SNPs In Exon Research Articles

Lactoferrin gene polymorphism of exons 8 and 13 in Murrah buffalo

Lactoferrin is one of the important candidate genes for mastitis resistance in dairy animals. The gene is located on chromosome BBU21 and consists of 17 exons spanning over 32.95 kb of genomic DNA. The present study was undertaken with the objectives to identify allelic variants in exon 8 and 13 of lactoferrin gene and to analyze their association with incidence of clinical mastitis in Murrah buffalo. A total of 200 lactating Murrah buffaloes, grouped as mastitis affected and non-affected, were included in the study. Genomic DNA was isolated from the whole blood sample of each animal. Two primer sets were used to amplify exon 8 and 13 of lactoferrin gene, which yielded respective amplicons of 216 bp and 211 bp. The PCR-RFLP analysis of lactoferrin gene revealed monomorphic pattern in exon 8 and polymorphic pattern in exon 13. Hpy 188I -RFLP for exon 13 exhibited polymorphism with three genotypes: AA, AB and BB with respective frequencies of 0.20, 0.58 and 0.22 whereas, frequencies for A and B alleles were estimated as 0.49 and 0.51. Comparison of nucleotide sequence of exonic region of lactoferrin gene in Murrah buffalo with that of Bos taurus cattle revealed a total of 5 mutations out of which 2 were transition and 3 were transversion. The SNPs in exon 8 were found to be non synonymous and revealed two amino acid changes in exon 8 of Murrah buffalo as compared to Bos taurus cattle. Chi-square (χ2) analysis indicated non significant association between genetic variants of exon 13 and incidence of clinical mastitis.

A novel locus (Bnsdt2) in a TFL1 homologue sustaining determinate growth in Brassica napus

BackgroundThe determinate growth habits is beneficial for plant architecture modification and the development of crops cultivars suited to mechanized production systems. Which play an important role in the genetic improvement of crops. In Brassica napus, a determinate inflorescence strain (4769) has been discovered among doubled haploid (DH) lines obtained from a spring B. napus × winter B. napus cross, but there are few reports on it. We fine mapped a determinate inflorescence locus, and evaluated the effect of the determinate growth habit on agronomic traits.ResultsIn this study, we assessed the effect of the determinate growth habit on agronomic traits. The results showed that determinacy is beneficial for reducing plant height and flowering time, advancing maturity, enhancing lodging resistance, increasing plant branches and maintaining productivity. Genetic analysis in the determinate (4769) and indeterminate (2982) genotypes revealed that two independently inherited recessive genes (Bnsdt1, Bnsdt2) are responsible for this determinate growth trait. Bnsdt2 was subsequently mapped in BC2 and BC3 populations derived from the combination 2982 × 4769. Bnsdt2 could be delimited to an approximately 122.9 kb region between 68,586.2 kb and 68,709.1 kb on C09. BLAST analysis of these candidate intervals showed that chrC09g006434 (BnaC09.TFL1) is homologous to TFL1 of A. thaliana. Sequence analysis of two alleles identified two non-synonymous SNPs (T136C, G141C) in the first exon of BnaC09.TFL1, resulting in two amino acid substitutions (Phe46Leu, Leu47Phe). Subsequently, qRT-PCR revealed that BnaC09.TFL1 expression in shoot apexes was significantly higher in NIL-4769 than in 4769, suggesting its essential role in sustaining the indeterminate growth habit.ConclusionsIn this study, the novel locus Bnsdt2, a recessive genes for determinate inflorescence in B. napus, was fine-mapped to a 68,586.2 kb - 68,709.1 kb interval on C09. The annotated genes chrC09g006434 (BnaC09.TFL1) that may be responsible for inflorescence traits were found.

Wx alleles in rice: relationship with apparent amylose content of starch and a possible role in rice domestication

The Waxy locus of rice is a highly polymorphic region embedded with microsatellite repeats in the 5'UTR leader intron 1 region, 23-bp duplication (wx motif) in exon 2, SNPs in exons 4, 6 and 10, p-Sine-r2 element in intron 1 and TnR-1 element in inton 13. Of the 80 polymorphic sites detected on the Wx gene, 24 are located in p-Sine-r2 and TnR-1 elements, revealing a higher substitution rate of bases in these two regions. All the cultivars with chalky endosperm had the 5'-AGTTATA-3' haplotype in intron 1 and 'A' to 'G' substitution at ?497 in exon 4. The AAC of starch from grains of all the accessions showed strong correlation (r=0.967) with GBSS-I activity in the grains. Based on the polymorphic sites of the Waxy locus and the GBSS-I activities, six allelic variants were defined which included wx, Wxop, Wxb, Wxin, Wxa2 and Wxa1, respectively, corresponded to glutinous, very low, low, intermediate, highII and highI amylose classes. Phylogenetic tree developed from alignment matrix of nucleotide sequences of the Waxy locus identified wx, Wxb and Wxin alleles with japonica lineage of Oryza sativa and the Wxop, Wxa2 and Wxa1 with indica lineage.

Polymorphisms in Genes Involved in Steroidogenesis in the Development of Severe Acne

Background: Acne is a multifactorial disease, in the pathogenesis of which one of the leading factors is the excessive effect of androgens on the hair follicles (HFs) and sebaceous glands (SGs), along with hypersecretion of sebum, pathological follicular hyperkeratosis and an inflammatory response. The search for genotypic markers in patients with varying severity of acne is a difficult task due to the multifactorial pathogenesis and the role of trigger factors in the formation of acne. The aim of this study was to determine SNPs within 3 genes involved in steroidogenesis (MVK, ARPC1B, and CA2) in patients with severe acne. Methods and Results: The study included 70 patients (42 men and 28 women) aged between 15 and 46 years (the median age - 22.1 years). The main group (MG) included 50 patients (29 men and 21 women) with severe acne. The control group (CG) consisted of 20 apparently healthy individuals (13 men and 7 women). Molecular-genetic diagnostics was carried out by the method of high-throughput DNA sequencing (next-generation sequencing). Our study showed that severe acne is associated with 12 polymorphic loci of the MVK gene (4 SNPs in exons and 8 SNPs in introns), 7 SNPs of the ARPC1B gene (2 SNPs in exons and 5 SNPs in introns), and 9 SNPs of the CA2 gene (3 SNPs in exons and 6 SNPs in introns). Conclusion: The revealed features of the SNPs within the MVK, ARPC1B, and CA2 genes in patients with severe acne probably indicate a hereditary determination of steroidogenesis in acne.

Identification of single nucleotide polymorphism c.957A>C of PLAG1 gene and its association with growth traits in Bali cattle

The PLAG1 gene is one of the genes that affect the growth traits located on chromosome 14 in cattle. This study aims to obtain SNP of the PLAG1 gene in exon 1 and exon 2 and their association with growth traits in Bali cattle. The number of samples used was 52 samples of Bali cattle, 10 samples of Peranakan Ongole (PO), and 8 samples of Limousine cattle. Identification of SNPs PLAG1 gene was analyzed by direct sequencing method and genotyping of selected SNPs was carried out using PCR-RFLP. Association of genotypes of SNP c.957A>C with growth using t-test. There were 7 SNPs in exon 2 of the PLAG1 gene, namely SNP c.339A>G, c.489C>T, c.795A>G, c.957A>C, c.1023C>T, c.1056A>G, and c.1353A>G. SNP c.957A>C was validated by PCR-RFLP using TaqI enzyme and obtained three genotypes, namely genotypes AA, AC, and CC with allele frequency A and C, respec-tively 0.10 and 0.90 in Bali cattle, while in PO and Limousine cattle were monomorphic. Genotype association of SNP c.957A>C PLAG1 gene were not associated with birth weight (BW0), weaning weight at 205 days of age (WW205), yearling weight at 365 days of age (YW365), yearling weight at 730 days of age (YW730), and average daily gain (ADG). SNP c.957A>C as a specific SNP for Bali cattle needs to be investigated in further research as a candidate marker for growth traits in Bali cattle.

Genetic, epigenetic, and environmental factors controlling oxytocin receptor gene expression

BackgroundThe neuropeptide oxytocin regulates mammalian social behavior. Disruptions in oxytocin signaling are a feature of many psychopathologies. One commonly studied biomarker for oxytocin involvement in psychiatric diseases is DNA methylation at the oxytocin receptor gene (OXTR). Such studies focus on DNA methylation in two regions of OXTR, exon 3 and a region termed MT2 which overlaps exon 1 and intron 1. However, the relative contribution of exon 3 and MT2 in regulating OXTR gene expression in the brain is currently unknown.ResultsHere, we use the prairie vole as a translational animal model to investigate genetic, epigenetic, and environmental factors affecting Oxtr gene expression in a region of the brain that has been shown to drive Oxtr related behavior in the vole, the nucleus accumbens. We show that the genetic structure of Oxtr in prairie voles resembles human OXTR. We then studied the effects of early life experience on DNA methylation in two regions of a CpG island surrounding the Oxtr promoter: MT2 and exon 3. We show that early nurture in the form of parental care results in DNA hypomethylation of Oxtr in both MT2 and exon 3, but only DNA methylation in MT2 is associated with Oxtr gene expression. Network analyses indicate that CpG sites in the 3′ portion of MT2 are most highly associated with Oxtr gene expression. We also identify two novel SNPs in exon 3 of Oxtr in prairie voles and a novel alternative transcript originating from the third intron of the gene. Expression of the novel alternative transcript is associated with genotype at SNP KLW2.ConclusionsThese results identify putative regulatory features of Oxtr in prairie voles which inform future studies examining OXTR in human social behaviors and disorders. These studies indicate that in prairie voles, DNA methylation in MT2, particularly in the 3′ portion, is more predictive of Oxtr gene expression than DNA methylation in exon 3. Similarly, in human temporal cortex, we find that DNA methylation in the 3′ portion of MT2 is associated with OXTR expression. Together, these results suggest that among the CpG sites studied, DNA methylation of MT2 may be the most reliable indicator of OXTR gene expression. We also identify novel features of prairie vole Oxtr, including SNPs and an alternative transcript, which further develop the prairie vole as a translational model for studies of OXTR.

Association analysis of novel SNPs in exon 10 of the growth hormone receptor gene with growth traits in Indian sheep

The growth hormone receptor (GHR) gene encodes the type I cytokine receptor that helps in joining the growth hormone to this receptor, thus promoting receptor-dimerization, leading to up-regulating growth. The ovine GHR gene located on chromosome 16, which consists of 10 exons and 9 introns, along with untranslated regions on either side, comprise a total size of 178.09 kb. However, earlier reports about polymorphism have mainly dealt with exon 10 which is also a larger fragment of this gene comprising 1102 bp. Hence, this study was carried out to detect polymorphism in exon 10 of the GHR gene and its association with growth traits. Genomic DNA was isolated from blood samples of Madras Red and Mecheri sheep breeds from India. Part of exon 10 (895 bp) of the GHR gene was amplified and sent for sequencing. The sequence analysis revealed transition of nucleotide G>A at loci G177624A and G177878A in both sheep breeds. Populations were screened by Tetra-primer ARMS-PCR. The genotype frequencies of GG, GA and AA were 0.276, 0.519 and 0.205 at 177624 G>A, and 0.307, 0.444 and 0.149 at 177878 G>A in Madras Red sheep; whereas in Mecheri they were 0.476, 0.372 and 0.152 at 177624 G>A, and 0.629, 0.314 and 0.057 at 177878 G>A, respectively. Likewise the estimated allele frequencies of G and A were 0.5355 and 0.4645 at 177624 G>A, and 0.5790 and 0.4210 at 177878 G>A in Madras Red sheep; whereas in Mecheri they were 0.6620 and 0.3380 at 177624 G>A, and 0.7860 and 0.2140 at 177878 G>A, respectively. The effect of sex was significant for birth, six and nine month weight; but non-significant for three and 12 month weight in Mecheri sheep. However, in the Madras Red breed the effect of sex was significant for all body weights except weaning weight. The effect of variations on growth traits, viz., birth weight, weight at weaning, and weight at six, nine and twelve months in both breeds were analysed for their association, and they were found non-significant. Since these SNPs are salient findings of GHR gene polymorphism in Indian sheep breeds, further investigation is required into the significant effects of these novel SNPs, which could be useful for genetic improvement based on marker assisted selection.

Novel Mutation of Exon 5 Prolactin Gene in IPB-D1 Chicken

<p class="JITV-ReferenceIsi">The prolactin gene (PRL) is a gene that controls the incubation and egg production in laying chickens. The nature of incubation will reduce egg production and disrupt the reproductive system in local chickens. The purpose of this study was to identify the polymorphism of prolactin genes in IPB-D1 chickens using the direct sequencing method. The polymorphism of the exon 5 prolactin gene was carried out on 46 samples of IPB-D1 chicken DNA which was a collection of the Division of Animal Genetics and Breeding, Faculty of Animal Science IPB. DNA sequences as a reference for designing exon 5 primers were obtained from the National Center for Biotechnology Information (NCBI) with the GenBank access code: AF288765.2. DNA extraction was carried out using the phenol-chloroform technique. DNA amplification resulted in a PCR product with a size of 557 bp. In this study, the genotype frequency, allele frequency, heterozygosity value and Hardy-Weinberg equilibrium were calculated. The results of the study found 5 SNPs in exon 5, namely g.7823A>G, g.7835A>G, g.7886T>A, g.8052T>C, and g.8069T>C. All SNPs are polymorphic and in Hardy-Weinberg equilibrium except g.8052T>C. The g.7823A>G, g.7835A>G, g.8052T>C SNPs are synonymous mutations that do not change amino acids, while the g.7886T>A and g.8069T>C SNPs are non-synonymous that change amino acids. Both g.7886T>A and g.8069T>C SNPs are potential as a marker assisted selection for the characteristics of egg production in IPB-D1 chickens.</p>

Novel alterations of CC2D1A as a candidate gene in a Turkish sample of patients with autism spectrum disorder

Background Autism spectrum disorder (ASD) is a neurodevelopmental disorder with large genetic background, but identification of pathogenic variants has proceeded slowly because hundreds of loci are involved in this complex disorder. CC2D1A gene firstly associated with the intellectual disability (ID) in a family with a large deletion. We aimed to contribute to the literature by sequencing this gene and by this way we report novel CC2D1A variations in patients with ASD. Methods Forty families who have a child with a diagnosis of ASD were enrolled to the study. DNA samples were obtained from each family member. Bidirectional CC2D1A gene sequencing was performed with CEQ Cycle Sequencing Kit, and the products were analyzed on the Beckman CEQ 8000. All of the genetic analysis was conducted in Erciyes University Genome and Stem Cell Center (GENKOK). Results According to the sequencing results, we defined new alterations in this gene with two SNPs in exon 15 and 19 (rs747172992 and rs1364074600) in our patients. We found a pathogenic variant in one patient. This variant was located in the acceptor region. Six of the variants were missense mutations. Additionally, six different benign variants were detected in 30 patients; however, they were not associated with ASD. Two patients carried multiple rare variants. Conclusion In vitro and in vivo functional analysis with this gene will help to understand its contribution to ASD pathogenesis. Future studies may help to elucidate the underlying biological mechanisms of these variants leading to the autism phenotype.

DNA sequence analysis and Jk blood group genotype-phenotype assessment.

BackgroundThe Kidd (JK) blood group is critical for clinical blood transfusion. Various methods for Jk typing have been commonly used, including urea hemolysis, serological test, and genotyping. However, the application of molecular methods has so far been restricted to selected samples and not been applied to the population-scale analysis.MethodsOne hundred eighty-three blood samples, containing 174 samples collected from voluntary blood donors of Chinese Han individuals, together with 3 Jk (aw+b-) and 6 Jk (a-b-) samples, were investigated by standard serology urea hemolysis test and Sanger-sequencing. Complete coverage of exons 4-11 and intron-exon borders have been sequenced.ResultsWe report the frequencies of three SNPs in exon 4, 7, and intron 9. Besides, sequence analysis revealed the simultaneous DNA variants of intron 7 (-68) and exon 9 (838) found in all samples, suggesting the co-inheritance of these SNPs—taking the observed SNPs frequencies into account. Further, we discuss the potential of the sequencing technique for high-resolution genotyping.ConclusionsThe described sequencing method for Jk exons delivers a genotyping technique for Jk molecular characterization. According to the co-inheritance of these DNA variants in intron 7 (-68) and exon 9 (838), and their regularity linkage with Jk phenotypes, these two sites offer a potential sequencing target for rapid and far more simplified Jk typing that can supplement routine serology and urea hemolysis tests.

Intronic TP53 Polymorphisms Are Associated with Increased Δ133TP53 Transcript, Immune Infiltration and Cancer Risk.

We investigated the influence of selected TP53 SNPs in exon 4 and intron 4 on cancer risk, clinicopathological features and expression of TP53 isoforms. The intron 4 SNPs were significantly over-represented in cohorts of mixed cancers compared to three ethnically matched controls, suggesting they confer increased cancer risk. Further analysis showed that heterozygosity at rs1042522(GC) and either of the two intronic SNPs rs9895829(TC) and rs2909430(AG) confer a 2.34-5.35-fold greater risk of developing cancer. These SNP combinations were found to be associated with shorter patient survival for glioblastoma and prostate cancer. Additionally, these SNPs were associated with tumor-promoting inflammation as evidenced by high levels of infiltrating immune cells and expression of the Δ133TP53 and TP53β transcripts. We propose that these SNP combinations allow increased expression of the Δ133p53 isoforms to promote the recruitment of immune cells that create an immunosuppressive environment leading to cancer progression.

A Potential Role of Bone Morphogenetic Protein 7 in Shell Formation and Growth in the Razor Clam Sinonovacula constricta.

Bone morphogenetic proteins (BMPs) not only play essential roles in bone development but also are involved in embryonic growth, organogenesis cell proliferation and differentiation. However, the previous studies on the functions of shellfish BMPs genes are still very limited. To better understand its molecular structure and biological function, BMP7 of the razor clam Sinonovacula constricta (Sc-BMP7) was cloned and characterized in this study. The full length of Sc-BMP7 is 2252 bp, including an open reading frame (ORF) of 1257 bp encoding 418 amino acids. The protein sequence included a signal peptide (1–32 aa), a prodomain (38–270 aa) and a TGF-β domain (317–418 aa). The quantitative expression of eleven adult tissues showed that Sc-BMP7 was significantly higher expressed in the gill, foot, and mantle (P < 0.05), but lower in hemocytes and hepatopancreas. In the early development stages, low expression was detected in the stages of unfertilized mature eggs, fertilized eggs, 4-cell embryos, blastula, gastrulae, whereas it increased after the stage of trochophore and demonstrated the highest expression in umbo larvae (P < 0.01). In shell repair experiment, Sc-BMP7 showed increasing expression level after 12 h. The higher expression of Sc-BMP7 was detected while Ca2+ concentration was reduced in seawater. After inhibiting Sc-BMP7 expression using RNA interference (RNAi) technology, expression of Sc-BMP7 mRNA and protein were significantly down-regulated (P < 0.05) in the central zone of mantle (nacre formation related tissue) and the pallial zone of mantle (prismatic layer formation related tissue). Association analysis identified two shared SNPs in exon of Sc-BMP7 gene from 246 individuals of two groups. These results indicated that BMP7 might be involved in shell formation and growth. These results would contribute to clarify the role of Sc-BMP7 in the regulation of growth and shell formation, and provide growth-related markers for molecular marker assisted breeding of this species.

Polymorphism of CD1B Gene and Its Association with Yolk Immunoglobulin (IgY) Concentration and Newcastle Disease Antibody Titer in IPB-D1 Chicken

The CD1B gene has an important role in the immune system of poultry by mediating antibody induction. The study aimed to identify the CD1B gene polymorphism and its association with the concentration of IgY and ND antibody titers in IPB-D1 chicken. As many as 111 of IPB-D1 chickens at 21 weeks old were used in this study. Polymorphism identification of the CD1B gene was made using the PCR Sequencing method, while the IgY and ND antibody titers were done using the ELISA and HI test, respectively. The associations of gene polymorphism with IgY and ND antibody titers were analyzed using the General Linear Model (GLM) procedure and Duncan`s Multiple Range test. The results show that there are 4 SNPs in exon 3, i.e., c.550 G>A, c.562 T>A, c.588 A>G, and c.612 C>G. All the SNPs are missense, silent mutations, and polymorphic. The c.550 G>A and c.562 T>A SNPs were in Hardy Weinberg’s equilibrium and heterozygosity (0.054-0.252) condition, while the c.588 A>G and c.612 C>G SNPs were not in equilibrium and their heterozygosity was low (0.072-0.252). The combination of 4 SNPs generated 8 haplotypes, i.e., haplotypes 1, 2, 3, 4, 5, 6, 7, and 8. Haplotypes 1, 2, and 8 had high frequencies (17.6%-23.5%). The c.588 A>G and c.612 C>G mutations were significantly associated (p A were significantly associated (p G and c.612 C>G mutations had higher IgY concentration and ND antibody titers (p<0.05) compared to the other haplotypes. In conclusion, this study has identified the CD1B gene as a polymorphic and is associated with IgY concentration and ND antibody titers in IPB-D1 chicken.

Candidate gene mapping reveals VrMLO12 (MLO Clade II) is associated with powdery mildew resistance in mungbean (Vigna radiata [L.] Wilczek)

Loss/reduction of function of Mildew Locus O (MLO) genes clade V and MLO clade IV has been shown to be responsible for powdery mildew (PM) resistance in several plant species. Mungbean (Vigna radiata) genome possesses 18 MLO genes, VrMLO1 - VrMLO18. A previous study using mungbean F2 and BC1F1 populations derived from a cross between “CN60″ (susceptible) and “RUM5″ (resistance) demonstrated that QTL qPMRUM5−3 is a major QTL for PM resistance caused by Erysiphe polygoni and is the same with major QTL qPMV4718−3 that confers PM resistance in “V4718″ (resistance). In this study, bioinformatics analysis revealed VrMLO12 locates in the qPMRUM5−3 region. Fine mapping in the F2 and BC1F1 populations using newly developed DNA markers including gene-specific markers demonstrated association between VrMLO12 and the PM resistance. Sequence analyses of VrMLO12 revealed that compared to susceptible mungbeans, RUM5 and V4718 possess SNPs in exon 10 and exon 13. The SNPs caused amino acid changes of VrMLO12, A387S and A476 G, respectively. The change occurred in transmembrane 6 domain and calmodulin binding domain (CaMBD) of the VrMLO12 protein, respectively. qRT-PCR showed that transcript expression level of VrMLO12 in RUM5 challenged with and without by E. polygoni was significantly higher than that in CN60. Phylogenetic analysis revealed that in contrast to previous findings that MLO proteins associated with PM resistance belong to MLO clade V and MLO clade IV, VrMLO12 belongs to MLO clade II. The result suggested that VrMLO12 may function differently from the other MLOs that associated with PM susceptibility. Our findings provide insight into the PM resistance in mungbean and tools for mungbean breeding.

NLRP3 Inflammasome Gene Polymorphisms Variably Associated with its Serum Levels in Acute Myocardial Infarction

Nucleotide-binding domain, leucine-rich-containing family, pyrin domain-containing-3 (NLRP3) plays a pivotal role in initiation of inflammation. genetic variation in NLRP3 gene have been proposed to predispose several inflammatory diseases. This study aimed to evaluate the risk of NLRP3 (exon 3) gene polymorphisms and its relation with serum NLRP3 among myocardial infarction. Case-control study involved 69 patients with Myocardial Infarction and 53 controls, from each subject 3 mL were collected and used for DNA extraction then the amplified exon 3 genes were sequenced by Sanger method. Serum NLRP3 was quantified using sandwich ELISA. According to the results Q705K found to possess a 16.21 times risk for MI incidence compared with controls. In addition, 44 novel single nucleotide polymorphisms have been identified at the position 14347, 14261, 14240 and 14229 and their allelic variants as a risk factor for MI incidence as 3.92, 8.6, 2.04 and 4.57 when compared with their relevant allele in controls respectively. Statistically high level of serum NLRP3 (1.7 ng mL-1) among MI patients compared to controls (0.71 ng mL-1). The 0.75 ng mL-1 considered as a good predictor for MI with ECG findings. Only Q705K and 14229 genetic variant alleles were significantly associated with high NLRP3 protein serum level among MI patients. Four novel SNPs in exon 3 of NLRP3 gene in addition to previously reported Q705K conferring risk for development of MI among Iraqis. Only variants allele of Q705K and gene position 14229 was associated with elevated serum NLRP3 protein among MI patients.

Mutation Identification in the Complete Myostatin Sequence in Indonesian Kampung Chicken

Kampung chicken, an Indonesian native chicken, has a potential for meat producers to improve national food security, as well as supplying highly favored meat to Indonesian consumers. To improve the meat production of Kampung chicken, the selection at the genomic level could be a promising approach. Myostatin is well known as a negative regulator of skeletal muscle growth. The objective of this study was to identify DNA mutation in the complete sequence of the myostatin gene in Kampung chicken. Genotyping was performed in 6 Kampung chickens by PCR and direct sequencing using 24 primer pairs covering 8.3 Kb of complete myostatin sequence. A total of 95 mutations discovered in the myostatin gene, including 24 in promoter, in 5’UTR, 5 in exon 1, 19 in intron 1, 37 in intron 2, 1 in exon 3, and 8 in 3’UTR. Among all mutations, 93 mutations were classified as point mutations, and 2 mutations were classified as indels. A total of 88 of 95 mutations (92%) were novel mutations. Six point mutations were found in the coding region, including 5 SNPs in exon 1, 1 SNP in exon 3, and no mutations in exon 2. No amino acid changes within all SNPs in coding region. Further studies in a larger population are needed to confirm this potential and novel mutations and their association with growth and meat production of Kampung chicken.

Fine mapping of the qHTB1-1QTL, which confers heat tolerance at the booting stage, using an Oryza rufipogon Griff. introgression line.

The qHTB1-1 QTL, controlling heat tolerance at the booting stage in rice, was fine mapped to a 47.1kb region containing eight candidate genes. Two positional candidate genes showed significant changes in expression levels under heat stress. High-temperature stress at the booting stage has the potential to significantly limit rice production. An interspecific advanced backcrossed population between the Oryza sativa L. cultivar R53 and the wild Oryza rufipogon Griff accession HHT4 was used as the source material to develop a set of chromosome segment introgression lines to elucidate the genetic mechanism of the qHTB1-1 QTL in heat tolerance. A single-chromosome-segment introgression line, IL01-15, was used to develop secondary populations for the mapping of qHTB1-1 on chromosome 1 for heat tolerance at the booting stage. Using the BC5F2, BC5F3, and BC5F4 populations, we first confirmed qHTB1-1 and validated the phenotypic effect. The qHTB1-1 QTL explained 13.1%, 16.9%, and 17.8% of the phenotypic variance observed in the BC5F2, BC5F3, and BC5F4 generations, respectively. Using homozygous recombinants screened from larger BC6F2 and BC6F3 populations, qHTB1-1 was fine mapped within a 47.1kb region between markers RM11633 and RM11642. Eight putative predicted genes were annotated in the region, and six genes were predicted to encode expressed proteins. The expression patterns of these six genes demonstrated that LOC_Os01g53160 and LOC_Os01g53220 were highly induced by heat stress in IL01-15 compared to R53. Sequence comparison of the gene-coding regions of LOC_Os01g53160 and LOC_Os01g53220 between R53 and IL01-15 revealed one synonymous and two nonsynonymous SNPs in exons, respectively. Our results provide a basis for identifying the genes underlying qHTB1-1 and indicate that markers linked to the qHTB1-1 locus can be used to improve the heat tolerance of rice at the booting stage by marker-assisted selection.

59 Genome activation in intracytoplasmic sperm injection-derived horse embryos

During pre-implantation development, embryos go through a critical period of embryonic genome activation (EGA). The timing of EGA is species specific, but little is known in horse embryos. Here, we aimed to characterise EGA in equine embryos produced by intracytoplasmic sperm injection. Embryos were produced by intracytoplasmic sperm injection of oocytes from 3 mares. Two embryos from each mare at each of 8 developmental stages (MII, zygote, 2-cell, 4-cell, 8-cell, 16-cell, morula, and blastocyst) were individually analysed by RNA-seq. Differential expression was evaluated using binomial Wald tests with an absolute logFC (fold change) threshold of 1 in the DESEqn 2R package. We found that EGA occurred in a two-step fashion. Minor EGA took place during the 2-cell to 4-cell transition, and featured up-regulation of 751 genes and discrete down-regulation of 60 genes in 4-cell embryos compared with 2-cell embryos. Differentially upregulated genes were enriched in gene ontology terms related to transcriptional activator activity, homeobox domains, and nucleosome assembly. Major EGA occurred during the 4-cell to 8-cell transition and included the largest number of differentially expressed genes (n=2,023) between consecutive stages. This period also featured the first massive transcript downregulation (n=816). Upregulated genes were enriched in gene ontology terms related to ribosomal assembly, translation, and RNA modification. Additionally, we observed that the number of intronic sequences was significantly higher from the 4-cell stage onward, indicating active transcription in comparison to oocytes, zygotes, and 2-cell embryos. To evaluate the timing of paternal genome activation, we used whole-genome sequencing data from the parents (average genome coverage of 19×) to quantify allele-specific expression. The average number of informative SNPs in exons, i.e. SNPs with alternative homozygous genotypes from the sire (AA mare - BB sire), was 26 128 per mare, corresponding to 7696 genes. Parental-specific transcript abundance was determined for each embryo, with an average of 1,911±865 informative SNPs detected per sample. Paternal alleles were considered expressed when they reached 10% of the maternal count. Across development, paternal transcripts became appreciable at the 4-cell stage, with 14.15±7.60% of the informative SNPs exhibiting paternal expression, and increased thereafter until reaching a maximum of 96.34% at the blastocyst stage. Overall, this work demonstrates that EGA in horse embryos starts at the 4-cell stage and achieves its main activation at the 8-cell stage. Further analysis will be performed to detail paternal vs. maternal gene expression at the different embryonic stages.

Whey protein polymorphisms in Sudanese goat breeds.

The aim of the present study was to assess genetic variation that is characteristic for Sudanese goat breeds in the milk whey protein genes (LALBA and BLG). Four Sudanese goat breeds were screened for variability in LALBA and BLG genes at the DNA level by comparative sequencing of five animals per breed. Sixteen SNPs were identified in LALBA: seven in the upstream region, six synonymous, and three in the 3´-UTR. Three novel synonymous SNPs in exon 2 (ss5197800003, ss5197800012, and ss5197800004) were found in Nubian, Desert, and Nilotic, but not in Taggar goats. One SNP in the promoter of LALBA (rs642745519) modifies a predicted transcription factor binding site for Tcfe2a. The SNPs in the 3'-UTR (rs657915405, rs641559728, and rs664225585) affect predicted miRNA target sites. With respect to haplotypes in the exonic region, haplotype LALBA-A is most frequent in Nubian, Desert, and Nilotic goats, while haplotype LALBA-D is prevalent in Taggar goats. In BLG, 30 SNPs were detected: eight in the upstream gene region, two synonymous, 17 intronic, and three in the 3'-UTR. Among the 30 identified SNPs, 15 were novel. Four of these novel SNPs were located in the upstream gene region, one was synonymous, and ten were intronic. The novel synonymous SNP (ss5197800017), located in exon 2, was only found in Nubian and Nilotic goats. The SNPs ss5197800010 and rs635615192 in the promoter are located in predicted binding sites of transcription factors (M6097, Elk3, Elf5, and GABPA). Among seven haplotypes detected in the coding region, haplotype BLG-A is most frequent in Nubian and Nilotic goats while haplotype BLG-B is most frequent in Desert and Taggar goats. The high variability in regulatory gene regions among Sudanese goats could potentially affect the quality and yield of whey proteins in goat milk and provide a wide resource for genetic improvement of milk production and milk technology characteristics.

Genome Data Allow in silico Pharmacogenetic Studies Using the Genetic Makeup of Both the Individual Patient As Well As the Disease

Pharmacogenomics is becoming more and more important due to personalized medicine. To understand the association between the genetic background of a patient and the response to a tailored treatment would strongly influence the choice of therapy and reduce trial-and-error strategies. Various screening assays have been established, e.g. to test the impact of acquired gene mutations in cancer on the response to different agents and combinations in cell culture models. In the era of genome data, in silico analyses are becoming an interesting tool and the hope is growing to predict a patient's response to therapy by association studies using the genetic makeup of both, the individual patient as well as the disease. The aim of this study was to get insights into the genetic background of AML patients refractory to treatment and to investigate associations of polymorphisms (SNPs) in cancer treatment target genes and disease associated mutations contributing to an altered response to treatment. The cohort consisted of 247 AML patients diagnosed by cytomorphology following the WHO classification. All patients were treated intensively with a standard chemotherapy protocol such as 7+3 and response to treatment was assessed after first and second induction. Following ELN guidelines patients were grouped into responder (group 1), showing cytomorphological complete remission after first induction (n=186), and treatment failure (group 2) with only partial remission or progressive disease after second induction (n=61). Whole genome sequencing (WGS) was performed with 90x coverage for all samples at diagnosis to assess their mutational profiles as well as their germline background. In depth analyses were restricted to exonic SNPs of 217 drug target genes (DrugBank 5; Schärfe et al., 2017) associated with cancer treatments and 20 recurrently mutated genes in AML. Morphologic subtypes as well as mutation frequencies in AML genes were similar in the responder and treatment failure groups. In total 9,742 unique SNPs were found with a median of 1,603 per patient. 37% of these SNPs were found in single patients only and another 27% were found in less than 5% of the cases. SNPs and somatic variants for the addressed genes were combined into a single matrix and co-occurrences of two variants were assessed by matrix multiplication for each group. First, we were interested in group-specific co-occurrences of SNPs and somatic variants. Here we found considerably less co-occurrences in group 1 compared to group 2. All identified co-occurrences in group 1 were unique whereas 772 co-occurrences could be found in more than one patient in group 2. For group 2 a co-occurrence network was created based on recurrent combinations of somatic variants and missense germline variants (figure A). 51% of the patients from group 2 were involved in these interactions, while group 1 did not show any recurrent co-occurrences. It was interesting to note that IDH2, KMT2A-PTD, SF3B1 and TP53 recurrently co-occurred with multiple missense SNPs. Second, we addressed the association of two SNPs in the investigated drug target genes. Looking at SNPs co-occurring in at least 10% of the patients again reflected the heterogeneity of group 1, showing no recurrently co-occurring SNPs, while group 2 showed 6 SNP pairs associated with MAPK family signaling cascades (figure B). Last, it was noteworthy that multiple non-coding SNPs were found in the 3'UTR of the drug target genes. These variants showed a higher frequency in group 2 compared to group 1. SNPs in the 3'UTR region might modify mRNA - miRNA interactions by creating, abolishing, or changing the miRNA-binding sites (figure C). Recently multiple miR-SNPs have been described that were prognostic for treatment outcome, suggesting a potential as predictive biomarkers. In conclusion, WGS data allow comprehensive genome wide but also targeted association analyses. We found three different mechanisms potentially altering treatment sensitivity based on the genetic makeup of individual patients. 1) Association of somatic mutations and SNPs located in drug target genes, 2) association of two SNPs, and 3) SNPs in the 3'UTR modulating miRNA interaction. All three mechanisms occurred only in the refractory AML cases and not in the responder group. This study shows the power of genomic data and in silico analyses that are the basis for future tools to predict response to treatment for individual patients. Disclosures Meggendorfer: MLL Munich Leukemia Laboratory: Employment. Walter:MLL Munich Leukemia Laboratory: Employment. Hutter:MLL Munich Leukemia Laboratory: Employment. Baer:MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.