Ovine KRTAP36-2: A New Keratin-Associated Protein Gene Related to Variation in Wool Yield.

Keratin-associated proteins (KAPs) are structural components of wool fibres. High-glycine/tyrosine (HGT)-KAPs are a subset of the KAP family, and their abundance in fibres varies. In this study, we report the discovery of an ovine HGT-KAP gene to which we assigned the name KRTAP36-2. Polymerase chain reaction and single-strand conformation polymorphism (PCR-SSCP) analyses revealed four variants of this gene in a screening population of 170 sheep from a variety of breeds. The DNA sequencing of the variants revealed four single-nucleotide polymorphisms (SNPs) and a dinucleotide deletion. Three of these SNPs were in the coding region, and one of these was non-synonymous and potentially led to the amino acid substitution p.Cys27Gly near the middle of the protein. The remaining SNP was located near the putative TATA box, and the di-nucleotide deletion was near the putative transcription initiation site. The effect of this variation in KRTAP36-2 was investigated in 274 Southdown × Merino lambs that were the progeny of five sires. Variation was only found to be associated with wool yield, that is, the proportion of the greasy fleece that remained as clean fleece upon scouring (expressed as a percentage). This may have some value in increasing wool production.

A missense SNP in the proopiomelanocortin (POMC) gene is associated with growth traits in Awassi and Karakul sheep.

This study was conducted to assess the association between proopiomelanocortin (POMC) gene and growth traits in Awassi and Karakul sheep. PCR-single strand conformation polymorphism (SSCP) method was utilized to assess the polymorphism of POMC PCR amplicons with body weight and length, wither and rump height, chest and abdominal circumference measured at birth, 3, 6, 9, and 12 months intervals. Only one missense SNP (rs424417456:C > A) was detected in exon-2, in which glycine was converted to cysteine in the 65th position in POMC (p.65Gly > Cys). rs424417456 SNP showed significant associations with all growth traits in the third, sixth, ninth, and twelfth months. At the age of 3 months onward, lambs with CC genotype showed higher body weight, body length, wither and rump heights, and chest and abdominal circumferences than lambs with CA and AA genotypes, respectively. Prediction analyses indicated a deleterious effect of p.65Gly > Cys on POMC structure, function, and stability. Owing to the strong correlation between rs424417456:CC and better growth characteristics, this genotype is proposed as a promising marker to enhance growth traits in Awassi and Karakul sheep. The predicted damaging effects caused by rs424417456:CA and rs424417456:AA genotypes may entail a putative mechanism through which lambs with these genotypes exhibit lower growth traits.

Variation in the ovine FOXP3 gene and its effect on growth traits in Egyptian Barki sheep.

The aim of the present study was to detect the FOXP3 gene polymorphisms in Barki sheep at a variable region covering exon 13, intron 13 and the coding sequence in exon 14 and to test the association of these polymorphisms with growth traits. 122 Barki lambs were phenotyped for various growth traits, viz., birth weight (BW), weaning weight (WW), pre-weaning daily gain in weight (ADG1), post-weaning daily gain in weight (ADG2) and marketing bodyweight (MW). The polymerase chain reaction - single-strand conformational polymorphisms (PCR-SSCP) and DNA sequencing methods were used to identify the genetic variants in the FOXP3 gene. The associations between the variation in FOXP3 gene and growth traits were tested using a general linear model. Two variants (F1 and F2 with gene frequencies of 0.64 and 0.36, respectively), and three genotypes (F1F1, F1F2 and F2F2 with frequencies of 0.37, 0.53 and 0.10, respectively) were detected. The association of FOXP3 genotype was significant (p < 0.05) with ADG2 and MW. It is concluded that FOXP3 genotype might be helpful for sheep breeders to produce fast-growing lambs. However, further studies are needed in a large population to confirm the association we found.

Variants of the SCD gene and their association with fatty acid composition in Awassi sheep.

BACKGROUND: Genetic factors affect the variability of fatty acid composition in ruminant products. Thus, this study aimed to investigate the association between the variations of the SCD gene and fatty acid composition in Awassi sheep. METHODS AND RESULTS: A total of 100 Awassi rams between the ages of one and two and a half years old were used in this study. Blood samples were taken at abattoirs in Babylon, and from each animal, longissimus dorsi (LD) muscle samples were taken to measure the fatty acid composition. DNA samples were isolated from each blood sample, then PCR-single strand conformation polymorphism (PCR-SSCP) experiments were conducted for genotyping followed by sequencing reactions. The study identified two genotypes (TT and TA) of the SCD gene (exon 3). Several novel variants were discovered in the amplified fragments of the SCD gene. CONCLUSIONS: The TA genotype resulted in increased intramuscular fat and monounsaturated fatty acids compared to the TT genotype. Breeding for the TA genotype could be used for producing meat containing less saturated fatty acids and more monounsaturated fatty acids, making meat more favorable for human consumption.

Major Histocompatibility Complex (MHC) Diversity of the Reintroduction Populations of Endangered Przewalski's Horse.

Major histocompatibility complex (MHC) genes are the most polymorphic in vertebrates and the high variability in many MHC genes is thought to play a crucial role in pathogen recognition. The MHC class II locus DQA polymorphism was analyzed in the endangered Przewalski's horse, Equus przewalskii, a species that has been extinct in the wild and all the current living individuals descend from 12 founders. We used the polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) to detect the polymorphism within the MHC DQA in 31 Przewalski's horses from two reintroduced populations. Consequently, only seven alleles were identified, with only four presenting in each population. In comparison with other mammals, the Przewalski's horse demonstrated less MHC variation. The nucleotide genetic distance of the seven ELA-DQA alleles was between 0.012 and 0.161. The Poisson corrected amino acid genetic distance of the founded alleles was 0.01-0.334. The allele and genotype frequencies of both reintroduced populations of Przewalski's horse deviated from the Hardy-Weinberg equilibrium. Specific MHC DQA alleles may have been lost during the extreme bottleneck event that this species underwent throughout history. We suggest the necessity to detect the genetic background of individuals prior to performing the reintroduction project.

Genetic diversity analysis of grapevine rupestris stem pitting-associated virus from grapevine by colony PCR-SSCP and -RFLP.

We have developed methods for detecting the genetic diversity of grapevine rupestris stem pitting-associated virus (GRSPaV) based on restriction fragment length polymorphism (RFLP) and single stranded conformational polymorphism (SSCP) in the 905 nt 3' sequence. The amplicons were cloned from six grapevine cultivars, and colony polymerase chain reaction (colony PCR) using recombination bacteria was subsequently analyzed by RFLP and SSCP. Four haplotypes of SSCP and six haplotypes of Sac I RFLPs were defined. The two methods had a 40% discrepancy rate in showing the degree of diversity. All clones were sequenced and were used to construct a phylogenetic tree with seven previously reported GRSPaV sequences. In the tree, all the newly acquired sequences were divided into three clusters, I, II, and III, which corresponded to haplotypes I, II, and III of SSCP, respectively. Haplotype IV of SSCP was grouped into cluster II. A recombination analysis showed that haplotype IV has undergone a recombination event. Together, these results indicate that the SSCP assay is useful for the rapid identification of genetic diversity of GRSPaV. This is the first report of an analysis of the large fragment of GRSPaV by colony PCR-SSCP. Keywords: grapevine; grapevine rupestris stem pitting-associated virus (GRSPaV); RFLP; SSCP; genetic diversity analysis.

Variation in caprine KRTAP1-3 and its association with cashmere fibre diameter.

Keratin-associated proteins (KAPs) are components of cashmere fibres. The gene encoding the KAP1-3 protein (KRTAP1-3) has been described in goats, but little is known about sequence variation in this gene and if it affects cashmere fibre traits. In this study, we used a polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) technique to screen for nucleotide sequence variation in caprine KRTAP1-3 in 327 Longdong cashmere goats, then analysed association between the genetic variation that was revealed and some cashmere fibre traits. Six PCR-SSCP patterns representing six different variant sequences of KRTAP1-3 (named A to F) were revealed. Among these variant sequences, seven single nucleotide polymorphisms (SNPs) were detected, with two of them being non-synonymous. Goats with genotype AC had higher mean fibre diameter (MFD) than those with genotype AB (P < 0.001), while goats with genotype AB had higher MFD than those with AA (P < 0.001). The presence of C (P < 0.001) and B (P = 0.006) in a genotype was associated with increased MFD, and together this suggests that variation in caprine KRTAP1-3 affects the key fibre trait of MFD.

Whole-genome resequencing to investigate the determinants of the multi-lumbar vertebrae trait in sheep.

Multi-lumbar vertebrae trait is a beneficial mutation that can significantly improve livestock meat production. However, the genetic basis of the multi-lumbar vertebrae in sheep is still unclear. Here, we analysed the number of lumbar vertebrae of Duolang sheep and found three different traits of lumbar vertebrae number. Compared with the normal sheep, the length and weight of animal carcass from the multi-lumbar vertebrae sheep increased by 2.21 cm and 0.78 kg, respectively. We performed high-throughput genome resequencing on multi-lumbar vertebrae (n = 18) and normal (n = 11) Duolang sheep and obtained a total of more than 528.87 GB data. We found that the most significantly selective region were located in the 49.68-49.74 MB of chromosome 4 by selective-sweep analysis. We annotated this region and found that it contains SFRP4 which is known to regulate bone development. We further used the PCR-SSCP technology to detect the single nucleotide polymorphism (SNP) of the putative candidate SFRP4 and found that the two SNPs (rs600370085:C > T and rs415133338: A > G) of this gene were significantly associated with the multi-lumbar vertebrae of Duolang sheep. Our study indicates that the SFRP4 may be a potential major gene that affects the number of lumbar vertebrae in Duolang sheep, and has the potential to be utilized for sheep breeding in the future.

Evaluation the Presence of SERPINA5 (Exon 3) and FTO rs9939609 Polymorphisms in Papillary Thyroid Cancer Patients.

BACKGROUND: A few researches evaluated the association of polymorphisms at SERPINA5 and fat mass and obesity-associated protein (FTO) genes with papillary thyroid cancer (PTC) globally. Here, we examined the presence of genetic variations within coding exon 3 of SERPINA5 gene and FTO rs9939609 polymorphism in Iranian PTC patients. METHODS: A total of 122 patients (42 cases for SERPINA5 and 80 cases for FTO gene) and 120 healthy subjects (40 subjects or SERPINA5 and 80 subjects for FTO gene) were recruited. The genetic variation within coding exon 3 of SERPINA5 gene was evaluated by reaction-single-strand conformation polymorphism (PCR-SSCP) and FTO rs9939609 polymorphism was evaluated by RFLP-PCR assay. RESULTS: The PCR-SSCP technique detected two rs6115G>A and rs6112T>C genetic variations within coding exon 3 of SERPINA5 gene and approved also by direct sequencing. For rs6112T>C polymorphism seven patients was heterozygous and for rs6115G>A seven PTC patients were heterozygous and two patients were homozygous. CONCLUSION: This study indicated that SERPINA5 rs6115G>A and rs6112T>C polymorphisms might be a novel susceptibility locus for PTC in Iranian patients. However, our findings do not support an association between FTO rs9939609 polymorphism and PTC risk.

A review of methods for detecting single-nucleotide polymorphisms in the Toll-like receptor gene family.

The Toll-like receptors play an essential role in immunity through targeting the pathogen-associated molecular patterns. Nucleotide variations in TLR genes, especially single-nucleotide polymorphisms, have been shown to alter host immune susceptibility to several infections and diseases. Since TLR genes' polymorphisms can be a promising biomarker, ongoing investigations aim to develop, optimize and validate SNP detection methods. This review discusses various TLR SNP detection methods, either used extensively or occasionally, but having a vast potential in high-throughput settings. Methods such as PCR-restriction fragment length polymorphism, TaqMan® assay, direct sequencing and matrix-assisted laser desorption ionization - time of flight mass spectroscopy MS are frequently used methods whereas Illumina GoldenGate® assay, reverse hybridization technology, PCR-confronting two-pair primers, KBiosciences KASPar® SNP assay, SNP stream®, PCR-fluorescence hybridization and SNaPshot® are powerful but sporadically used methods. We suggest that, for individual laboratories, the detection method of choice depends on a combination of factors such as throughput volume, reproducibility, feasibility and cost-effectiveness.

Single-strand conformation polymorphism-based genetic characterization of the Cyclospora cayetanensis strains collected from different provinces in Turkey.

INTRODUCTION AND OBJECTIVE: Cyclospora cayetanensis, a coccidian protozoan species, has been recently found to cause diarrhea in all age groups in immunocompetent and immunocompromised individuals in most regions of the world. This study aimed to conduct the molecular detection of C. cayetanensis and to determine the genetic diversity of the 18S ribosomal RNA (rRNA) gene sequence of C. cayetanensis isolated from individuals living in different provinces in Turkey by using PCR-single-strand conformation polymorphism (SSCP). MATERIAL AND METHODS: A total of 22 subjects were included in the study. Fourteen of the subjects were female and eight were male, with ages ranging between 7-65 years. Stool specimens were examined using wet mount and modified acid-fast staining methods, which revealed the presence of oocysts in the samples. The 18S rRNA ITS-1 Ccits37f-GCTTGCTATGTTTTAGCATGTGG and Ccits501r-GCACAATGAATGCACACACA gene regions were used as primers. The PCR products were analyzed by agarose gel electrophoresis and visualized on a UV transilluminator. For the SSCP, the PCR products were denatured with formamide, run for 16 h in 6% (49:1) polyacrylamide gel, and then imaged with silver staining. RESULTS: SSCP assay was performed given that the DNA strands demonstrated different folds; the DNA strands contain different nucleotides based on the PCR-SSCP results for the Cyclospora strains collected in 4 provinces. Moreover, 3 different band profiles were observed in the investigated samples. A slight mutation difference was observed among the strains collected. CONCLUSIONS: Further comprehensive studies involving more C. cayetanensis-positive specimens and utilizing different mutation screening methods are warranted to demonstrate mutation differences in Cyclopora strains in Turkey.

Cloning, characterisation and expression of the SERPINB14 gene, and association of promoter polymorphisms with egg quality traits in layer chicken.

1. Ovalbumin (SERPINB14) is the most abundant protein present in egg white contributing about 54% of the total egg protein. In this study, the objectives were to clone and characterise the coding sequence of the SERPINB14 gene, to explore its expression profile, identify polymorphisms in the promoter of the gene and explore any association with egg quality traits in White Leghorn chickens.2. SNPs and mRNA expression of SERPINB14 in White Leghorn chicken lines were detected by PCR-single strand conformation polymorphism (SSCP) along with sequencing and qPCR. The open reading frame (ORF) was cloned in an expression plasmid vector and sequenced.3. The ORF of this gene was 1161 bp encoding a peptide of 386 amino acids. There were three SNPs observed in the coding region of the gene, one of which was of the mis-sense type, having c562G>A transition which resulted in substitution of alanine to threonine at position 188 in the protein sequence. In both the lines, an increase in expression of the gene was observed after onset of egg production with peak expression at the 40th week of age compared to before onset of lay. The SERPINB14 gene was expressed in the magnum, but not in ovary and infundibulum, tissues of each White Leghorn line. The promoter region of the gene showed SNPs with three haplotypes; H1, H2, and H3. The haplo groups were associated with the egg weight and age at sexual maturity in the IWI line and Haugh unit and albumin index in the IWK line.4. It was concluded that the ORF of SERPINB14 gene in White Leghorn chicken lines is polymorphic. The promoter region of the gene is also polymorphic and has significant (P < 0.05) association with Haugh unit and egg weight in IWK and IWI chicken lines, respectively.

Frequency and characterization of RHD variant alleles in a population of blood donors from southeastern Brazil: Comparison with other populations.

BACKGROUND: The correct determination of D antigen could help to avoid alloimmunization in pregnant women and patients receiving blood transfusions. However, there are limitations in the identification of D variants as the partial and weak D phenotypes make the determination of D antigen a great challenge in the transfusion routine.' STUDY DESIGN AND METHODS: The molecular characterization of D variants was performed on blood donors from southeastern Brazil with atypical D typing. Furthermore, the serological profile of all RHD variant alleles identified was analyzed using different Anti-D clones. The prevalence of RHD alleles and genotypes found was compared with those described in other countries and in other regions from Brazil. RESULTS: Atypical serologic D typing occurred in 0.79 % of blood donors. The majority of RHD variant alleles (88 %) were first characterized by multiplex PCR and PCR-SSP as RHD*weak partial 4 (47 %), followed by RHD*weak D type 3 (29.9 %), RHD*weak D type 2 (3.9 %) and RHD*weak D type 1 (3.1 %). Genomic DNA sequencing characterized the RHD*weak partial 4 variants found in RHD*DAR1.2 (weak 4.2.2) (22 %), RHD*DAR3 (weak 4.0.1) (2.4 %), RHD*DAR3.1 (weak 4.0) (22 %) and RHD*DAR4 (weak 4.1) (0.8 %). RHD variant alleles associated with partial D, such as, RHD*DAU-4 (1.6 %), RHD*DAU-5 (2.4 %), RHD*DAU-6 (1.6 %), RHD* DIII type 8 (1.6 %), RHD*DVII (3.9 %) and RHD* DMH (0.8 %) were also observed. CONCLUSION: The prevalence of RHD variant alleles observed in this cohort differ from those found in other populations, including Brazilians from other regions. RHD allele distribution in specific regions should be considered for implementation of algorithms and genotyping strategies aiming at a more effective and safe transfusion.

Sensitivity and applications of the PCR Single-Strand Conformation Polymorphism method.

PCR Single-Strand Conformation Polymorphism is a method used to identify and detect mutations and is now well known for its many applications on living beings. This paper will discuss the experimental details, limitations and sensitivity of the PCR Single-Strand Conformation Polymorphism method in relation to all existing literature available to us until today. Genomic DNA extraction, PCR amplification and Single-Strand Conformation Polymorphism conditions (concentration of polyacrylamide slab gel electrophoresis, dissociation treatment of double- stranded DNA) and comparison with PCR Restriction Fragment Length Polymorphism are presented. Since its discovery in 1989, there have been many variations, innovations, and modifications of the method, which makes it very easy, safe, fast and for this reason widely applied in clinical diagnostic, forensic medicine, biochemical, veterinary, microbiological, food and environmental laboratories. One of the possible applications of the method is the diagnosis and identification of mutations in new strains of coronaviruses, because science needs more tools to tackle the problem of this pandemic. The PCR Single-Strand Conformation Polymorphism method can be applied in many cases provided that control samples are available and the required conditions of the method are achieved.

D variants in the population of D-negative blood donors in the north-eastern region of Croatia.

OBJECTIVES: The aim of this study was to determine RHESUS D GENE (RHD) allelic variants among Croatian D-negative blood donors and compare our results with respective data from other European countries. BACKGROUND: Altered or reduced D antigen expression can result in D variants, which can be mistyped and can lead to the alloimmunisation of the blood recipient. RHD genotyping can distinguish D variants: weak D, partial D and DEL, thus preventing alloimmunisation. MATERIAL/METHODS: A total of 6523 samples obtained from D-negative Croatian donors were screened for the presence of RHD using the real-time polymerase chain reaction (PCR) method. PCR-SSP was performed for D variant genotyping by using commercial genotyping kits (Inno-Train, Kronberg, Germany). Genomic DNA sequencing for all 10 exons of the RHD was performed when the genotyping kits failed to assign a D variant. RESULTS: RHD molecular screening revealed 23 (0.35%) RHD-PCR positive samples, all C/E positive, in decreasing frequency: 11 hybrid RHD-CE (2-9) D-CE variants, 4 weak partial D type 11 and 2 weak D type 2. Six samples remained unresolved and were sequenced. For 12 of 23 samples (excluding large hybrids), an adsorption/elution of anti-D serum was performed, confirming that all 12 were RhD+. The calculated frequency of clinically significant D alleles in RhD-negative blood donors was 1:543 (0.18%) or 1:53 (1.89%) in C/E blood donors. CONCLUSION: Data on the significant frequency of D variants among serologically D-negative blood donors in the north-eastern region of Croatia could help in introducing RHD molecular screening of blood donors in a routine workflow.

Nucleotide Sequence Variation in the Insulin-Like Growth Factor 1 Gene Affects Growth and Carcass Traits in New Zealand Romney Sheep.

Insulin-like growth factor 1 (IGF1) is a mediator of the effects of growth hormone and polymorphism in the IGF1 gene (IGF1) is reported to affect fat deposition in some livestock species. In this study, nucleotide sequence variation in three regions of ovine IGF1 (part of the 5' flanking region, the exon 3 region, and the exon 4 region) was investigated in 848 New Zealand Romney lambs using PCR-single strand conformation polymorphism (SSCP) analyses to ascertain if single nucleotide polymorphisms (SNPs) existed. Six SNPs were identified across these three regions. The effect of the sequence variation in the exon 3 and exon 4 regions on growth and carcass traits were investigated. One of the PCR-SSCP sequence variants in the exon 3 region was associated with variation in hot carcass weight, carcass fat depth at the 12th rib measured using video imaging and the percentage proportion of leg lean meat, whereas the other was associated with variation in growth rate to weaning. No associations were detected for the other gene regions analyzed. The results suggest that polymorphism in exon 3 of ovine IGF1 has potential for use as a gene-marker for some carcass and growth traits.

Two co-inherited novel SNPs in the MC4R gene related to live body weight and hormonal assays in Awassi and Arabi sheep breeds of Iraq.

Melanocortin-4 receptor (MC4R) gene plays a key role in the regulation of body weight and energy homeostasis. This study aims to evaluate the association of single nucleotide polymorphisms (SNPs) of the MC4R gene with live body weight and hormonal assays in two breeds of sheep that differ in productive performance, Awassi and Arabi. All known coding sequences of the MC4R gene were covered in this study. DNA samples from 150 animals (Awassi and Arabi breed) were genotyped by PCR-single-strand conformation polymorphism (PCR-SSCP) to assess their pattern of genetic variation. Concerning exon 1, clear heterogeneity was detected with three different SSCP-banding patterns. The sequencing reactions confirmed these variations by detecting the presence of the two novel SNPs, 107G/C and 138A/C, and three genotypes, GC, AC and AA. The 107G/C SNP was detected in GC genotype, while the 138A/C was detected on both GC and AC genotypes. The other SSCP-banding pattern (AA genotype) did not show any detectable unique variation. Both SNPs were closely and strongly linked in both breeds (D' and r2 values were 1.00), which signifies that both loci were co-inherited as one unit. Association analysis indicated that both breeds with GC/AC haplotype showed higher live body weight (37.250 ± 0.790) relative to the GG/AA (30.244 ± 0.968) and CC/CC (47.231 ± 1.230) haplotypes (p < .05). Concerning the genotyping of exon 2, only 362 bp showed heterogeneity with a missense mutation, with no significant association (p > .05) with the measured traits. In conclusion, the two novel SNPs (107G/C and 138 A/C) were highly associated with live body weight in both breeds. Haplotype analysis confirmed that these two novel SNPs were in strong linkage disequilibrium (LD) and could be used as genetic markers for sheep phenotypic trait improvement.

p53 Gene Mutation in Blood of Zebu Cattle with Squamous Cell Carcinoma of the Horn.

Horn cancer is one of the most important diseases in Zebu castrated male cattle. The objective of this study was to investigate the presence of p53 gene mutation in the blood of affected cattle and its value for early diagnosis and prognosis. The study was conducted on blood samples from 20 affected cattle and six healthy control cattle from Western India. Plasma samples were evaluated for the presence of p53 gene mutation using the polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP) technique and the results were correlated with the stage of cancer. Five of the 20 cases had stage I neoplasms, nine stage II and six stage III, based on histopathological examination. PCR-SSCP analysis revealed an aberrant pattern of DNA migration on polyacrylamide gel electrophoresis of DNA extracts from blood samples of six animals with stage II and stage III cancer. No mutation was identified in blood from cattle with stage I cancer or from healthy control cattle. These results suggest that PCR-SSCP detection of p53 gene mutation in blood has potential diagnostic and prognostic value, and indicate the need for further large-scale investigation.

Inactivating mutations of class II transactivator (CIITA) gene in gastric and colorectal cancers.

Expression of MHC class II, which is important against cancer immunity, depends on the transcactivator CIITA. These data suggest a possibility that CIITA gene might be inactivated in cancers. In this study, we studied inactivating mutation status of CIITA gene in gastric (GC) and colorectal (CRC) cancers by analyzing the C7 repeat in the CIITA (exon 11) gene. We found frameshift mutations in 3 GCs and 6 CRCs in cancers with high microsatellite instability (MSI-H) (9/113), but not in those with microsatellite-stable (MSS) (0/90) (P = 0.004). They were all deletion or duplication of one base in the C7 repeat that would result in truncation of amino acid synthesis. Immune therapy is now a major option in cancer therapy and our results on the genetic alterations of MHC II-related CTIITA in MSH-H GC and CRC might provide useful information for the treatment of MSI-H cancers.

The development of species-specific AFLP-derived SCAR and SSCP markers to identify mantis shrimp species.

Mantis shrimp has become commercially valuable in many countries, while the commercially aquaculture still unsuccessful. The stable supply of the species-specific markers for precise identification can play a key role of foods authentication as well as restoring/enhancing mantis shrimp stocks in future. The aim of this research was to identify species-specific markers for Squillid and Harpiosquillid mantis shrimp taxa using Amplified fragment length polymorphism-Single strand conformation polymorphism (AFLP-SSCP) approaches. Selective amplification would be substituted as a total of 40 primer combinations was performed using either three-base (i.e., EcoRI+3 and MseI+3 in 20 primer combinations) or two-base (i.e., EcoRI+2 and MseI+2 in 20 primer combinations) selective primers. These had been size-fractionated via 6% denaturing polyacrylamide gel electrophoresis, ten AFLP fragments exhibiting species or genus-specific characteristics were cloned, sequenced, and GenBank interrogated. A primer pair was designed and their specificity was tested versus the genomic DNA of various species. Results show that the primer E+2-13/M+2-13Hr158 generated PCR products for just H. harpax, while E+3-14/M+3-2HhHr151 and E+2-13/M+2-13Hh150 generated PCR products for both H. harpax and H. raphidea and not others (i.e., M. nepa, O. oratoria, and E. woodmasoni). SSCP was then applied in order to differentiate between H. harpax and H. raphidea. These SSCP results indicate that species can be differentiated based on polymorphic fragment nucleotides. Indeed, primers E+2-13/M+2-13Hr158, E+3-14/M+3-2HhHr151, and E+2-13/M+2-13Hh150 were all successfully confirmed as present in processed mantis shrimp samples (i.e., saline-preserved and heat-dried). These results provide new species-specific markers for mantis shrimp identification.