Identification of barley accessions from the VIR collection carrying the mlo11(cnv2) powdery mildew resistance allele

Background. The search for barley (Hordeum vulgare L.) genotypes that carry effective genes for resistance to powdery mildew agent Blumeria graminis f. sp. hordei is a present-day issue for Russian plant breeding. The mlo11 allele that confers long-term protection of barley against the pathogen is rarely found among the varieties, approved for cultivation in the territory of Russia. There is no information on the occurrence among Russian varieties of another effective allele, mlo11 (cnv2), therefore, the search for its source is a current necessity. Materials and methods. Seven barley accessions from Ethiopia and 7 accessions from Japan have been tested for resistance to the northwestern population of the powdery mildew agent in the field and in laboratory conditions. To identify of the Mlo gene alleles, nucleotide sequences of the Stowaway-MITE (Miniature Inverted-repeat Transposable Elements) and the adjacent promoter fragments were determined. Results. Phytopathological tests in the field and greenhouse conditions, as well as molecular markers were used to study 14 barley accessions from Ethiopia and Japan. According to the preliminary tests, plants were resistant to powdery mildew. The highly effective allele of powdery mildew resistance mlo11 (cnv2) was for the first time identified in four barley accessions from Ethiopia, k-20087, k-20523, k-20524 and k-28126. Under field conditions, adult plants were resistant, and in the greenhouse they were moderately damaged by powdery mildew (1-2 points). The disease symptoms were similar to those described for the sample Eth295, a carrier of the mlo11(cnv2) allele variant: single pustules and the absence of necrotic spots on the leaves. The fragments of Stowaway-MITE and adjacent Mlo 5' promoter sequences were amplified in all 14 accessions. The amplicons were cloned and sequenced. The unique marker SNPs within the MITE and Mlo 5’ promoter sequences, i.e. the substitutions of cytosine by thymine in positions 262 and 452, were found only in k-20087, k-20523, k-20524 and k-28126. These accessions belong to different botanical varieties and differ from each other in a number of morphological features, i.e. they are not duplicates. Conclusions. The genotypes selected as a result of the study can serve as a source of the mlo11(cnv2) allele in breeding powdery mildew-resistant barley varieties.

Molecular screening of bovine β-casein (CSN2) A2 variant in Indonesian Holstein dairy cattle as attempt to produce digestive friendly milk

Cow’s milk-intolerance is a digestive problem on people who not able to digest milk. This problem may relate to the variant of (3-casein (CSN2), especially A1, suggested due to (3-casomorphins (BCM-7) formation during enzymatic digestion, for that selecting cattle free BCM-7 become a concern to produce digestive friendly milk. This study aimed to differentiate A1 and A2 allele variant of CSN2 gene in selected population of Indonesian Holstein cattle. In total 70 cows DNA were collected, and fragment of CSN2 exon 7 which contain Single Nucleotide Polymorphism (SNP) rs43703011 and rs43703013 were amplified. Variant analysis was done by mutation site analysis using PCR-Restriction Fragment Length Polymorphism (PCR-RFLP) with Mspl restriction enzyme and DNA sequence for confirmation. Result shows 2 allele variants of (3-casein that are B type, representing A1 family variant, and A2 in mutation site rs43703013C>G. We found A2 allele in the studied population is superior in frequency than A1 (0.916 vs. 0.084). Of that, 8.6% cattle were heterozygotes that is BA2 and 91.4% were homozygotes A2A2. Cattle which carry A1 allele variant should be excluded from dairy cattle breeding program for further milk production free of BCM-7.

A Unique Allele Variant at STR Locus D2S1338 in a Paternity Testing Case

Background: The relationship testing through DNA profiling may undesirably be affected by the rare allele variants, tri-allelic pattern and null alleles. Therefore, it is vital to report such anomalies. We report a paternity testing in a sexual assault case studied at Punjab Forensic Science Agency, Lahore Pakistan showing a unique allele variant in mother and child. Methods: DNA was extracted from the buccal swabs of reference samples using organic extraction method and DNA profiling was done for 15 autosomal STRs and amelogenin using Identifiler Plus kit. Results: A novel out of marker range (OMR) allele variant between STR Loci D16S539 and D2S1338 was observed in the DNA profiles of victim (mother) as well as the child. At STR locus D2S1338 Twenty one different allele variant are listed at STRBase ranging from 11 to 28. The allele variant observed in this case study was appeared at less than marker range (< D2S1338) with a size of 297.50 bp. The novel variant OMR allele at D2S1338 was labeled as allele 13, when compared to the other allele in allelic ladder. Moreover, the PFSA DNA database was searched for this unique allelic variation and it was found that this was present in only two other samples of distinct cases. Conclusion: The overall frequency of this unique allele variant was 3 in 10,125 unrelated individuals with frequency of occurrence of 0.0296. According to our limited knowledge it is the first report of a novel OMR allele variant at D2S1338 in Pakistani Population.

POLYMORPHISM OF THE BETA-CASEIN GENE IN HOLSTEIN COWS

Представлены результаты исследований частоты встречаемости аллельных вариантов А1 и А2 гена бета-казеина (CSN2) у животных голштинской породы (n=510), принадлежащих пяти племенным хозяйствам Российской Федерации. Анализ ДНК проводился методом полимеразной цепной реакции с искусственно созданным сайтом рестрикции (ACRS-ПЦР). В среднем, по всему исследованному поголовью частота генотипов составила: A1A1—15% (n=78), A1A2—41 % (n=210), A2A2—44% (n=222). Частота желательного аллельного варианта А2 в среднем по всем стадам достигла 0,641 и превысила частоту аллеля А1 (0,359). Генотип А2А2 чаще встречается в группе племенных животных, импортированных из Дании. Им обладают 77% особей из 40 исследованных. Частота аллеля А2 в стаде исследуемого племенного хозяйства Курской области в 7 раз превысила частоту аллеля А1 и достигла 0,875. На 2-ом месте по частоте аллеля А2 находится маточное поголовье, завезенное из США. В племенном хозяйстве Московской области 42% животных из 354 исследованных имеют желательный генотип А2А2, частота аллеля А2 составила 0,640. В Камчатском крае отмечена сходная частота аллеля А2 (0,635) у племенных особей голштинской породы североамериканской селекции. В группе скота венгерской селекции, принадлежащей племенному хозяйству Рязанской области, частота аллеля А2 гена CSN2 ниже (0,611). В другом хозяйстве этого региона у голштинской породы канадского происхождения частота аллеля А1 (0,522) превысила частоту аллеля А2 (0,478). По исследованному поголовью оценки наблюдаемой (Ho) и ожидаемой (He) гетерозиготности имеют сходные значения и составляют 0,430 и 0,460 соответственно. The results of studies of the frequency of occurrence of allelic variants A1 and A2 of the beta-casein gene (CSN2) in Holstein animals (n=510) belonging to five breeding farms of the Russian Federation are presented. DNA analysis was performed by polymerase chain reaction with an artificially created restriction site (ACRS-PCR). On average, the frequency of genotypes for the entire studied population was: A1A1-15% (n=78), A1A2—41% (n=210), A2A2—44% (n=222). The frequency of the desired A2 allele variant reached 0.641 on average across all herds and exceeded the frequency of the A1 allele (0.359). The A2A2 genotype is more common in a group of breeding animals imported from Denmark. It is possessed by 77% of the 40 individuals studied. The frequency of the A2 allele in the herd of the studied breeding farm of the Kursk region was 7 times higher than the frequency of the A1 allele and reached 0.875. On the 2nd place in the frequency of the A2 allele is the breeding stock imported from the United States. In the breeding farm of the Moscow region, 42% of the 354 animals studied have the desired genotype A2A2, the frequency of the A2 allele was 0.640. In the Kamchatka Territory, a similar frequency of the A2 allele (0.635) was observed in cows of the Holstein breed of North American selection. In the group of Hungarian-bred cattle belonging to the Ryazan Region breeding farm, the frequency of the A2 allele of the CSN2 gene is lower (0.611). In another farm in this region, the Holstein breed of Canadian origin had the frequency of the A1 allele (0.522) higher than the frequency of the A2 allele (0.478). For the studied livestock, the estimates of observed (Ho) and expected (He) heterozygosity have similar values and are 0.430 and 0.460, respectively.

Multi locus sequence typing of clinical Burkholderia pseudomallei isolates from Malaysia

Background Melioidosis is a neglected tropical disease with rising global public health and clinical importance. Melioidosis is endemic in Southeast Asia and Northern Australia and is of increasing concern in Malaysia. Despite a number of reported studies from Malaysia, these reports are limited to certain parts of the country and do not provide a cohesive link between epidemiology of melioidosis cases and the nation-wide distribution of the causative agent Burkholderia pseudomallei. Methodology/principle findings Here we report on the distribution of B. pseudomallei sequence types (STs) in Malaysia and how the STs are related to STs globally. We obtained 84 culture-confirmed B. pseudomallei from confirmed septicaemic melioidosis patients from all over Malaysia. Prior to performing Multi Locus Sequence Typing, the isolates were subjected to antimicrobial susceptibility testing and detection of the YLF/BTFC genes and BimA allele. Up to 90.5% of the isolates were sensitive to all antimicrobials tested while resistance was observed for antimicrobials typically administered during the eradication stage of treatment. YLF gene cluster and bimABp allele variant were detected in all the isolates. The epidemiological distribution patterns of the Malaysian B. pseudomallei isolates were analysed in silico using phylogenetic tools and compared to Southeast Asian and world-wide isolates. Genotyping of the 84 Malaysian B. pseudomallei isolates revealed 29 different STs of which 6 (7.1%) were novel. ST50 was identified as the group founder followed by subgroup founders ST376, ST211 and ST84. A low-level diversity is noted for the B. pseudomallei isolates described in this study while phylogenetic analysis associated the Malaysian STs to Southeast Asian isolates especially isolates from Thailand. Further analysis also showed a strong association that implicates agriculture and domestication activities as high-risk routes of infection. Conclusions/significance In conclusion, MLST analysis of B. pseudomallei clinical isolates from all states in Malaysia revealed low diversity and a close association to Southeast Asian isolates.

27 The role of ligands of activatory receptor NKG2D in the immune-dependent pathogenesis and evolution of inflammatory bowel disease (IBD)

BackgroundLong-term inflammation in IBD is mediated by several immune cells, including T lymphocytes and natural killer (NK) cells, through the engagement of NK group 2D (NKG2D) receptors. Allelic variations of NKG2D ligands (NKG2Dls, MICA/B, ULBP1-3) influence differential levels and localization of protein expression or the release of soluble isoforms. The affinity of interaction with NKG2D can be also affected, modulating the cytotoxic activity of the target cell. Evaluation of these molecular pathways and soluble ligands presents the potential use a clinical biomarker for patient outcomes.MethodsGut tissue biopsies (left and right sides) and peripheral blood were collected from patients. 10 pediatric and 11 adult patients with IBD, 10 patients with gut malignancies and history of IBD were included in the study. Plasma form IBD patients and 10 healthy donors as controls, was used to quantify soluble NKG2DLs (sNKG2DLs) by ELISA (R&D Systems Duo Set). Nucleic acids were extracted from gut biopsies using the BioMasher II (Kimble) and All Prep DNA/RNA universal kit (Qiagen). Single nucleotide polymorphisms (SNPs, N=26) and relative gene expression of NKG2DL genes were conducted by qPCR using Taqman assays.Results9/11 adult patients had diagnosis of ulcerative colitis, compared to 3/10 pediatrics. 5/10 pediatrics had Crohn’s disease and 2/10 unclassified IBD. A trend of prevalence of some allelic variants was detected for most of NKGD2Ls.In addition, mRNA encoding for NKG2DLs was detected commonly, although with heterogeneous quantifications, in all the tissues, including the retrospectively collected malignancies with history of IBD. Interestingly, the levels of sNKG2DLs were higher in pediatric (p<0.001) as compared to adult patients. No or low levels of sNKG2DLs were detectable in healthy donors. Moreover 3/5 patients with the highest level (700–1500 pg/ml) of sMICA had homozygosity at least in one of the rs1051792 or rs1051794 polymorphic site (GG allele variant MICA-129Val or MICA-250Val) that have been reported to be associated with soluble form of MICA.ConclusionsThese results, although preliminary and further investigations are ongoing, suggest the relevance of NKG2D/NKG2DL pathway in the development and evolution of IBD. sNKG2DLs could be detected in most of patients, with different levels and highest concentrations in pediatric patients. In some cases, the presence of sNKG2DLs in the plasma could be associated with defined polymorphisms in genes encoding for these proteins.Ethics ApprovalThis study was approved by Sidra Medicine and Hamad Medical Corporation Ethics Boards; approval number 180402817 and MRC-02-18-096, respectively.

PERAN POLIMORFISME GEN MATRIKS METALLOPROTEINASE-9 DENGAN KEJADIAN PENYAKIT PARU OBSTRUKTIF KRONIK

Excessive secretion of MMP-9 is one of the cause of COPD is which can cause undesirable lung tissue degradation. Increased expression of MMP-9 is associated with increased gene promoter activity. The purpose of this study was to determine the role of mettaloproteinase-9 matrix gene polymorphisms with COPD events. This was a case-control study. With a total sample of 60 patients consisting of 30 COPD patients and 30 non-COPD patients. Samples were amplified by PCR (Polymerase Chain Reaction) The results of DNA amplification were digested with SphI restriction enzyme to see the allele variant of MMP-9 C-1562T. Visualization of restriction results with 2% agarose gel electrophoresis will show 2 bands on the T allele (188bp, 247bp), 1 band on the C allele (435bp). The results obtained were homozygous CC variants in COPD 20 (66.67%) and non-COPD 21 (70%). Heterozygous variant of CT 10 (33.33%) in COPD and 9 (30%) in non-COPD. TT variant is not found at all. Based on the results obtained by the most CC homozygous variants in COPD (66.67%) and non-COPD (70%) and no association was found between MMP-9 polymorphism and COPD events (p> 0.05). Based on the results of this study, there is no significant relationship that we can conclude.

ABO genetic variation in Neanderthals and Denisovans

1AbstractVariation at the ABO locus was one of the earliest sources of data in the study of human population identity and history, and to this day remains widely genotyped due to its importance in blood and tissue transfusions. Here, we look at ABO blood type variants in our archaic relatives: Neanderthals and Denisovans. Our goal is to understand the genetic landscape of the ABO gene in archaic humans, and how it relates to modern human ABO variation. We found two derived Neanderthal variants of the O allele in the Siberian Neanderthals (O1 and O2), one of these variants is shared with an European Neanderthal, who is a heterozygote for this O1 variant and a rare cis-AB variant. The Denisovan individual is heterozygous for two ancestral variant of the O1 allele, similar to variants found widely in modern humans. Perhaps more surprisingly, the O2 allele variant found in Siberian Neanderthals can be found at low frequencies in modern Europeans and Southeast Asians, and the O1 allele variant found in Siberian and European Neanderthal is also found at very low frequency in modern East Asians. Our genetic distance analyses suggest both alleles survive in modern humans due to inbreeding with Neanderthals. We find that the sequence backgrounds of the surviving Neanderthal-like O alleles in modern humans retain a higher sequence divergence than other surviving Neanderthal genome fragments, supporting a view of balancing selection operating in the Neanderthal ABO alleles by retaining highly diverse haplotypes compared to portions of the genome evolving neutrally.

The synergistic effect of bone mineral density and methylenetetrahydrofolate reductase (MTHFR) polymorphism (C677T) on fractures

A functional polymorphism in methylenetetrahydrofolatereductase (MTHFR) has been identifi ed at codon677 (C677T). The T-allele variant (valine type) has lowerenzyme activity than the wild type (C-allele or alaninetype), resulting in a slightly elevated homocysteine level,which has been recently recognized as a risk factor for fracture.However, whether subjects bearing the T allele havehigher susceptibility to fractures is still controversial. Wehave investigated the effects of MTHFR polymorphism onfracture susceptibility in Japanese postmenopausal women.A total of 502 postmenopausal ambulatory Japanese womenwere followed up for 5.1 ± 3.4 (mean ± SD) years, and atotal of 155 patients with incident fractures (121 patientswith vertebral fractures and 34 cases with fractures at othersites) were recorded. When compared with the patientswithout any fractures, the patients with incident fractureswere older, had more prevalent fractures, had higher urinarylevels of bone turnover markers as well as plasma homocysteinelevel, but were shorter in body height and had lowerbone mineral density. The prevalence of the TT genotypeof MTHFR was signifi cantly higher in the patients withincident fractures compared to the other genotypes. Thesubjects with the TT genotype had a higher incidence rateof fracture and higher plasma level of homocysteine thanthe subjects bearing the non-TT genotype. This relationshipwas observed in both osteoporotic and nonosteoporoticgroups. The hazard ratio for TT genotype without osteoporosis,non-TT genotype with osteoporosis, and TT genotypewith osteoporosis was 1.49 (0.91–2.45), 3.64 (2.50–5.29),and 7.21 (4.34–11.97), respectively, compared to the non-TTgenotype without osteoporosis. A higher hazard ratio forthe TT genotype with osteoporosis was still apparent afteradjustment for age, body size, and number of prevalentvertebral fractures. These results indicate that the TT genotypeof MTHFR may be a risk factor for future fracture inaddition to the traditional risk factors.

PARK16 rs708730 polymorphism decreases Parkinson’s disease risk in European ancestry population: a meta-analysis

Parkinson’s disease (PD) is a complex fatal chronic neurodegenerative disease most common in elderly people. The early genome-wide association studies (GWAS) found that the minor allele variant of PARK16 rs708730 polymorphism is a significant protective factor for PD in Caucasian populations. However, these results cannot be repeated by the following studies in Caucasian populations and other populations. We considered that the inconsistency of the findings may be caused by the small-scale samples or the heterogeneity among different populations. Therefore, in this study, we synthesized the previous related GWAS studies through three authoritative sources, and used the large-scale samples (10,645 PD cases and 30,499 controls) to reevaluate the association between rs708730 polymorphism and PD. The results showed that there is no association between them in Asian ancestry population. While, in European ancestry population, we found that the minor allele variant (G) of rs708730 polymorphism is significantly associated with a decreased risk of PD. Collectively, our findings further verified the association of rs708730 with PD and show its genetic heterogeneity among different populations, which can help to develop a better understanding of the PD’s pathogenesis.