Development and validation of a novel 30-plex STR assay for canine individual identification and parentage testing.

Domestic dogs are helpers in outdoor human work and companions for families; thus, individual canine identification and parentage testing are crucial in certain fields, including forensics and breeding programs. In this study, a six-dye fluorescent labeling multiplex amplification system containing 29 canine short tandem repeats (STRs) and the sex-determining marker DAmel was developed. The system was called the Tronfo Canine 30-plex STR Kit and was further validated according to the Scientific Working Group on DNA Analysis Methods and the Organization of Scientific Area Committees for Wildlife Forensics guidelines, including tests for PCR conditions, precision, species specificity, sensitivity, stability, repeatability and reproducibility, a population study, and a study of 16 paternity test cases. The results indicated that the novel canine STR assay was accurate, specific, reproducible, stable, and robust. Complete profiles were obtained with 31.25 pg of canine DNA. Additionally, 500 unrelated canine individuals were investigated using this novel system, and the combined power of discrimination and exclusion values were 0.999999999999999999 and 0.999996451039850, respectively. These results suggest that the Tronfo Canine 30-plex STR Kit is highly polymorphic, informative, and suitable for individual canine identification and parentage testing.

Developmental validation of a novel multiple genotyping assay with 24 Canine STR loci.

Canine individual identification and parentage testing are essential in various fields, including forensics and breeding programs. This study aimed to develop and validate the Canine 25 A kit, a multiplex polymerase chain reaction (PCR) system designed to address these critical requirements. This novel system enables the simultaneous amplification of 24 canine autosomal short tandem repeat (STR) loci and one sex-determining marker. Validation of the Canine 25 A kit was conducted following the Scientific Working Group on DNA Analysis Methods (SWGDAM) guidelines, demonstrating significant sensitivity, high inhibitor tolerance, canine specificity within a mixture, species specificity, and precision in genotype determination. The Canine 25 A kit was crucial in resolving several forensic cases, such as casework samples from a dog attack incident and parentage determination. Its effectiveness in genotyping these samples highlights its significance in forensic applications. Population genetic parameter analysis revealed a high discriminatory power, as indicated by the calculated combined discrimination power (CDP) values for each breed exceeding 0.999 999 999 999, while the combined power of exclusion (CPE) surpassed 0.9999. Overall, the Canine 25 A kit offers a precise and dependable tool for canine individual identification and parentage determination.

Genetic Diversity and Signatures of Selection in 15 Chinese Indigenous Dog Breeds Revealed by Genome-Wide SNPs.

There are dozens of recognized indigenous dog breeds in China. However, these breeds have not had extensive studies to describe their population structure, genomic linkage disequilibrium (LD) patterns, and selection signatures. Here, we systematically surveyed the genomes of 157 unrelated dogs that were from 15 diverse Chinese dog breeds. Canine 170K SNP chips were used to compare the genomic structures of Chinese and Western dogs. The genotyping data of 170K SNP chips in Western dogs were downloaded from the LUPA (a European initiative of canine genome project) database. Chinese indigenous dogs had lower LD and shorter accumulative runs of homozygosity (ROH) in the genome. The genetic distances between individuals within each Chinese breed were larger than those within Western breeds. Chinese indigenous and Western dog breeds were clearly differentiated into two separate clades revealed by the PCA and NJ-tree. We found evidence for historical introgression of Western dogs into Chinese Kazakhstan shepherd and Mongolia Xi dogs. We suggested that Greenland sledge dog, Papillon, and European Eurasier have Chinese dog lineages. Selection sweep analysis identified genome-wide selection signatures of each Chinese breed and three breed groups. We highlighted several genes including EPAS1 and DNAH9 that show signatures of natural selection in Qinghai-Tibetan plateau dogs and are likely important for genetic adaptation to high altitude. Comparison of our findings with previous reports suggested RBP7, NMNAT1, SLC2A5, and H6PD that exhibit signatures of natural selection in Chinese mountain hounds as promising candidate genes for the traits of endurance and night vision, and NOL8, KRT9, RORB, and CAMTA1 that show signals of selection in Xi dogs might be candidate genes influencing dog running speed. The results about genomic and population structures, and selection signatures of Chinese dog breeds reinforce the conclusion that Chinese indigenous dogs with great variations of phenotypes are important resources for identifying genes responsible for complex traits.

A comprehensive survey of copy number variation in 18 diverse pig populations and identification of candidate copy number variable genes associated with complex traits.

BACKGROUND: Copy number variation (CNV) is a major source of structural variants and has been commonly identified in mammalian genome. It is associated with gene expression and may present a major genetic component of phenotypic diversity. Unlike many other mammalian genomes where CNVs have been well annotated, studies of porcine CNV in diverse breeds are still limited. RESULT: Here we used Porcine SNP60 BeadChip and PennCNV algorithm to identify 1,315 putative CNVs belonging to 565 CNV regions (CNVRs) in 1,693 pigs from 18 diverse populations. Total 538 out of 683 CNVs identified in a White Duroc × Erhualian F2 population fit Mendelian transmission and 6 out of 7 randomly selected CNVRs were confirmed by quantitative real time PCR. CNVRs were non-randomly distributed in the pig genome. Several CNV hotspots were found on pig chromosomes 6, 11, 13, 14 and 17. CNV numbers differ greatly among different pig populations. The Duroc pigs were identified to have the most number of CNVs per individual. Among 1,765 transcripts located within the CNVRs, 634 genes have been reported to be copy number variable genes in the human genome. By integrating analysis of QTL mapping, CNVRs and the description of phenotypes in knockout mice, we identified 7 copy number variable genes as candidate genes for phenotypes related to carcass length, backfat thickness, abdominal fat weight, length of scapular, intermuscle fat content of logissimus muscle, body weight at 240 day, glycolytic potential of logissimus muscle, mean corpuscular hemoglobin, mean corpuscular volume and humerus diameter. CONCLUSION: We revealed the distribution of the unprecedented number of 565 CNVRs in pig genome and investigated copy number variable genes as the possible candidate genes for phenotypic traits. These findings give novel insights into porcine CNVs and provide resources to facilitate the identification of trait-related CNVs.

A genome-wide investigation of expression characteristics of natural antisense transcripts in liver and muscle samples of pigs.

Natural antisense transcripts are endogenous transcripts that are complementary to the sense-strand of DNA. These transcripts have been identified in various eukaryotic species and are involved in a broad range of regulatory events and biological processes. However, their general biological functions, expression characteristics and regulatory mechanisms are still unclear. In this study, 497 liver and 586 muscle samples were harvested from a White Duroc×Erhualian F(2) resource population. The expression profiles of sense and antisense transcripts were determined by tag-based RNA sequencing. We identified 33.7% and 20.4% of transcripts having both sense and antisense expression, and 12.5% and 6.1% of transcripts only expressing antisense transcripts in liver and muscle, respectively. More than 32.2% of imprinting or predicted imprinting genes in the geneimprint database were detected with both sense and antisense expression. The correlations between sense and antisense expression in sense-antisense pairs were diverse in both liver and muscle, showing positive, negative or absent correlation. Antisense expression increases gene expression variability. More interestingly, compared to eQTL mapping of sense transcripts in which more than one eQTL was mapped for a transcript, only one eQTL was identified for each antisense transcript, and the percentage of cis-eQTL in antisense eQTL was higher than that in sense eQTL. This suggests that the expressions of antisense transcripts tend to be cis-regulated by a single genomic locus. To our knowledge, this study is the first systematical investigation of antisense transcription in pigs. The findings improve our understanding of the complexity of porcine transcriptome.