Genetic diversity of the prion protein gene (<Emphasis Type="Italic">PRNP</Emphasis>) coding sequence in Czech sheep and evaluation of the national breeding programme for resistance to scrapie in the Czech Republic

Of 34 breeds kept in the Czech Republic 45,604 sheep were genotyped for codons 136, 154 and 171 in the prion protein gene (PRNP) during the years 2006–2014. In this cohort, haplotypes ARR, ARQ, ARH, AHQ, VRQ, AHR and ARK were detected. The haplotype AF₁₄₁RQ associated with susceptibility to atypical scrapie was observed in nine out of 30 breeds analysed for this purpose. In addition, six rare nonsynonymous substitutions producing haplotypes AT₁₃₇RQ, AN₁₃₈RQ, AG₁₅₁RQ, AH₁₅₁RQ, ARL₁₆₈Q and ARQE₁₇₅ were identified in various breeds. Due to their low frequencies, these polymorphisms are of no potential importance for the breeding programme. With regard to their genetic particularity, Sumavka, Valachian and Cameroon breeds were screened for additional polymorphisms. Further haplotypes, AR₁₄₃RQ and AS₁₄₆RQ, were found in Sumavka and Cameroon, and in Valachian sheep, respectively. Frequencies of the ARR (resistance-associated), VRQ (susceptibility-associated) haplotypes, and of the most resistant ARR/ARR genotype calculated for sheep born in the years 2001–2003 and 2011–2013 documented effects of the 10 year-lasting national breeding programme. The total frequency of ARR doubled from 36.8 to 75.8 %, while the frequency of VRQ decreased from 4 to 0.7 %. The total frequency of the ARR/ARR genotype increased from 17.7 to 59 %. These data show that the national scrapie resistance breeding programme has had an important desirable effect on haplotype and genotype frequencies of PRNP in Czech sheep.     

Polymorphism of the prion protein gene (<Emphasis Type="Italic">PRNP</Emphasis>) in two Chinese indigenous cattle breeds

Prion protein (PRNP) gene has been located at position q17 of chromosome 13 in cattle. The polymorphisms of PRNP gene might be associated with BSE susceptibility. In the present work, we investigated the polymorphisms of PRNP gene, including SNP in exon 3, 23-bp indel in promoter region, 12-bp indel in intron 1 in 2 Chinese indigenous cattle breeds of northeast China. Eighty-six animals from Yanbian (34) and Chinese Red Steppes (52) were genotyped at PRNP locus by analyzing genomic DNA. A total of 4 single nucleotide polymorphism (SNP) sites were revealed in the PRNP gene exon 3 of the 2 cattle breeds investigated. Three of these SNPs were non-synonymous mutations that resulted in the amino acid exchanges (K119N, S154N, and M177V), and one is silent nucleotide substitutions (A234G). The two amino acid mutations of S154N and M177V were detected only in Yanbian with a very low frequency (0.0147), and they appears to be absent in Chinese Red Steppes. The average gene heterozygosity (H _e), effective allele numbers (N _e), Shannon’s information index (I) and polymorphism information content (PIC) were 0.3088, 1.5013, 0.3814 and 0.2000 in Yanbian, respectively, being relatively higher than that of Chinese Red Steppes (0.2885, 1.4985, 0.3462 and 0.1873, respectively). In 23-bp indel and 12-bp indel loci, three different genotypes were identified in both Yanbian and Chinese Red Steppes breeds. Based 23- and 12-bp indels, four haplotypes was constructed in the 2 Chinese cattle breeds, of which the 23-bp (−)/12-bp (−) was main haplotypes accounting for more than 50% of the total in both Yanbian and Chinese Red Steppes breeds. These results might be useful in understanding the genetic characteristics of PRNP gene in Chinese indigenous cattle breeds.     

Prion‐like Doppel gene polymorphisms and scrapie susceptibility in portuguese sheep breeds

The establishment of an association between prion protein gene (PRNP) polymorphisms and scrapie susceptibility in sheep has enabled the development of breeding programmes to increase scrapie resistance in the European Union. Intense selection for PRNP genotype may lead to correlated selection for genes linked to PRNP. We intended to investigate if any association exists between genetic variation in prion‐like protein Doppel gene (PRND) and scrapie susceptibility, determined through PRNP genotyping. Sampling included 460 sheep from eight Portuguese breeds and the PRND gene coding region was analysed by multiple restriction fragment‐single strand conformation polymorphism (MRF‐SSCP), whereas PRNP genotyping was carried out by primer extension. A synonymous substitution (c.78G>A) was detected in codon 26 of the PRND gene, in all breeds except Churra Mondegueira. Linkage disequilibrium was found between the PRND and PRNP loci (P = 0.000). Specifically, PRND was monomorphic in the 45 animals with the more resistant ARR/ARR PRNP genotype (P = 0.003), whereas a higher frequency of PRND heterozygotes (GA) was associated with ARQ/AHQ (P = 0.029). These results constitute preliminary evidence of an association between a polymorphism in the PRND gene and scrapie susceptibility, and indicate that the possibility of undesirable consequences from widespread selection for PRNP genotype on genetic diversity and reproduction traits needs to be further investigated.     

Lack of Prion Accumulation in Lymphoid Tissues of PRNP ARQ/ARR Sheep Intracranially Inoculated with the Agent of Scrapie

Sheep scrapie is a transmissible spongiform encephalopathy that can be transmitted horizontally. The prion protein gene (PRNP) profoundly influences the susceptibility of sheep to the scrapie agent and the tissue levels and distribution of PrP^Sc in affected sheep. The purpose of this study was to compare the survival time and PrP^Sc tissue distribution in sheep with highly resistant and highly susceptible PRNP genotypes after intracranial inoculation of the agent of scrapie. Five sheep each of genotype VRQ/VRQ, VRQ/ARR or ARQ/ARR were inoculated. Sheep were euthanized when clinical signs of scrapie became severe. Clinical signs, microscopic lesions, and western blot profiles were uniform across genotypes and consistent with manifestations of classical scrapie. Mean survival time differences were associated with the 171 polymorphic site with VRQ/VRQ sheep surviving 18 months, whereas VRQ/ARR and ARQ/ARR sheep survived 60 and 56 months, respectively. Labeling of PrP^Sc by immunohistochemistry revealed similar accumulations in central nervous system tissues regardless of host genotype. Immunoreactivity for PrP^Sc in lymphoid tissue was consistently abundant in VRQ/VRQ, present but confined to tonsil or retropharyngeal lymph node in 4/5 VRQ/ARR, and totally absent in ARQ/ARR sheep. The results of this study demonstrate the susceptibility of sheep with the ARQ/ARR genotype to scrapie by the intracranial inoculation route with PrP^Sc accumulation in CNS tissues, but prolonged incubation times and lack of PrP^Sc in lymphoid tissue.     

Polymorphism of the prion protein gene (<Emphasis Type="Italic">PRNP</Emphasis>) in Polish cattle affected by classical bovine spongiform encephalopathy

Recent attempts to discover genetic factors affecting cattle resistance/susceptibility to bovine spongiform encephalopathy (BSE) have led to the identification of two insertion/deletion (indel) polymorphisms, located within the promoter and intron 1 of the prion protein gene PRNP, showing a significant association with the occurrence of classical form of the disease. Because the effect of the polymorphisms was studied only in few populations, in this study we investigated whether previously described association of PRNP indel polymorphisms with BSE susceptibility in cattle is also present in Polish cattle population. We found a significant relation between the investigated PRNP indel polymorphisms (23 and 12 bp indels), and susceptibility of Polish Holstein-Friesian cattle to classical BSE (P < 0.05). The deletion variants of both polymorphisms were related to increased susceptibility, whereas insertion variants were protective against BSE.     

The PrP genotype of sheep of the improved Valachian breed

In a worldwide majority of sheep breeds an excessive susceptibility to scrapie associated with the PrP gene alleles coding for valine (V; at the 136 codon) and glutamine (Q; at the 171 codon) (e.g., VRQ/VRQ, VRQ/ARQ, or ARQ/ARQ) was demonstrated. Particularly the PrPVRQ allele is closely associated with the high-risk development of the disease; the PrPARQ allele can also fulfill this function but under certain limited conditions. Polymorphism in the PrP gene sequences (conclusively related to the increased susceptibility of sheep to scrapie) of improved Valachian sheep from two Slovak regions, Orava and Spis, was determined. Examination of 735 sheep showed that ARR/ARQ was the most frequent genotype (45.2%). High-risk genotypes were determined in 32.4% of sheep (ARQ/ARQ 19.3, ARR/VRQ 9.0, ARR/VRQ 3.5, VRQ/VRQ 0.3, ARR/VRR 0.3). Low-risk genotypes were found in 67.7% of sheep (ARR/ARQ 45.2, ARR/ARR 10.9, ARR/AHQ 5.7, ARQ/ARQ 4.9, AHQ/AHQ 0.7, ARR/AHR 0.3). Despite the geographically distant flocks of improved Valachian sheep investigated no difference in the occurrence of individual PrP genotypes was observed.     

Linkage disequilibrium across six prion gene regions spanning 20 kbp in U.S. sheep

Single nucleotide polymorphisms (SNPs) and haplotype alleles within the prion gene (PRNP) coding sequence of domestic sheep (Ovis aries) are associated with genetic predisposition to scrapie, a transmissible spongiform encephalopathy disease of sheep. This report describes regions of linkage disequilibrium (LD) throughout the PRNP gene region in U.S. sheep and provides a genetic framework for identifying additional PRNP determinants associated with scrapie resistance. Four sequence tagged sites (i.e., STS or amplicons) totaling 3869 bp and spanning 20 kbp of genomic PRNP sequence were sequenced in a diverse panel of 90 sires representing ten popular U.S. breeds of sheep. Analysis of these sequences identified 36 previously unreported polymorphisms. In combination with two previously characterized STS, 62 polymorphisms were analyzed in a 20-kbp PRNP region in this panel of U.S. sheep. Two regions of strong LD and ten common haplotypes were identified. The haplotype encoding amino acid residues A, R, and Q at codons 136, 154, and 171, respectively, was observed on nine larger haplotypes spanning PRNP from the promoter region to the 3′ untranslated region. The haplotype encoding VRQ was observed on two larger haplotypes, whereas ARR, ARH, and AHQ were each present on a single haplotype. The existence of multiple haplotypes encoding ARQ raises the question of whether sheep bearing these different haplotypes are equally susceptible to scrapie. The haplotype structure within the 20-kbp region of PRNP identified in this study is important for higher-resolution analysis of genetics contributions to scrapie susceptibility. Nucleotide sequence data reported are available in the DDBJ/EMBL/GenBank databases under the accession number DQ077504.     

Assessment of the genetic susceptibility of sheep to scrapie by protein misfolding cyclic amplification and comparison with experimental scrapie transmission studies

The susceptibility of sheep to scrapie is influenced mainly by the prion protein polymorphisms A136V, R154H, and Q171R/H. Here we analyzed the ability of protein misfolding cyclic amplification (PMCA) to model the genetic susceptibility of sheep to scrapie. For this purpose, we studied the efficiency of brain homogenates from sheep with different PrP genotypes to support PrP^Sc amplification by PMCA using an ARQ/ARQ scrapie inoculum. The results were then compared with those obtained in vivo using the same sheep breed, genotypes, and scrapie inoculum. Genotypes associated with susceptibility (ARQ/ARQ, ARQ/AHQ, and AHQ/ARH) were able to sustain PrP^Sc amplification in PMCA reactions, while genotypes associated with resistance to scrapie (ARQ/ARR and ARR/ARR) were unable to support the in vitro conversion. The incubation times of the experimental infection were then compared with the in vitro amplification factors. Linear regression analysis showed that the efficiency of in vitro PrP^Sc amplification of the different genotypes was indeed inversely proportional to their incubation times. Finally, the rare ARQK₁₇₆/ARQK₁₇₆ genotype, for which no in vivo data are available, was studied by PMCA. No amplification was obtained, suggesting ARQK₁₇₆/ARQK₁₇₆ as an additional genotype associated with resistance, at least to the isolate tested. Our results indicate a direct correlation between the ability of different PrP genotypes to undergo PrP^C-to-PrP^Sc conversion by PMCA and their in vivo susceptibility and point to PMCA as an alternative to transmission studies and a potential tool to test the susceptibility of numerous sheep PrP genotypes to a variety of prion sources.     

Deletion/insertion polymorphism of the prion protein gene (<Emphasis Type="Italic">PRNP</Emphasis>) in Polish Red cattle,Polish White-backed cattle and European bison (<Emphasis Type="Italic">Bison bonasus</Emphasis> L., 1758)

The aim of the present study was to identify deletion/insertion polymorphism of the bovine prion protein (PRNP) gene within the promoter sequence (23 bp indel), intron 1 (12 bp indel) and the 3′ end untranslated region (14 bp indel). The experiment was performed on three groups of animals protected under a genetic resources conservation program: 139 Polish Red (PR) cows, 79 Polish White-backed cows and 50 European bison (Bison bonasus L., 1758). White-backed cattle were characterized by a higher frequency of ins/del heterozygotes and a relatively lower frequency of ins/ins homozygotes within the promoter sequence region (23 bp indel), compared to Polish Red cattle. At the polymorphic locus of intron 1 (12 bp indel) the genetic structure of both cattle populations was similar. Monomorphism, expressed by the occurrence of one genotype variant in each of the analyzed sequence regions, was observed in European bison. Five haplotypes were found in Polish White-backed cows, four haplotypes in Polish Red cows and only one in analyzed group of bison. Differences between the observed and expected number of PRNP haplotypes were recorded in Polish Red cattle. The article is published in the original.     

新疆地区绵羊品种朊蛋白基因136、154和171位点的多态性

绵羊痒病是一种渐进性和致死性中枢神经系统疾病,绵羊朊蛋白基因(Prion protein gene,PRNP)多态性与痒病的易感或抗性有关,其中PRNP136位(V/A)、154位(H/R)和171位(H/Q/R)的基因多态性与该病发生最相关。为评价新疆地区主要绵羊品种对痒病的易感性,文章对新疆地区10个绵羊品种(阿勒泰、巴士拜、巴音布鲁克、多浪、和田、策勒黑、中国美利奴、德国肉用美利奴、特克赛尔和萨福克羊)共746只个体PRNP基因的136位(V/A)、154位(H/R)和171位(H/Q/R)的遗传多态性进行分析,检测到了ARQ、ARR、ARH、ARK、VRQ、AHR、AHQ、AHH8种等位基因,其中ARQ和ARR等位基因存在于所有品种中,且ARQ在所有品种中的基因频率最高。ARH存在于除萨福克和德国肉用美利奴羊外的8个绵羊品种中。仅在新疆地方品种阿勒泰、巴音布鲁克、巴士拜和多浪羊中检测到ARK等位基因。而VRQ、AHR、AHQ和AHH4种等位基因只在中国美利奴羊上存在,且频率极低。在10个品种中共检测到了ARQ/ARQ、ARQ/ARK、ARR/ARR、ARH/ARH、ARQ/ARR、ARH/ARQ、ARH/ARR、ARK/ARK、ARH/ARK、ARQ/VRQ、ARQ/AHQ、ARQ/AHR和ARH/AHH13种基因型,其中中度易感的ARQ/ARQ基因型频率最高,而抗性最强的ARR/ARR基因型仅存在于巴音布鲁克、策勒黑、中国美利奴、特克赛尔和德国肉用美利奴羊,且频率较低。文章首次在中国美利奴羊上发现了易感性很强的VRQ/ARQ基因型。上述结果提示新疆的主要绵羊品种对痒病的抗性较弱。     

Prion protein gene polymorphisms in sheep in the state of Paraná, Brazil

To determine the polymorphisms of the prion protein gene in sheep from the state of Paraná, Brazil, 323 animals of meat breeds (Suffolk, Hampshire Down, Texel, Ile de France, Dorper, Dorset, Santa Inês and crossbreds) were genotyped by restriction fragment length polymorphism (RFLP) analysis. The most frequent allele was ARQ, with a frequency of 0.61, followed by ARR (0.30). VRQ and AHQ alleles were present at very low frequencies (0.13 and 0.05 respectively), and the ARH allele was not found. Seven genotypes were identified (ARR/ARR, ARR/ARQ, ARQ/ARQ, ARR/VRQ, ARR/AHQ, ARQ/VRQ and ARQ/AHQ), of which ARQ/ARQ was the most frequent (0.41). The Santa Inês breed and crossbred animals showed the highest genotypic variability.     

Prion protein (PrP) gene polymorphism in Red Maasai and Black Head Persian sheep breeds in Tanzania: Consistent profile regardless of locations

The status of scrapie in Africa is largely unknown. The susceptibility to scrapie and its pathology is determined by amino acid polymorphisms at positions 136 (A/V), 154 (R/H) and 171 (Q/R/H) of the ovine PrP gene (PrP genotype) of the animals. Despite the widely studied PrP gene polymorphisms worldwide, limited data is available on PrP genotypes of sheep from the African continent. Previously, we have reported six PrP genotypes derived from four different alleles in Red Maasai and Black Herd Persian (BHP), the ARQ/ARQ genotype being more frequent in the Red Maasai than in the BHP sheep. The highly susceptible VRQ allele was not found in any of the sheep breeds; the ARR allele was absent in the Red Maasai or occurred at a low frequency in the BHP. The lack of the highly susceptible VRQ alleles among Tanzanian sheep examined, necessitated further examinations on genetic susceptibility in sheep of the same breeds, but originating from an entirely different part of Tanzania. Consistently, ARQ/ARQ genotype was observed in 88% of Red Maasai and 64% of BHP sheep, ARQ/AHQ genotype was present in 12% in Red Maasai and 36% in BHP. Neither the highly resistant (ARR/ARR), nor the highly susceptible (VRQ/VRQ) genotypes were found. The ARQ and AHQ were the only alleles observed. The ARQ allele constituted 94%, 82% and 67% in the Red Maasai, BHP and cross-bred sheep, respectively. The AHQ constituted 6%, 18% and 33%, respectively. Data reported here, provide additional information on genetic susceptibility of the Red Maasai, BHP and their crosses; they may be helpful in policy formulation for future prevention of scrapie.     

Detection of Insertions/Deletions Within <Emphasis Type="Italic">SIRT1</Emphasis>, <Emphasis Type="Italic">SIRT2</Emphasis> and <Emphasis Type="Italic">SIRT3</Emphasis> Genes and Their Associations with Body Measurement Traits in Cattle

Growth traits are complex quantitative traits controlled by numerous candidate genes, and they can be well-evaluated using body measurement traits. As the members of the nicotinamide adenine dinucleotide-dependent family of histone deacetylases, class I sirtuin genes (including SIRT1, SIRT2 and SIRT3) play crucial roles in regulating lipid metabolism, cellular growth and metabolism, suggesting that they are potential candidate genes affecting body measurement traits in animals. Hence, the objective of this work aimed to detect novel insertions/deletions (indels) of SIRT1, SIRT2 and SIRT3 genes in 955 cattle belonging to five breeds, as well as to evaluate their effects on body measurement traits. Herein, the novel 12-bp indel of SIRT1 gene, the 7-bp indel of SIRT2 gene and the 26-bp indel of SIRT3 gene were firstly reported, respectively. The association analysis indicated that the indels within SIRT1 and SIRT2 genes were significantly associated with body measurement traits such as body weight, chest circumference, height at hip cross, hip width, body height, etc. (P?<?0.05 or P?<?0.01). Therefore, based on these findings, the two novel indel variants within bovine SIRT1 and SIRT2 genes could be considered as potential molecular markers for growth traits in cattle selection practices and breeding.     

Review: A review on classical and atypical scrapie in caprine: Prion protein gene polymorphisms and their role in the disease

Scrapie is a naturally occurring transmissible spongiform encephalopathy in sheep and goat. It has been known for ~250 years and is characterised by the accumulation of an abnormal isoform of a host-encoded prion protein that leads to progressive neurodegeneration and death. Scrapie is recognised in two forms, classical and atypical scrapie. The susceptibility to both types of scrapie is influenced by polymorphisms of the prion protein gene (PRNP). Sheep susceptibility or resistance to classical scrapie is strongly regulated by the polymorphisms at codons 136, 154 and 171 of the PRNP. The genetic role in atypical scrapie in sheep has been defined by polymorphisms at codons 141, 154 and 171, which are associated with different degrees of risk in the occurrence of the ovine disease. Progress has been achieved in the prevention of scrapie in sheep due to efficient genetic breeding programmes based on eradication and control of the disease. In Europe, the success of these programmes has been verified by applying eradication and genetic selection plans. In general terms, the ovine selection plans aim to eliminate and reduce the susceptible allele and to enrich the resistant allele ARR. During outbreaks all susceptible animals are slaughtered, only ARR/ARR resistant rams and sheep and semi-resistant females are preserved. In the occurrence of scrapie positive goats a complete cull of the flock (stamping out) is performed with great economic loss and severe risk of extinction for the endangered breeds. The ability to select scrapie-resistant animals allows to define new breeding strategies aimed to boost genetic progress while reducing costs during scrapie outbreaks. Allelic variants of PRNP can be protective for caprine scrapie, and the knowledge of their distribution in goats has become very important. Over the past few years, the integration of genetic information on goat populations could be used to make selection decisions, commonly referred to as genetic selection. The objective of this review was to summarise the main findings of polymorphisms of the caprine prion protein (PrP) gene and to discuss the possible application of goat breeding schemes integrating genetic selection, with their relative advantages and limitations.     

Epidemiological investigation of <Emphasis Type="Italic">eaeA</Emphasis>-positive <Emphasis Type="Italic">Escherichia coli</Emphasis> and <Emphasis Type="Italic">Escherichia albertii</Emphasis> strains isolated from healthy wild birds

Escherichia coli has commonly been associated with diarrheal illness in humans and animals. Recently, E. albertii has been reported to be a potential pathogen of humans and animals and to be carried by wild birds. In the present study, the prevalence and genetic characteristics of intimin-producing E. coli and E. albertii strains were evaluated in wild birds in Korea. Thirty one of 790 Enterobacteriaceae strains from healthy wild birds were positive for the intimin gene (eaeA) and twenty two of the 31 strains were identified as atypical enteropathogenic E. coli (aEPEC) that did not possess both EAF and bfpA genes. A total of nine lactose non-fermenting coliform bacterial strains were identified as E. albertii by PCR and sequence analysis of housekeeping genes. A total of 28 (90.3%) eaeA-positive strains were isolated from waterfowl. Fifteen aEPEC (68.2%) and two E. albertii (22.2%) strains had a β-intimin subtype and 14 aEPEC strains harboring β-intimin belonged to phylogenetic group B2. AU eaeA-positive E. albertii and 3 aEPEC strains possessed the cytolethal distending toxin gene (cdtB). The eaeA-positive E. coli and E. albertii strains isolated from healthy wild birds need to be recognized as a potential pathogroup that may pose a potential threat to human and animal health. These findings indicate that eaeA-positive E. coli as well as E. albertii can be carried by wild birds, posing a potential threat to human and animal health.     

Polymorphisms in the <Emphasis Type="Italic">HSP90AA1</Emphasis> 5′ flanking region are associated with scrapie incubation period in sheep

Susceptibility to scrapie is mainly controlled by point mutations at the PRNP locus. However, additional quantitative trait loci (QTL) have been identified across the genome including a region in OAR18. The gene which encodes the inducible form of the cytoplasmic Hsp90 chaperone (HSP90AA1) maps within this region and seems to be associated with the resistance/susceptibility to scrapie in sheep. Here, we have analyzed several polymorphisms which were previously described in the ovine HSP90AA1 5′ flanking region and in intron 10 in two naturally scrapie infected Romanov sheep populations. First, we have studied 58 ARQ/VRQ animals pertaining to the sire family where the QTL influencing scrapie incubation period in OAR18 was detected. We have found a significant association between polymorphisms localized at −660 and −528 in the HSP90AA1 5′ flanking region and the scrapie incubation period. These two polymorphisms have also been studied in a second sample constituted by 62 VRQ/VRQ sheep showing an extreme incubation period. Results are concordant with the first dataset. Finally, we have studied the HSP90AA1 expression in scrapie and control animals (N = 41) with different HSP90AA1 genotypes by real time PCR on blood samples. The HSP90AA1 expression rate was equivalent in CC₋₆₀₀AA₋₅₂₈ and CG₋₆₀₀AG₋₅₂₈ scrapie resistant animals (ARR/ARR) and was higher in their CC₋₆₀₀AA₋₅₂₈ than in their CG₋₆₀₀AG₋₅₂₈ scrapie susceptible counterparts (VRQ/VRQ). Our results support the hypothesis that the ovine HSP90AA1 gene acts as a modulator of scrapie susceptibility, contributing to the observed differences in the incubation period of scrapie infected animals with the same PRNP genotype.     

The porcine <Emphasis Type="Italic">ANG</Emphasis>, <Emphasis Type="Italic">RNASE1</Emphasis> and <Emphasis Type="Italic">RNASE6</Emphasis> genes: molecular cloning,polymorphism detection and the association with haematological parameters

Angiogenin (ANG) [also known as ribonuclease, RNase A family, 5 (RNASE5)], ribonuclease, RNase A family, 1 (pancreatic) (RNASE1) and ribonuclease, RNase A family, k6 (RNASE6) are three members of the RNase A superfamily. It has been suggested that these three genes play important roles in host defense. In this study, we obtained the whole open reading frame (ORF) of each gene and found the deduced proteins contain some similar structures harboring a catalytic triad and an invariant “CKXXNTF” signature motif. One single nucleotide polymorphism (SNP) was detected in each gene (g. 149G>T polymorphism in the porcine ANG gene, which resulted in an amino acid change from glycine to valine, g. 296A>G polymorphism in the porcine RNASE1 gene and g. 389C>T polymorphism in the porcine RNASE6 gene). Association analyses revealed the significant associations (P < 0.05) between the porcine ANG g. 149G>T polymorphism and mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean platelet volume (MPV) and platelet-large cell ratio (P-LCR) measured on 0-day-old pigs and MCV measured at 32 days after birth. The porcine RNASE6 g. 389C>T polymorphism was significantly associated (P < 0.05) with MCV, MCH and neutrophil percentage (NEI %) measured on 0-day-old pigs, respectively. Our current findings, if confirmed by other studies, might shed some light on the roles of the investigated genes in host defense.     

Introgression molecular analysis of a leaf rust resistance gene from <Emphasis Type="Italic">Coffea liberica</Emphasis> into <Emphasis Type="Italic">C. arabica</Emphasis> L.

Leaf rust caused by the fungus Hemileia vastatrix is the most devastating disease of arabica coffee (Coffea arabica). Therefore, developing leaf rust-resistant varieties has been a breeding objective of the highest priority in many countries. The purpose of the present work was to gain insight into the mechanism of introgression into C. arabica of a leaf rust resistance gene from C. liberica (i.e. S_H3 resistance factor) and to identify associated molecular markers. An F₂ progeny (i.e. 101 individuals) derived from a cross between Matari, an arabica accession and liberica-introgressed line S.288, was evaluated for resistance against three different races of H. vastatrix. The progeny segregated for the S_H3 gene in a 3:1 ratio, as expected for a single dominant gene. Amplified fragment length polymorphism analysis of a population subset using 80 different primer combinations revealed that at least half of the total polymorphism observed in the population is associated with introgression of C. liberica chromosome fragments. Furthermore, 15 primer combinations generating candidate marker bands associated with the S_H3 resistance gene were used to analyse the whole F₂ population. A total of 34 marker bands originating from S.288 and attributable to introgression were scored. None exhibited segregation distortion. Linkage analysis revealed only three distinct introgressed fragments corresponding to a total length of 52.8 cM. Twenty-one markers were strongly associated (LOD score >14) with the S_H3 gene and were grouped together in a single linkage group of 6.3 cM. The results are discussed in relation to the efficient use of genetic resources in arabica breeding.     

<Emphasis Type="Italic">S</Emphasis> locus-linked F-box genes expressed in anthers of <Emphasis Type="Italic">Hordeum bulbosum</Emphasis>

Diploid Hordeum bulbosum (a wild relative of cultivated barley) exhibits a two-locus self-incompatibility (SI) system gametophytically controlled by the unlinked multiallelic loci S and Z. This unique SI system is observed in the grasses (Poaceae) including the tribe Triticeae. This paper describes the identification and characterization of two F-box genes cosegregating with the S locus in H. bulbosum, named Hordeum S locus-linked F-box 1 (HSLF1) and HSLF2, which were derived from an S ₃ haplotype-specific clone (HAS175) obtained by previous AMF (AFLP-based mRNA fingerprinting) analysis. Sequence analysis showed that both genes encode similar F-box proteins with a C-terminal leucine-rich repeat (LRR) domain, which are distinct from S locus (or S haplotype-specific) F-box protein (SLF/SFB), a class of F-box proteins identified as the pollen S determinant in S-RNase-based gametophytic SI systems. A number of homologous F-box genes with an LRR domain were found in the rice genome, although the functions of the gene family are unknown. One allele of the HSLF1 gene (HSLF1-S ₃) was expressed specifically in mature anthers, whereas no expression was detected from the other two alleles examined. Although the degree of sequence polymorphism among the three HSLF1 alleles was low, a frameshift mutation was found in one of the unexpressed alleles. The HSLF2 gene showed a low level of expression with no tissue specificity as well as little sequence polymorphism among the three alleles. The multiplicity of S locus-linked F-box genes is discussed in comparison with those found in the S-RNase-based SI system. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Nucleotide sequence data reported are available in the DDBJ/EMBL/GenBank databases under the accession numbers AB511822–AB511825 and AB511859–AB511862.