ĐA HÌNH MICROSATELLITE LIÊN KẾT VỚI GEN HEPCIDIN/HAMP TIỀM NĂNG TRONG CHỌN GIỐNG CÁ RÔ PHI VẰN KHÁNG BỆNH DO Streptococcus iniae

Bệnh xuất huyết do vi khuẩn Streptococcus sp là mầm bệnh truyền nhiễm chính gây thiệt hại đáng kể đến sản lượng cá rô phi toàn cầu. Hepcidin/HAMP ở cá đã được báo cáo có liên quan đến miễn dịch bẩm sinh chống lại các mầm bệnh vi khuẩn. Trong nghiên cứu này, chúng tôi tiến hành phân tích mối quan hệ giữa tính đa hình microsatellites/SSRs liên kết với gen hepcidin/HAMP và khả năng kháng bệnh do Streptococcus iniae trên cá rô phi vằn dòng NT1 (Đài Loan). 17 chỉ thị SSRs và cặp mồi đặc hiệu đã được thiết kế dựa trên WebSat. Kết quả đánh giá trên 95 cá thể cho thấy 9/17 chỉ thị SSRs có tính đa hình cao và tuân theo định luật Hardy-Weinberg. Các chỉ thị này sẽ được sử dụng để đánh giá khả năng kháng vi khuẩn S. iniae. 29 cá rô phi NT1 thế hệ thứ nhất (khối lượng 23,59 ± 5,388 g/con) đã được cảm nhiễm với vi khuẩn S. iniae 89353 bằng phương pháp tiêm, với liều tiêm LD50 là 1,3x105 cfu/mL. Kết quả phân tích cho thấy, có sự sai khác có ý nghĩa thống kê về kiểu gen và tần số alen giữa nhóm sống và nhóm chết sau cảm nhiễm vi khuẩn ở 3 chỉ thị SSRs (SSR7, SSR9 và SSR16) (p<0,05). Đây là marker có tiềm năng cho chọn giống cá rô phi Đài Loan kháng bệnh do S. iniae. ABSTRACT Streptococcus has been recognized as a major infectious disease-causing significant economic loss in tilapia aquaculture in many countries. The hepatic antimicrobial peptide hepcidin/HAMP was reported to be associated with innate immunity which defends against various bacterial pathogens and viruses. In this study, we analyzed the corelation between the microsatellites/SSRs polymorphism in the hepcidin/HAMP genes and the resistance to Streptococcus iniae in the NT1strain (tilapia strain in Taiwan). Seventeen of hepcidin/HAMP-related SSRs and 17 SSR-specific PCR primer were designed using WebSat. The result showed that 9/17 hepcidin/HAMP-related SSRs were polymorphic markers and there is significant deviation from Hardy-Weinberg Equilibrium (HWE) (p<0,05). These SSRs were examined for disease resistance to S. iniae. Twenty-nine the First generation (G1) tilapia of NT1 strain (average weight of 23,59 ± 5,388g/fish) were challenged with virulent S. iniae 89353 through intraperioneal injection at dose of LD50 (1,3x105 cfu/ml). In this study, the genotype and the allele frequency in three SSRs (SSR7, SSR9 và SSR16) were significantly different between two groups (death fish with infected signals of S. iniae and alive fish infected with S. iniae) (p<0,05). Three SSRs (SSR7, SSR9 và SSR16) are considered as potential molecular markers for selective breeding of Taiwanese tilapia which resists to S. iniae.

Molecular S-genotyping of sweet cherry (Prunus avium L.) genetic resources

Sweet cherries are self-incompatible, which is determined by a gametophytic self-incompatibility system (GSI). The self-incompatibility is controlled by a multi-allelic S-locus. Knowledge about the S-allele constitution of the cultivars is essential for fruit growers and breeders. Recently, molecular PCR-based methods have been developed to distinguish all S-alleles in sweet cherries. In our work, we analysed S-locus genotypes by 13 universal and allele-specific PCR primer combinations within 117 registered, old and local sweet cherry cultivars from the Czech genetic resources of the Research and Breeding Institute of Pomology in Holovousy, the Czech Republic. We confirmed the previous S-genotyping for 66 accessions except for Drogans Gelbe, Hedelfinger, Erika, Meckenheimer Frühe, Badeborner, Bing, Alfa, Gamma, Huldra, Rivan, Valerij Tschkalov, Viola and Winkler’s Frühe. It could be due to either mislabelling or mistakes in the previous analyses. Newly, S-genotyping was determined for 51 accessions in which we found 4 new S-loci combinations. We detected the S-locus combinations in 19 incompatibility groups. The most frequent incompatibility groups were III (S₃S₄), II (S₁S₃), IV (S₂S₃), and VI (S₃S₆) with 22, 20, 12 and 12 genotypes, respectively.

Development of a triplex real-time PCR for simultaneous detection of allergenic ingredients in processed food

SYBR Green real-time or quantitative PCR (Q-PCR) is a suitable system in which to establish a multiplex method to detect allergenic ingredients in food. In this study, a triplex Q-PCR method was developed to detect trace amounts of peanut, soybean and sesame in processed food. Specific PCR primer sets were designed and the concentration of the primers used in the triplex PCR was optimised. The triplex method showed high specificity and sensitivity which were similar to those of the simplex method, and it was applied for the detection of allergenic ingredients in commercially available processed food. The results demonstrate that the developed triplex Q-PCR is a quick, reliable and efficient method for the detection of allergenic ingredients in processed food.

A multi-tool approach to assess microalgal diversity in lichens: isolation, Sanger sequencing, HTS and ultrastructural correlations

Lichen thalli represent the most conspicuous examples of fungal-algal interactions. Studies that describe phycobiont diversity within entire thalli are based mainly on Sanger sequencing. In some lichen species, this technique could underestimate the intrathalline coexistence of multiple microalgae. In this study different multi-tool approaches were applied to two lichen taxa, Circinaria hispida and Flavoparmelia soredians, to detect algal coexistence. Here, we combined Sanger sequencing, a specific polymerase chain reaction (PCR) primer, 454-pyrosequencing, phycobiont isolation and ultrastructural characterization. Furthermore, we compared pyrenoid ultrastructural features of lichenized phycobionts with microalgae isolated in culture. An improved methodology was used to isolate and propagate phycobionts which, in combination with fast genetic identification, resulted in a considerable reduction in time and cost to complete the process. This isolation method, coupled with a specific PCR primer, allowed for the detection of coexisting algae in C. hispida (four Trebouxia lineages). 454-pyrosequencing detected only a fraction of such diversity, while Sanger sequencing identified only the primary phycobiont. Ultrastructural features of the isolated algae were observed by transmission electron microscopy; the maintenance of the pyrenoid characteristics suggested the existence of different Trebouxia lineages. In F. soredians a single Trebouxia lineage was identified using all these approaches.In cases of lichens with algal coexistence, a combination of different molecular and ultrastructural approaches may be required to reveal the underlying algal diversity within a single thallus. The approach proposed in this study provides information about the relationship between molecular and ultrastructural data, and represents an improvement in the delimitation of taxonomic features which is needed to recognize intrathalline Trebouxia diversity.

Array-based sequencing of filaggrin gene for comprehensive detection of disease-associated variants

ABSTRACTThe filaggrin gene (FLG) is essential for skin differentiation and epidermal barrier formation with links to skin diseases, however it has a highly repetitive nucleotide sequence containing very limited stretches of unique nucleotides for precise mapping to reference genomes. Sequencing strategies using polymerase chain reaction (PCR) and conventional Sanger sequencing have been successful for complete FLG coding DNA sequence amplification to identify pathogenic mutations but this time-consuming, labour intensive method has restricted utility. Next-generation sequencing (NGS) offers obvious benefits to accelerate FLG analysis but standard re-sequencing techniques such as oligoprobe-based exome or customized targeted-capture can be expensive, especially for a single target gene of interest. We therefore designed a protocol to improve FLG sequencing throughput using a set of FLG-specific PCR primer assays compatible with microfluidic amplification, multiplexing and current NGS protocols. Using DNA reference samples with known FLG genotypes for benchmarking, this protocol is shown to be concordant for variant detection across different sequencing methodologies. We applied this methodology to analyze cohorts from ethnicities previously not studied for FLG variants and demonstrate usefulness for discovery projects. This comprehensive coverage sequencing protocol is labour-efficient and offers an affordable solution to scale up FLG sequencing for larger cohorts. Robust and rapid FLG sequencing can improve patient stratification for research projects and provide a framework for gene specific diagnosis in the future.