA interference transgenic fruit and RG controls were inoculated with three important apple post‐harvest pathogens [Colletotrichum acutatum, Penicillium expansum and Neofabraea alba (synonym Phlyctema vagabunda)]. From results obtained over four seasons, we demonstrate that reduced (E,E)‐α‐farnesene is associated with decreased disease initiation rates of all three pathogens. In each case, the infection rate was significantly reduced 7 days post‐inoculation, although the size of successful lesions was comparable with infections on control fruit. These results indicate that (E,E)‐α‐farnesene production is likely to be an important factor involved in fungal pathogenesis in apple fruit.     

TMEM207 hinders the tumour suppressor function of WWOX in oral squamous cell carcinoma

The WW domain‐containing oxidoreductase (WWOX) functions as a tumour suppressor in oral carcinogenesis. As aberrant TMEM207 expression may lead to tumour progression by hampering the tumour suppressor function of WWOX in various cancers, we explored the expression and pathobiological properties of TMEM207, focusing on the WWOX‐mediated regulation of the HIF‐1α pathway in oral squamous cell carcinoma (OSCC). TMEM207 immunoreactivity was detected in 40 of 90 OSCC samples but not in neighbouring non‐tumorous epithelial tissues. Moreover, TMEM207 expression was significantly correlated with lymph node metastasis and poor prognosis. An in situ proximal ligation assay demonstrated the colocalization of TMEM207 and WWOX in invasive OSCC cells, especially glycogen‐rich ones. Enforced expression of TMEM207 abrogated the binding of WWOX to HIF‐1α, increased HIF‐1α and GLUT‐1 expression, even under normoxic conditions, and promoted tumour growth in a xenoplant assay using SAS tongue squamous cancer cells. In contrast, TMEM207 knockdown decreased GLUT‐1 expression in two OSCC cell lines. As a whole, our findings indicate that the aberrant expression of TMEM207 contributes to tumour progression in OSCC, possibly via promoting aerobic glycolysis.     

Multiple copies of eukaryotic translation initiation factors in Brassica rapa facilitate redundancy,enabling diversification through variation in splicing and broad‐spectrum virus resistance

Recessive strain‐specific resistance to a number of plant viruses in the Potyvirus genus has been found to be based on mutations in the eukaryotic translation initiation factor 4E (eIF4E) and its isoform, eIF(iso)4E. We identified three copies of eIF(iso)4E in a number of Brassica rapa lines. Here we report broad‐spectrum resistance to the potyvirus Turnip mosaic virus (TuMV) due to a natural mechanism based on the mis‐splicing of the eIF(iso)4E allele in some TuMV‐resistant B. rapa var. pekinensis lines. Of the splice variants, the most common results in a stop codon in intron 1 and a much truncated, non‐functional protein. The existence of multiple copies has enabled redundancy in the host plant's translational machinery, resulting in diversification and emergence of the resistance. Deployment of the resistance is complicated by the presence of multiple copies of the gene. Our data suggest that in the B. rapa subspecies trilocularis, TuMV appears to be able to use copies of eIF(iso)4E at two loci. Transformation of different copies of eIF(iso)4E from a resistant B. rapa line into an eIF(iso)4E knockout line of Arabidopsis thaliana proved misleading because it showed that, when expressed ectopically, TuMV could use multiple copies which was not the case in the resistant B. rapa line. The inability of TuMV to access multiple copies of eIF(iso)4E in B. rapa and the broad spectrum of the resistance suggest it may be durable.     

A plant RNA virus hijacks endocytic proteins to establish its infection in plants

Endocytosis and endosomal trafficking play essential roles in diverse biological processes including responses to pathogen attack. It is well established that animal viruses enter host cells through receptor‐mediated endocytosis for infection. However, the role of endocytosis in plant virus infection still largely remains unknown. Plant dynamin‐related proteins 1 (DRP1) and 2 (DRP2) are the large, multidomain GTPases that participate together in endocytosis. Recently, we have discovered that DRP2 is co‐opted by Turnip mosaic virus (TuMV) for infection in plants. We report here that DRP1 is also required for TuMV infection. We show that overexpression of DRP1 from Arabidopsis thaliana (AtDRP1A) promotes TuMV infection, and AtDRP1A interacts with several viral proteins including VPg and cylindrical inclusion (CI), which are the essential components of the virus replication complex (VRC). AtDRP1A colocalizes with the VRC in TuMV‐infected cells. Transient expression of a dominant negative (DN) mutant of DRP1A disrupts DRP1‐dependent endocytosis and supresses TuMV replication. As adaptor protein (AP) complexes mediate cargo selection for endocytosis, we further investigated the requirement of AP in TuMV infection. Our data suggest that the medium unit of the AP2 complex (AP2β) is responsible for recognizing the viral proteins as cargoes for endocytosis, and knockout of AP2β impairs intracellular endosomal trafficking of VPg and CI and inhibits TuMV replication. Collectively, our results demonstrate that DRP1 and AP2β are two proviral host factors of TuMV and shed light into the involvement of endocytosis and endosomal trafficking in plant virus infection.     

The prion‐related protein (testis‐specific) gene (PRNT) is highly polymorphic in Portuguese sheep

The objective of this study was to search for polymorphisms in the ovine prion‐related protein (testis‐specific) gene (PRNT). Sampling included 567 sheep from eight Portuguese breeds. The PRNT gene‐coding region was analyzed by single‐strand conformation polymorphism and sequencing, allowing the identification of the first ovine PRNT polymorphisms, in codons 6, 38, 43 and 48: c.17C>T (p.Ser6Phe, which disrupts a consensus arginine‐X‐X‐serine/threonine motif); c.112G>C (p.Gly38>Arg); c.129T>C and c.144A>G (synonymous) respectively. Polymorphisms in codons 6, 38 and 48 occur simultaneously in 50.6% of the animals, 38.8% presenting as heterozygous. To study the distribution of the polymorphism in codon 43, a restriction fragment length polymorphism analysis was performed. Polymorphic variant c.129C, identified in 89.8% of the animals with 32.8% presented as heterozygous, was considered the wild genotype in Portuguese sheep. Eight different haplotypes which have comparable distribution in all breeds were identified for the PRNT gene. In conclusion, the PRNT coding region is highly polymorphic in sheep, unlike the prion protein 2 dublet gene (PRND), in which we previously found only one synonymous substitution (c.78G>A), in codon 26. The absence or reduced number of PRND heterozygotes (c.78G>A) was significantly associated with three PRNT haplotypes (17C‐112G‐129T‐144A,17CT‐112GC‐129CT‐144AG and 17T‐112C‐129C‐144G), and the only three animals found homozygous at c.78A had the 17C‐112G‐129C‐144A PRNT haplotype. These results constitute evidence of an association between polymorphic variation in PRND and PRNT genes, as has already been observed for PRND and prion protein gene (PRNP).     

Efficacy of Acibenzolar‐S‐Methyl and β‐Aminobutyric Acid for Control of Downy Mildew in Greenhouse Grown Basil and Peroxidase Activity in Response to Treatment with these Compounds

Downy mildew, caused by the oomycete pathogen Peronospora belbahrii, is a devastating foliar disease of basil in the United States and worldwide. Currently there are very few chemistries or organic choices registered to control this disease. In this study, two systemic acquired resistance (SAR) inducers, acibenzolar‐S‐methyl (ASM) and β‐aminobutyric acid (BABA), were evaluated for their in vitro effects on the pathogen, for their potential to control basil downy mildew in greenhouses, and for changes in peroxidase activity in basil plants treated with these two SAR inducers. No significant inhibition of sporangial germination was detected in water agar amended with ASM at concentrations lower than 100 mg/l or with BABA at concentrations lower than 500 mg/l. Efficacy of ASM and BABA in greenhouses varied depending on the rate, method and timing of application. The area under the disease progress curve (AUDPC) of disease severity was significantly reduced compared to the non‐treated control when ASM was sprayed (in all experiments) or drenched (in one out of two experiments) pre‐, or pre‐ + post‐inoculation at rates of 25–400 mg/l. Three weekly post‐inoculation sprays of ASM at the rate of 50 mg/l reduced AUDPC by 93.0 and 47.2% when started 3 and 7 days after inoculation (DAI), respectively. The AUDPC of disease severity was also significantly reduced when BABA was sprayed pre‐ + post‐inoculation at rates of 125–500 mg/l. According to the prediction using a log‐logistic function, 50% maximum disease protection was achieved at a concentration of 27.5 mg/l of ASM. Basil plants treated with these two SAR inducers and challenged with the pathogen showed significantly higher peroxidase activity than the non‐treated control at 8 DAI. Temporally, the highest activity of peroxidase was detected at 8 DAI, decreased at 15 DAI and waned further at 23 DAI.     

Maternal and paternal genetic diversity of ancient sheep in Estonia from the Late Bronze Age to the post‐medieval period and comparison with other regions in Eurasia

Sheep were among the first domesticated animals to appear in Estonia in the late Neolithic and became one of the most widespread livestock species in the region from the Late Bronze Age onwards. However, the origin and historical expansion of local sheep populations in Estonia remain poorly understood. Here, we analysed fragments of the hypervariable D‐loop of mitochondrial DNA (mtDNA; 213 bp) and the Y‐chromosome SRY gene (130 bp) extracted from 31 archaeological sheep bones dated from approximately 800 BC to 1700 AD. The ancient DNA data of sheep from Estonia were compared with ancient sheep from Finland as well as a set of contemporary sheep breeds from across Eurasia in order to place them in a wider phylogeographical context. The analysis shows that: (i) 24 successfully amplified and analysed mtDNA sequences of ancient sheep cluster into two haplogroups, A and B, of which B is predominant; (ii) four of the ancient mtDNA haplotypes are novel; (iii) higher mtDNA haplotype diversity occurred during the Middle Ages as compared to other periods, a fact concordant with the historical context of expanding international trade during the Middle Ages; (iv) the proportion of rarer haplotypes declined during the expansion of sheep from the Near Eastern domestication centre to the northern European region; (v) three male samples showed the presence of the characteristic northern European haplotype, SNP G‐oY1 of the Y‐chromosome, and represent the earliest occurrence of this haplotype. Our results provide the first insight into the genetic diversity and phylogeographical background of ancient sheep in Estonia and provide basis for further studies on the temporal fluctuations of ancient sheep populations.     

Polymorphism in the neurofibromin gene,Nf1, is associated with antagonistic selection on wing size and development time in Drosophila melanogaster

In many invertebrates, body size shows genetically based clines, with size increasing in colder climates. Large body size is typically associated with prolonged development times. We consider variation in the CNS‐specific gene neurofibromin 1 (Nf1) and its association with body size and development time. We identified two major Nf1 haplotypes in natural populations, Nf1‐insertion‐A and Nf1‐deletion‐G. These haplotypes are characterized by a 45‐base insertion/deletion (INDEL) in Nf1 intron 2 and an A/G synonymous substitution (locus L17277). Linkage disequilibrium (LD) between the INDEL and adjacent sites is high but appears to be restricted within the Nf1 gene interval. In Australia, the frequency of the Nf1‐insertion‐A haplotype increases with latitude where wing size is larger, independent of the chromosomal inversion In(3R)Payne. Unexpectedly, the Nf1‐insertion‐A haplotype is negatively associated with wing size. We found that the Nf1‐insertion‐A haplotype is enriched in females with shorter development time. This suggests that the Nf1 haplotype cline may be driven by selection for development time rather than size; females from southern (higher latitude) D. melanogaster populations maintain a rapid development time despite being relatively larger, and the higher incidence of Nf1‐insertion‐A in Southern Australia may contribute to this pattern, whereas the effects of the Nf1 haplotypes on size may be countered by other loci with antagonistic effects on size and development time. Our results point to the potential complexity involved in identifying selection on genetic variants exhibiting pleiotropic effects when studies are based on spatial patterns or association studies.