BOLA-DRB3 gene with mastitis prevalence in the investigated breeds. This supports previous studies that the BOLA-DRB3 gene represents an important locus affecting resistance to mastitis in cattle. These results contribute to an increase in knowledge and available data regarding the genetic variability of SNPs for udder health in cattle.In order to improve animals’ resistance to mastitis in subsequent generations and to reduce disease prevalence, one feasible approach could be the genomic selection of cows with the most potentially protective immune responses. In this study, 298 cattle were genotyped for 89 single nucleotide polymorphisms through the Kompetitive Allele Specific PCR. Our findings reveal a significant association of a molecular marker in the CXCR2, CXCL8, TLR4, BRCA1, LTF, BOLA-DRB3), which were found to be associated with genetic resistance or susceptibility to mastitis. A total of 298 cattle (250 Romanian Spotted and 48 Romanian Brown) were genotyped by Kompetitive Allele Specific PCR (KASP) and a chi-squared test was used for genetic association studies with clinical mastitis. A total of 35 SNPs (39.3%) among the selected 89 SNPs were successfully genotyped, of which 31 markers were monomorphic. The polymorphic markers were found in two genes: TLR4 (rs460053411) and BOLA-DRB3 . The polymorphic SNPs with MAF > 5% and call rates > 95% were used for the association study. The results showed that rs110124025 in the BOLA-DRB3 gene was significantly associated with mastitis prevalence (p ≤ 0.05) in both investigated breeds. Current results show that the SNP rs110124025 in the BOLA-DRB3 gene can be used as a candidate genetic marker in selection for mastitis resistance in Romanian dairy cattle.Mastitis is the most common production disease in the dairy sector worldwide, its incidence being associated with both cows’ exposure to bacteria and the cows’ genetic make-up for resistance to pathogens. The objective of our study was to analyse 89 missense SNPs belonging to six genes ( Globally, mastitis is a prevalent disease that impairs animal health and welfare, resulting in serious economic losses ,2. In caBos taurus genome, provided the opportunity to identify single nucleotide polymorphisms (SNPs) in various genes that could possibly be utilized as genetic markers for resistance or susceptibility to mastitis. Researchers and livestock breeders are therefore trying to utilize genetic variation in resistance to mastitis to manage this pressing issue [Intense selection in recent decades in dairy cows and the use of elite sires to maximize milk yields has had detrimental effects on other functional traits, such as the incidence of clinical mastitis ,5. At thng issue . Many cang issue ,11,12,13ng issue .LTF) gene maps to Bos taurus autosome (BTA) 22, which is a member of the transferrin gene family, consists of 17 exons and 2722 variant alleles, has an antibacterial, antiviral, antifungal and anti-inflammatory activity [TLR4) gene maps to BTA8, consists of four exons and the protein encoded by this gene is a member of the toll-like receptor (TLR) family that plays an important role in pathogen recognition and activation of innate immunity [IL8/CXCL8 (C-X-C motif chemokine ligand 8) gene maps to BTA6 and the protein encoded by this gene, which is referred to as interleukin-8 (IL-8), is an important mediator of the inflammatory response and acts on the CXCR1 and CXCR2 receptors [CXCR2 (C-X-C motif chemokine receptor 2) gene is located on BTA2 and encodes the cytokine receptors necessary for neutrophil migration to infection sites [BRCA1 (breast cancer 1) gene plays a crucial function in DNA repair of double-stranded breaks and in maintaining genomic stability [BOLA-DRB3  gene is located on BTA23 and consists of six exons, of which the second exon is the most polymorphic. This gene plays a central role in the immune system and resistance to pathogens [In accordance with other studies carried out so far, for the present study we have chosen six genes that have been previously associated with mastitis. The lactotransferrin  belongs to the Simmental strain, being a dual-purpose breed, with a current census of 376,000 cows, representing 36% of the breed structure in Romania . The RS The Romanian Brown cattle  belongs to the Braunvieh strain, having a census of 220,000 cows. The RB is the result of crossbreeding between the Braunvieh breed from Switzerland and local unimproved Mocănița and Grey cattle , with thGenomic selection for both RS and RB breeds has been introduced in the past five years in Romania, being used particularly in young bulls that are produced for AI stations. Although performance recordings and selection programmes are in place for both breeds, semen imports are occurring regularly in order to manage inbreeding rates and genetic gain.Having in mind the importance of udder health, this study aimed to analyse the polymorphism of 89 SNP markers belonging to six genes that were found to be associated with genetic resistance to mastitis through the use of the Kompetitive Allele Specific PCR technique in Romanian Spotted and Romanian Brown cattle breeds.The experimental design, sample collection protocols and procedures were approved by the Institutional Ethics Committee of the Research and Development Station for Bovine Arad belonging to the Academy for Agricultural and Forestry Sciences (Decision no. 51/ PN-II-RU-TE-1402). Blood samples were collected from animals by trained veterinarians. All research activities involved in the present study were performed in accordance with the Directive 2010/63/EU on the protection of animals used for scientific purposes.A total of 298 cattle reared at the Research and Development Station for Bovine Arad (46°10′16.2″ N 21°14′08.6″ E) in Romania, including 250 Romanian Spotted and 48 Romanian Brown, were included in the research herd. The cows were both primiparous and multiparous, and the entire herd was included in the Official Performance and Recording Scheme.® farm management software. Animals were fitted with AfiTag® pedometers . During the study, cattle were kept on deep straw bedding, with a space allowance of 9 m2 in the resting area and free access to outside paddocks, benefiting from a dry and clean environment. Cows from both breeds were housed in groups of 40 to 50 animals, according to lactation stage and productivity. Additionally, all cows had a feeding space allowance of about 75 cm/head, were fed twice per day and had unlimited access to water. The feeding of cows differed according to season. Daily individual diet consisted of 6 kg alfalfa hay, 6 kg concentrates and 35 kg maize silage during the winter and in the summer the silage was reduced to 18 kg and 45 kg of green fodder was added.Cows were milked twice per day in a ‘herringbone’ milking parlour (2 sides × 14 units), which employs the AfiFarm 5.4Milk yield per milking session and milk conductivity were recorded daily for a period of 20 months. Production levels and milk quality data were taken from the results of the Official Performance Control service. The individual milk samples were collected in the herd at intervals of 28 days in order to monitor udder health using somatic cell count (SCC) as a proxy for mastitis. A mastitis diagnostic was based on daily clinical observation of the udder during the 20-month trial. Clinical mastitis was initially identified by trained technicians on the basis of signs such as udder swelling, hardness of the affected quarter, abnormal milk and/or changes in the consistency or colour of the milk. In addition, data based on clinical signs were corroborated with SCC, where we used a threshold value of 285,000 somatic cells/mL of milk or greater. Only the cows that cumulatively met the two conditions were declared positive for clinical mastitis.LTF (lactotransferrin), TLR3 (toll-like receptor 4), IL8/CXCL8 (C-X-C motif chemokine ligand 8), BRCA1 (breast cancer 1), BOLA-DRB3  and CXCR2 (C-X-C motif chemokine receptor 2). The SNPs from the investigated candidate genes were collected by querying the Single Nucleotide Polymorphism Database maintained by the National Center for Biotechnology Information (NCBI) and Ensembl. The SNPs were generally selected to fall within the coding region of the gene and change the resulting codon (missense mutations). As a result, the final list of SNPs used in the study included 86 missense (non-synonymous) and 3 synonymous SNPs. Details regarding the SNP ID, gene symbol, chromosome position based on the ARS-UCD1.2 genome assembly of Bos taurus, alleles, synonymous/non-synonymous nature, position in coding region and amino acid change are presented in The decision of which single nucleotide polymorphism (SNPs) to be used in the present study for genotyping was conducted gradually, with the first step being the candidate gene selection, followed by SNP identification and selection. A total of 89 bi-allelic SNPs were chosen across the cattle genome, based on previous results from the literature ,23,24,25The bovine genomic DNA was extracted from total blood. Fresh blood (2 mL) was collected from the tail vein (vena cava) of the animals in vacutainers containing K3EDTA as an anticoagulant . Genomic DNA was isolated from 300 µL blood samples using a commercial DNA extraction kit , according to the manufacturer’s instructions. After isolation, DNA concentration and quality were assessed using a NanoDrop 2000 Spectrophotometer . Samples were subsequently diluted to a uniform concentration of 20 ng of DNA per sample for use in Kompetitive Allele Specific PCR (KASP) assays. All investigated DNA samples were sent to LGC Genomics  in order to perform the KASP genotyping for the bi-allelic discrimination of the panel of the 89 selected SNPs. Genotyping data were visualized as a cluster plot using the SNP Viewer software .p < 0.05) difference in genotype distribution within the same SNP.In order to identify the SNP genotypes, the raw allele call received from LGC Genomics was analysed using the KlusterCaller software from LGC Genomics. Considering that the aim of the present research was to investigate molecular markers for their use in designing future breeding programs, we conducted the analysis in order to see if polymorphic SNPs are suitable for the planned work by measuring the markers’ informativeness. Thereby, heterozygosity and the polymorphism information content (PIC) parameters were estimated. POPGENE 1.32  was usedn = 250) and Romanian Brown (n = 48). The KASP genotyping method was used to analyse the SNPs across the six genes. A total of 35 SNPs (39.3%) among the selected 89 SNPs were successfully genotyped across the two breeds  could not be genotyped through the KASP genotyping method. The following 31 SNPs were fixed: rs480434223, rs209319366, rs464576793, rs467207447, rs446793067 in CXCR2; rs134178216, rs468955241, rs448378725, rs466813222, rs453466519, rs452742998, rs445886432 in CXCL8; rs8193041, rs8193048, rs8193049, rs8193050, rs8193053, rs8193055, rs135659119, rs8193066 in TLR4; rs437880924, rs470945089, rs480362322, rs469103334 in BRCA1; rs460348489, rs461145277, rs476824727, rs447239896 in LTF and rs208667846, rs476406737, rs454104745 in BOLA-DRB3. The polymorphic SNPs were found on two genes: TLR4 (rs460053411) and BOLA-DRB3 . Thereby, a total of 35 markers (monomorphic and polymorphic) were analysed from a total of 10,430 genotypes assayed. The average call rate for all successfully genotyped SNPs was 95.98%. A total of 8 alleles and 11 genotypes were found at 4 polymorphic markers in the 298 cattle.Although the set of SNPs was selected from dbSNP, a large number of SNPs (BOLA-DRB3) had a minor allele frequency ≥ 5% (MAF ≥ 0.05) (TLR4) was a rare SNP (MAF < 0.01) and was removed from all further analyses. The graphical KASP results displayed three clusters, in addition to the non-template controls, corresponding to the two homozygous samples and to heterozygous samples. For the three SNPs  in the BOLA-DRB3 gene, grouping of the samples according to the genotypes was observed   and one observed A–C.The genotype and allele frequencies for the polymorphic SNPs in Romanian Spotted and Romanian Brown cattle are shown in 2χ) test showed that the populations were in Hardy–Weinberg equilibrium for all SNPs in the BOLA-DRB3 gene, except for rs208816121 (p < 0.05) in Romanian Spotted and rs460053411 in the TLR4 gene that was removed from all further analyses  and Romanian Brown (p = 0.0359 and p = 0.0500) breeds. A higher frequency of mastitis (occurring once) for the AA genotype was observed in both the Romanian Spotted and Romanian Brown cattle  compared to the GG genotype .Two traits were considered for mastitis, as follows: the frequency , as well as how many quarters were affected . The results showed that the most significant effects for frequency of mastitis and number of affected quarters were found in the case of rs110124025 in the Romanian Spotted , while the frequency for mastitis affecting all four quarters of the udder was more than two-fold higher . In Romanian Brown cows, the frequency for mastitis affecting two quarters of the udder was almost 9 times higher in AA compared to GG animals , while the frequency affecting all four quarters was 4.5 times higher .In the case of the number of affected quarters, the frequency for two and/or four quarters affected by mastitis was significantly higher in animals with the AA genotype for rs110124025 has a significantly higher risk of developing mastitis during lactation, as well as having significantly more quarters affected by mastitis compared to individuals carrying the GG genotype. No significant associations with mastitis (p > 0.05) were found for the other two polymorphic markers in the BOLA-DRB3 gene, rs42309897 and rs208816121.These results indicate that an individual carrying the The genetic architecture for resistance to mastitis in dairy cattle constitutes a great interest for current research, particularly in the last decade, given the intense selection and use of sires with high breeding value to maximize milk yield, which has had detrimental effects on functional traits such as udder health. The goal of the current research was to detect the genetic background involved in resistance to mastitis through analysis of SNP markers belonging to genes that were found to be associated with genetic resistance to mastitis, and evaluate the usefulness of the selected markers in Romanian Spotted and Romanian Brown cattle breeds. While considering that clinical mastitis shows low heritability ,30, the CXCR2, CXCL8, BRCA1, LTF, BOLA-DRB3 and TLR4) using Kompetitive Allele Specific PCR technology. Among the 89 SNPs, 54 (60.7%) failed and 35 (39.3%) were successfully genotyped, of which 31 SNPs were fixed. The KASP assay success rates in the present study were lower than in the literature, where success rates of 78.5% [The analyses were performed in Romanian Spotted and Romanian Brown cattle breeds on SNPs located in six genes (of 78.5%  and 94.8of 78.5%  were prep < 0.05). The rs110124025 SNP in the BOLA-DRB3 gene had a significant effect on mastitis prevalence. The genetic variant GA of rs110124025 was at a low frequency in both breeds. This might be attributed to the relatively small number of animals studied. The homozygous genotypes were more frequent than the heterozygous genotypes for the other polymorphic markers in the BOLA-DRB3 gene.Four of the investigated markers were polymorphic. Among polymorphic loci with MAF ≥ 5%, only the Romanian Spotted breed was in Hardy–Weinberg disequilibrium at the g.25475692C>T locus  in cattle, also known as bovine leukocyte antigen (BOLA), was found to play a critical role in the immune system, and due to its primary function in pathogen identification, has been used as a disease marker in cattle [BOLA-DRB3 is a highly polymorphic gene and has been studied as a potential candidate locus for mastitis susceptibility. This gene is located in the most polymorphic bovine MHC region, with more than 130 genetic variants [BOLA-DRB3 gene has been linked with resistance to different infectious diseases in cattle [The present study highlights that the n cattle . BOLA-DRvariants . It playn cattle ,37,41,42Current results agree with those published by Cziszter et al.  in a comBOLA-DRB3 genes with mastitis resistance in Romanian cattle are quite limited. The rs110124025 SNP in the BOLA-DRB3 was confirmed to be associated with mastitis resistance in our study. Therefore, our findings validate this SNP as a potential candidate for selection for udder health in Romanian dairy cattle.Data regarding the associations of CXCR2, CXCL8, TLR4, BRCA1, LTF, BOLA-DRB3) and identified one marker (rs110124025) in BOLA-DRB3 that was associated with frequency of mastitis and with the number of affected quarters. Our findings support the use of BOLA-DRB3 alleles in future research on cattle genetic resistance for mastitis, as well as use as a candidate for future genomic selection programs. The current work contributes to an increase in knowledge regarding the genetic variability of SNPs for udder health in cattle.The Kompetitive Allele Specific PCR genotyping assay was used to identify SNP markers in already known candidate genes associated with genetic resistance or susceptibility to mastitis. We have analysed the missense SNPs belonging to six genes (


Solanum tuberosum L.) is an important crop in China, and it is widely cultivated from Northeast China to South China. Thousands of varieties are bred by breeding institutions or companies, and distinguishing the different varieties based on morphological characteristics is difficult. Using DNA fingerprints is an efficient method to identify varieties that plays an increasingly important role in germplasm identification and property rights protection. In this study, the genetic diversity and population structure of 135 autotetraploid potatoes were evaluated using specific-locus amplified fragment sequencing (SLAF-seq) methods. A total of 3,397,137 high-quality single-nucleotide polymorphisms (SNPs), which were distributed across 12 chromosomes, were obtained. Principal component analysis (PCA), neighbour-joining genetic trees, and model-based structure analysis showed that these autotetraploid potato subpopulations, classified by their SNPs, were not consistent with their geographical origins. On the basis of the obtained 3,397,137 SNPs, 160 perfect SNPs were selected, and 71 SNPs were successfully converted to penta-primer amplification refractory mutation (PARMS-SNP) markers. Additionally, 190 autotetraploid potato varieties were analysed using these 71 PARMS-SNP markers. The PCA results show that the accessions were not completely classified on the basis of their geographical origins. The SNP DNA fingerprints of the 190 autotetraploid potato varieties were also constructed. The SNP fingerprint results show that both synonyms and homonyms were present amongst the 190 autotetraploid potatoes. Above all, these novel SNP markers can lay a good foundation for the analysis of potato genetic diversity, DUS  testing, and plant variety protection.The autotetraploid potato ( Solanum tuberosum L.) are the world’s fourth most important crop after rice, wheat, and maize. According to FAO data, the production of potatoes was 359 million tons across the world in 2021 (https://www.fao.org/faostat/en/#data/QI). The potato has an important role in food security, poverty alleviation, and improved health status because potatoes yield more food per unit of cropland in less time than any other major crop and are not at risk for the ill effects of speculative activity, unlike major cereal commodities [Potatoes  cluster tree of 135 potatoes was constructed based on 339,713,771 SNPs . The resAfter the 3,397,137 SNPs were filtered using (1) minor allele frequency (MAF) > 0.4, (2) miss rate < 0.2, (3) heterozygosity < 0.4, and (4) no sequence variation in the 100 bp flanking region, they were selected as perfect SNP candidates . A totalAfter PCR amplification was conducted, the fluorescence values of FAM (5-carboxyfluorescein) and HEX (hexachlorofluorescein succinimide ester) were detected. The genotype was analysed using the fitploy R package on the basis of dosage score. Out of the 71 PARMS-SNP markers, 2 (StSNP63 and StSNP139) had one dosage class, while 6 PARMS-SNP markers had zero, one, two, three, four, and five dosage classes. Most of the PARMS-SNP markers had three dosage classes . The meaThe StSNP13 locus, with 48 samples, was detected using Sanger sequencing to assess the accuracy of the genotype result. The results show that the dosage score obtained in this manner may not give the correct genotype, as demonstrated . HoweverAfter SNP loci were selected in accordance with PIC values > 0.60, a total of 21 SNP loci were selected to construct the SNP fingerprints of the 190 autotetraploid potatoes. The results show that “Longshu No. 6” and “Longshu No. 16” were identical, whereas the other 188 potatoes were uniquely identifiable .Different potato varieties could easily be mixed during transportation and conservation due to vegetative propagation. So, identifying variety authenticity is necessary. The UPGMA dendrogram shows that “Longshu No. 6” and “Longshu No. 16” were clustered together and that their SNP fingerprints were identical. The leaf morphological characteristics of the two samples were analysed to further verify the results and no differences were found between them, suggesting that the two samples were synonyms .There were three samples named “Longshu No. 7” . However, the pairwise genetic distances between these samples were 6.61 (A12 and H17), 3.09 (A12 and C17), and 6.14 (C17 and H17). In addition, the UPGMA dendrogram shows that A12, C17, and H17 did not cluster together. The SNP fingerprints of the three potatoes were different A. The poGenotyping-by-sequencing (GBS) is a powerful approach to identify SNPs and analyse crop genetic diversity ,39,40,41GBS and SNP arrays are effective approaches to analyse genetic diversity, but they are costly. Several PCR-based methods, such KASP, TaqMan, and PARMS, are also effective in detecting SNPs. In the present study, 160 perfect SNPs were selected on the basis of 135 GBS data. However, only 43% of these perfect SNPs were successfully converted to SNP-PARMS markers. The low successful conversion ratio may be due to limited read depths (average sequencing depth is 10×) and the complex autotetraploid genome. Uitdewilligen et al.  showed tPCR-based SNP identification methods, such as KASP, are widely used in diploids , but PCRIn this study, the genetic diversity of 190 autotetraploid potatoes genotyped using 69 SNP markers was analysed. The PCA results show that these potato clusters were not completely classified on the basis of their geographical origins. This result is consistent with the GBS result from the data of 135 potatoes. The pairwise genetic distance shows that the genetic diversity of the 190 potatoes was narrow because some potato varieties share the same parent or female relationships. So, extended genetic diversity is very important for autotetraploid potato breeding.Autotetraploid potatoes are conserved or shared by tubers in the field or tissue culture seedlings in the lab, easily leading to confusion due to mislabelling and mechanical mixture . DNA finDue to the complex breeding process, about 10 years is needed to develop new potato varieties. So, preventing unauthorised use of new varieties and support breeding activities is very important. Within the plant variety protection system, DUS testing is necessary. The current DUS testing process is labour-intensive, time-consuming, and environment-sensitive. Using DNA markers to supplement DUS testing is simple and efficient. These results could lay a solid foundation for potato DUS testing and plant variety protection.−1 for SLAF-seq and 20 ng μL−1 for PARMS-SNP genotyping.A total of 135 commercial autotetraploid potato cultivars  were colHae III-Hpy166II, GBS was performed for 135 cultivars following the protocol of Sun et al. [After the DNAs were digested using n et al. . The libn et al.  by usinghttps://itol.embl.de). PCA was conducted using Plink2 (https://www.cog-genomics.org/plink/2.0/).On the basis of the 3,397,137 identified SNPs, the genetic distances amongst 135 potatoes were analysed using neighbour-joining and polygenetic trees, which were constructed using Phylip . The polwww.snpway.com). The primers are listed in The detected SNPs were filtered using the following criteria: (1) MAF > 0.4, (2) miss rate < 0.2, (3) heterozygosity < 0.4, and (4) no sequence variation in the flanking region of 100 bp. SNPs that fulfilled these were selected as perfect SNP candidates . The priGenotyping tests were carried out with a 5 μL PCR reaction system and the thermal cycling program of PARMS in accordance with Lu et al. . PCR ampAfter the 190 potatoes were genotyped using 71 SNP markers, PCA was performed using the FactoMineR R package .Each SNP locus and variety were characterised by MA, OH, and PIC. MA was calculated as the proportion of the amount of “A” compared with the sum of all genotype calls for each sample, not including SNPs with no calls. OH was calculated as the proportion of heterozygous genotypes  compared with the sum of all genotype calls for each sample, not including SNPs with no calls. PIC was analysed following the description of Sasaki . DendrogIn this study, the genetic diversity of autotetraploid potatoes was analysed using GBS and SNP markers. The results demonstrate that SNP markers are a powerful tool to detect specific loci and/or alleles in autotetraploid potatoes. On the basis of the GBS data, 69 SNP markers were developed, and the genetic diversity of 190 autotetraploid potatoes was analysed using the 69 SNP markers. The SNP fingerprints of the 190 autotetraploid potatoes were also constructed. These novel 69 SNP markers could lay a solid foundation for the analysis of potato genetic diversity, DUS testing, and plant variety protection.


Growth and fatness traits are economically important in pig farming. The exploration of underlying genetic architecture for vital phenotypes is helpful in speeding up the process of genetic improvement for different pig breeds. Hence, for Diannan small-ear (DSE) pig, eight phenotypic traits including six body measurement traits and two fatness traits were examined. Based on the Geneseek Porcine 50K SNP Chip data, single nucleotide polymorphisms (SNPs) were detected in a DSE pig population. Through a genome-wide association study, some candidate genes were detected potentially related to the traits of interest. These findings help to understand the genetic basis of porcine growth traits could be used in future pig breeding schemes.VIPR2, SLC10A2, NUCKS1, MCT1, CHCHD3, SMOX, and GPR1, which are mainly involved with adipocyte differentiation, lipid metabolism, skeletal muscle development, and average daily weight gain. Our work offers novel insights into the genetic architecture of economically important traits in swine and may play an important role in breeding using molecular markers in the DSE breed.In the livestock industry, the growth and fatness traits are directly related to production efficiency and economic profits. As for Diannan small-ear (DSE) pigs, a unique indigenous breed, the genetic architecture of growth and fatness traits is still elusive. The aim of this study was to search the genetic loci and candidate genes associated with phenotypic traits in DSE pigs using GWAS based on the Geneseek Porcine 50K SNP Chip data. A total of 22,146 single nucleotide polymorphisms (SNPs) were detected in 265 DSE pigs and used for Genome-wide association studies (GWAS) analysis. Seven SNPs were found to be associated with back height, chest circumference, cannon bone circumference, and backfat thickness at the suggestive significance level. Based on gene annotation results, these seven SNPs were, respectively, mapped to the following candidate genes,  Growth and fatness traits are economically important in the global pig breeding industry. Pig growth and fatness traits, such as backfat thickness, body weight, body length, and chest circumference, are the key traits of interest when making a breeding program. This is because they have an important relationship with productivity. For instance, backfat thickness was regarded as the evaluation indicator for carcass lean percentage . A largehttps://www.animalgenome.org/cgi-bin/QTLdb/SS/index, accessed on 21 October 2022). It is noteworthy that a list of important QTLs for body growth and fat deposition traits on Sus scrofa chromosome (SSC)1, 2, 4, 6, and 7 were explored in multiple populations [HMGA1, GRM4, and PLAG1 were identified at the mapped loci [During the last decade, breeders specifically focused on improving the individual size and decreasing backfat thickness to elevate production efficiency. Genome-wide association studies (GWAS) represent a powerful and ubiquitous tool for investigating the genetic basis for phenotypic traits of interest, and it was widely used to assess the genetic architecture of growth and fatness traits in various swine populations, including Chinese local breeds such as Erhualian  and Bamaulations ,12,13,14ped loci ,15. ThesThe objective of the current work was to clarify the genetic architecture of growth and backfat traits in DSE pigs at the finishing stage. To this end, eight phenotypic traits including body weight (BW), body height (BOH), body length (BL), back height (BAH), chest circumference (CC), cannon bone circumference (CBC), abdominal circumference (AC), and backfat thickness (BF) were measured for 265 DSE pigs. Subsequently, using the Geneseek Porcine SNP50 BeadChip array platform, single nucleotide polymorphisms (SNPs) were genotyped in this population. Then, the GWAS analysis was used to detect QTL and to identify candidate genes for those eight traits. Our results offered a foundation for the molecular marker-assisted breeding and improvement of the growth and fatness-related traits in pigs.®, Minneapolis, MN, USA) was used to determine BF at between 3rd and 4th last ribs  [In current study, all DSE pigs  were derived from the Sipsongpanna breeding farm . Animals were grouped into different commercial pens and bred under the same nutritional and management conditions. A total of 265 individuals between 390 and 420 days old were selected to measure the growth and fatness-related traits. The recorded phenotypic values were BW, BOH, BAH, CC, BL, CBC, AC, and BF. According to the method applied in previous research , six traat 5 cm) . BW was The MEANS procedure of SAS  was used to calculate descriptive statistics for eight traits. Phenotype distribution plots were visualized using R package “ggpubr”. The phenotypic correlations among the eight traits were estimated and visualized by using the “psych” and “corrplot” R packages, respectively .p-value > 10−5.Animal ear tissues were collected for genomic DNA extraction through a typical phenol-chloroform protocol . The conTo investigate the population structure of DSE pigs and determine whether principal components (PCs) should be added into GWAS model, we performed a principal component analysis (PCA) based on the filtered SNPs. Briefly, eigenvalues and eigenvectors were calculated by GCTA software , and PCAN  represents the polygenic effect; A is the kinship matrix achieved prior from the SNPs; and e~N  refers to the residual error. Bonferroni correction method was used for the multiple test correction. The threshold values in this study were set based on the Bonferroni correction method. Briefly, N  was the total number of filtered SNPs, the genome-wide significant threshold  and genome-wide suggestive level  were used as thresholds. GEMMA (version 0.98.5)  was usedhttp://asia.ensembl.org/biomart/martview/, accessed on 23 January 2023). In addition, the GO annotation of candidate genes was performed using Gene Ontology Consortium .To identify the candidate genes of each significant SNP, we searched for annotated genes within 100 Kb upstream or downstream of each significantly associated SNP on pig reference genome (Sscrofa 10.2) (p < 0.01), whereas BF was weakly correlated with CC , BW , and BL  (< 0.001) .After genomic mapping and SNP calling, we obtained 22,146 SNPs, in total, in all tested individuals via a series of filters. The density distribution plot of filtered SNPs across Sscrofa genome are shown in Traits BAH, BF, CC, and CBC had at least one chromosome-level significant locus , Table 2To verify whether the phenotype-associated SNPs in this study were located in any previously known QTLs, the overlap between our GWAS results and previously reported QTL data were examined using PigQTLdb . CompareThe results of functional annotation for candidate genes are shown in Considerable attention has been given to genomic selection in either western or Chinese indigenous pig breeds, which can improve populations for a set of important traits. In the world, pork accounts for 35% of meat consumption, representing a vital component of human daily diets . The demMCT1, also named SLC16A1) gene was closest to ALGA0000014, which plays an important role in the process of glycolysis [MCT1 is an important carrier of short-chain fatty acids and lactate in various tissues [(+/−) mice (C57BL/6J) with a high-fat diet (HFD) and found that MCT1 (+/−) mice had decreased weight gain, which was largely due to reduced fat accumulation (50.0% less after 9 months of HFD), especially in white adipose and liver tissue [MCT1 were significantly increased during adipocyte differentiation of three different adipocyte cell lines, accompanied by the elevated lactate flux capacity [CHCHD3) was the candidate gene located nearest to the SNP (WU_10.2_1_778943) on SSC18. Suppression of CHCHD3 could result in tissue undergrowth and cell proliferation defects, suggesting that CHCHD3 has the capacity of regulating tissue growth [MCT1 and CHCHD3 are important candidate genes related to fat deposition. Additionally, the results in this study indicated that these two SNPs could be used as key molecular markers for improving the meat quality and/or meat production of pigs.In this study, seven potential SNPs that surpassed the suggestive level related to four phenotypes . Body measurement trait is closely related to productivity, because it reflects the pig’s growth status. For example, BF is one of the most important fatness traits for pigs. Here, two suggestive SNPs on SSC4 (ALGA0000014) and 18 (WU_10.2_1_778943) were found to be related to BF. The monocarboxylate transporter 1  and ultrasonic measurements of L. dorsi muscle depth (UMD) of Esme sheep [SMOX was significantly associated with fiber size, skeletal muscle development, and muscular atrophy and hypertrophy in animals [CC is highly positively related to body weight and is an important indicator of thinness and fatness . The SNPme sheep . In addi animals ,37,38. TGPR1, CMKLR2) gene. A previous study showed that GPR1 was involved in the generation of adipocytes [GPR1 contributes to bone loss in mice [GPR1 might be related to the powerful leg strength and jumping ability of DSE pigs.CBC reflects the physical quality of the livestock, whether it is strong or not . The sugipocytes . In addiipocytes . Interes in mice ,42. We sVIPR2) gene, also known as VPAC2R, which was involved in the insulin secretion process [VIPR2-knockout could cause growth inhibition, a decrease in fat mass, and increase in lean mass, as well as a change of IGF-I levels, indicating that VIPR2 possesses multiple functions in metabolism [SLC10A2, also name as ASBT). As reported, SLC10A2 is associated with body weight gain and could improve insulin sensitivity [SLC10A2 decreased serum triglyceride (TG) and hepatic production of TG, hinting at the vital role in regulation of lipid metabolism [NUCKS1) gene was identified near the SNP WU_10.2_9_72689043. A recent GWAS study indicated that NUCKS1 is associated with increases in height [Among the three candidate SNPs related to BAH, WU_10.2_18_1597750 on SSC18 was identified to be close to the vasoactive intestinal peptide receptor 2 ( process . In a pr process . In part process . Moreovetabolism . Anothersitivity . It was tabolism . The nucn height . When exIn the current study, only seven candidate SNP loci were identified with suggestive thresholds by the genome-wide association study, indicating the limitations of this study such as limited sample size and genotyping platform. Compared with other genotyping methods, such as whole genome resequencing analysis, the SNP chip data can only offer limited information throughout the whole genome. The SNP markers of the Geneseek Porcine 50K SNP Chip were mainly designed based on the SNP markers identified from western pig breeds. Therefore, some important information in the Chinese native pig breed such as the DSE pig may be ignored in this platform because of the different genetic background among different breeds. Moreover, the statistical power for candidate gene discovery could have been limited by small sample size. Therefore, it is necessary to verify these candidate loci’s genetic effects in a larger samples and other pig breeds. Additionally, further investigation on the functional mechanism of these important candidate genes identified in this research is necessary. Other strategies, such as whole-genome sequencing and other SNP chips designed specifically for Chinese native breeds of pigs, will be meaningful to increase the statistic power of the GWAS and elucidate the underlying function of specific genes.MCT1, SMOX, and VIPR2, which are mainly involved with adipocyte differentiation, skeletal muscle development, and average daily weight gain. These results can contribute to understanding the genetic basis of pig growth and development and provide potential candidate molecular markers for DSE pig-breeding programs.To summarize, a total of seven SNPs were found to be significantly associated with BAH, BF, CC, and CBC in DSE pigs by GWAS at the suggestive significance level. Based on gene annotation analysis, these phenotype-associated SNPs were mapped to the important candidate genes, such as


Aplasia cutis congenita (ACC) is a rare genetic disorder characterized by the localized or widespread absence of skin in humans and animals. Individuals with ACC may experience developmental abnormalities in the skeletal and muscular systems, as well as potential complications. Localized and isolated cases of ACC can be treated through surgical and medical interventions, while extensive cases of ACC may result in neonatal mortality. The presence of ACC in pigs has implications for animal welfare. It contributes to an elevated mortality rate among piglets at birth, leading to substantial economic losses in the pig farming industry. In order to elucidate candidate genetic loci associated with ACC, we performed a Genome-Wide Association Study analysis on 216 Duroc pigs. The primary goal of this study was to identify candidate genes that associated with ACC.KRT71, KRT1, KRT4, ITGB7, CSAD, RARG, SP7, PFKL, TRPM2, SUMO3, and TSPEAR.This study identified nine significant SNPs associated with ACC. Further analysis revealed the presence of two quantitative trait loci, 483 kb  on SSC 5 and 159 kb  on SSC13. By annotating candidate genes within a 1 Mb region surrounding the significant SNPs, a total of 11 candidate genes were identified on SSC5 and SSC13, including The results of this study further elucidate the potential mechanisms underlying and genetic architecture of ACC and identify reliable candidate genes. These results lay the foundation for treating and understanding ACC in humans.The online version contains supplementary material available at 10.1186/s12864-023-09803-6. Aplasia cutis congenita (ACC) is a rare congenital disorder characterized by the localized or widespread absence of skin in humans and animals , 2. The Since the successful completion of the Human Genome Project in 2003, remarkable advancements in genome sequencing technology have led to a substantial reduction in sequencing costs . These bDue to the low probability of ACC occurrence, it took us 5 years (2017–2021) to collect a total of 116 ACC-affected piglets (58 from American Duroc population and 48 from Canadian Duroc population) and 100 healthy piglets (50 from American Duroc population and 50 Canadian Duroc population). Subsequently, we conducted a genome-wide association analysis on a total of 216 Duroc piglets to elucidate candidate genetic loci associated with ACC. The primary goal of this study was to identify candidate genes that contribute to the development of ACC, thus providing valuable insights into the genetic architecture underlying skin aplasia. By leveraging the pig as a model organism, our research aimed to unravel the fundamental mechanisms governing skin development disorders, ultimately offering valuable implications for treating of related human diseases.Sus scrofa chromosome (SSC) 6 exhibited the most uniform distribution of SNPs.ACC can occur in various parts of piglets' bodies  were approximately 500 kb . As presented in Fig. P = 2.42E-5) and were identified as associated with ACC. Two SNPs (13_207176753 (P = 2.25E-5) and13_207265708 (P = 2.25E-5)) on SSC13 were found to be the lead SNPs. They are in complete LD and located within a 159 kb haplotype block , which suggests that mutations near the potential quantitative trait locus (QTL) may have essential effect on ACC . For more comprehensive information, please consult Fig. SAfter analyzing the quality-controlled SNPs and phenotype data using the MLM model in GEMMA software , signifiP-value of the associated SNPs all dropped below the threshold line  and 5_18617273 (P = 6.37E-5), which are located within a 483 kb haplotype block   as a covariate into the MLM using GEMMA software , Sp1 transcription factor (SP1), phosphofructokinase, liver type (PFKL), transient receptor potential cation channel subfamily M member 2 (TRMP2), respectively (Table r2) dropped below 0.2 after a distance of approximately 500 kb . KRT71 exhibits differential expression in various human skin regions, including the buttocks, arms, abdomen, and legs (https://www.bgee.org/gene/ENSG00000139648). Studies have highlighted the pivotal role of KRT71 in hair formation, as it is involved in abnormal hair follicle morphology, focal hair loss, Male-pattern baldness, and abnormal hair cortex morphology [KRT1 gene is a member of the keratin gene family. Type II cytokeratins consist of acidic or neutral proteins that form heterotypic pairs of keratin chains and are co-expressed during the differentiation of simple and stratified epithelial tissues (https://www.ncbi.nlm.nih.gov/gene/3848). Notably, KRT1 has been implicated in various conditions, including abnormal skin morphology, impaired skin barrier function, abnormal epidermal layer morphology, and neonatal lethality with incomplete penetrance [KRT1, another candidate gene, KRT4, encodes a protein that belongs to the keratin gene family. It has been implicated in a range of skin and hair abnormalities, including epidermal hyperplasia, abnormal morphology of the suprabasal layer in the epidermis, impaired skin barrier function, skin inflammation, abnormal morphology of the dermal layer, and diluted coat color [Through gene function annotation, we identified seven candidate genes associated with ACC on SSC5, namely rphology –27. The netrance . Like KRat color –32. All RARG is not only associated with skin abnormalities, such as abnormal epidermal layer morphology, abnormal epidermis stratum corneum morphology, and impaired skin barrier function [SP7 also correlates with skeletal developmental abnormalities [CSAD gene on SSC5, the peak SNP (5_18403003) located within, is associated with neonatal lethality and incomplete penetrance [ITGB7, is implicated in abnormal immune system function in animals [Research findings suggest that ACC may be accompanied by developmental deficits or musculoskeletal deficiencies . The canfunction , but alsfunction –38. The malities –41. Due netrance . Further animals , 44. ThePFKL gene on SSC13, the lead SNP (13_207176753) located within, encodes the liver (L) subunit of an enzyme that catalyzes the conversion of D-fructose 6-phosphate to D-fructose 1,6-bisphosphate, which is a crucial step in glucose metabolism (glycolysis). Previous studies have linked the PFKL gene to hair follicle degeneration and abnormal hair cycle catagen phase [TRPM2, located near another lead SNP (13_207265708), is closely associated with the innate immune system and may contribute to increased susceptibility to bacterial infections, potentially leading to mortality [The en phase . Anotherortality –48.TSPEAR gene participates in ectodermal development and plays a crucial role in tooth and hair follicle morphogenesis by regulating the Notch signaling pathway [SUMO3 gene also influences the ectoderm development in embryos, resulting in embryonic growth arrest and delayed somite formation [During embryonic development, the ectoderm undergoes differentiation to form the skin, hair, and skeletal system. Research has demonstrated that the  pathway , 50. It  pathway . The SUMormation . In summKRT71, KRT1, KRT4, ITGB7, CSAD, RARG, SP7, PFKL, TRPM2, SUMO3, and TSPEAR. These findings shed light on the genetic underpinnings of ACC in pigs, contribute to the understanding and treatment of ACC and provide a foundation for future studies elucidating the underlying molecular mechanisms.The present study aimed to investigate the candidate genes and loci associated with ACC. GWAS analysis was conducted on a herd of 226 Duroc pigs, identifying nine significantly associated SNPs. Further investigation revealed the presence of a 483 kb QTL  on SSC 5 and a 159 kb QTL  on SSC13. By annotating candidate genes within a 1 Mb region surrounding the significant SNPs, a total of 11 candidate genes were identified on SSC5 and 13, including https://arriveguidelines.org) for the reporting of animal experiments.The animals and experimental methods used in this study follow the guidelines of the Ministry of Agriculture of China and the Use Committee of South China Agricultural University (SCAU). The ethics committee of SCAU  approved all animal experiments. The experimental animals were not anesthetized or euthanized in this study. We confirmed that all methods are reported in accordance with ARRIVE guidelines  American Duroc piglets and 98 recently farrowed  Canadian Duroc piglets were collected from breeding farms in Guangdong Wen’s Foodstuffs Co., Ltd. . The farrowing sows were raised under the same environmental conditions, and all affected piglets were identified on-site personnel.r2 value of 0.89. This ensured the reliability and robustness of our imputed data. The genotype quality control of the 216 pigs was conducted by PLINK v1.9 software [P < 10–6) and was unmapped or located on the sex chromosome was also removed. After quality control, the final 41,362 (50 K) and 14,942,886 (SWIM imputed) SNPs are retained for subsequent analysis, respectively.The genomic DNA necessary for this study was obtained by applying the standard phenol/chloroform method to isolate and extract DNA from the tail tissues of 216 piglets. Subsequently, rigorous quality control measures were implemented, including assessment of the light absorption ratios (A260/280 and A260/230), gel electrophoresis analysis, and quantification of DNA concentration at 50 ng/μL. GenoBaits Porcine 50 K Panel was used for genotyping. After genotyping, we enhanced the genotype data to the whole-genome sequence level using an imputation strategy. We employed the Swine Imputation (SWIM) Server tool  with defsoftware . IndividPCA and LD analysis were performed using the SNPs that passed quality control standards to investigate the population structure of the two Duroc pig populations. PCA was performed with GCTA software v1.92.4beta . The avey represents a vector of the phenotype, with affected piglets assigned as 1 and healthy individuals assigned as 0; W is the incidence matrix of covariates, including fixed effects of the sex, and the top five principal components from PCA analysis; α represents the vector of corresponding coefficients including the intercept; X is the vector of all marker genotypes; β specifies the corresponding effect size of the marker; u is the vector of random effects, with u ~ MVNn ; ε is the vector of random residuals, with ε ~ MVNn ; λ signifies the ratio between two variance components; τ−1is the variance of the residual errors; K is GRM; I is an n × n identity matrix and n refers to the number of pigs. In the 50 K chips GWAS, the Bonferroni method was used to determine the genome-wide significant threshold (0.05/N), where N is the number of SNPs. Since that is a stringent criterion, we set a more lenient threshold to detecting the suggestive SNPs (1/N) [In the present study, the GEMMA software v0.98.1  was usedPs (1/N) , 57. FurPs (1/N) . Based oPs (1/N) , 60.The PLINK v1.9  and HaplSus scrofa 11.1 genome (http://ensembl.org/Sus_scrofa/Info/Index). Functional gene annotation (v105) was downloaded in GIFF3 format from the Ensembl website (http://ftp.ensembl.org/pub/release-105/gff3/sus_scrofa/). The R package BioMart [P value < 0.05 were selected to explore further the genes invoked in pathway and biological processes [http://revigo.irb.hr) in conjunction with the Mus musculus database to eliminate GO term redundancy  and cluster the remaining terms in a 2D space [https://www.informatics.jax.org/), GeneCards (http://www.genecards.org/) and Ensembl (www.ensembl.org/biomart/martview) were used to query gene functions.Based on the LD decay distances of the American and Canadian Duroc populations, the candidate genomic regions were determined to 500 kb on either side of the significant SNPs. All SNPs refer to the latest version of the  BioMart  efficien BioMart  and GO a BioMart  to invesrocesses . Subsequ2D space , 66. ThiAdditional file 1: Fig S1. Photos of ACC-afflicted piglets.Additional file 2: Table S1. Distributions of SNPs after QC and the average SNPs on each chromosome for ACC trait.Additional file 3: Fig S2. Manhattan plots of Imputed GWAS for ACC in 216 pigs. Description: The x-axis represents the chromosomes, and the y-axis represents the -log10. The dashed lines indicate the thresholds for ACC (P =1.00E-6).Additional file 4: Table S2. Significant SNPs associated with ACC in Duroc pigs identified by imputed GWAS. Description: This file provides information about significant SNPs discovered in the GWAS with imputed data.Additional file 5: Fig S3. Manhattan plots of 50K GWAS using logistic mixed model for ACC in 216 pigs. Description: The x-axis represents the chromosomes, and the y-axis represents the -log10. The dashed lines indicate the suggestive thresholds for ACC (P =2.42E-5).Additional file 6: Table S3. Genes annotation within 1 Mb surrounding nine significant SNPs identified in GWAS. Description: This file provides the positional information of protein-coding genes within a 1 Mb region surrounding significant SNPs.Additional file 7: Table S4. Pathway Enrichment Analysis Reveals Significantly Enriched KEGG and GO Pathways Related to ACC. Description: This file provides the enrichment of protein-coding genes in KEGG and GO terms (P < 0.05).


The article is a comprehensive review concerning tetracyclic triterpene and steroid glycosides from sponges . The extensive oxidative transformations of the aglycone and the use of various monosaccharide residues, with up to six possible, are responsible for the significant structural diversity observed in sponge saponins. The saponins are specific for different genera and species but their taxonomic distribution seems to be mosaic in different orders of Demospongiae. Many of the glycosides are membranolytics and possess cytotoxic activity that may be a cause of their anti-predatory activities. All these data reveal the independent origin and parallel evolution of the glycosides in different taxa of the sponges. The information concerning chemical structures, biological activities, biological role, and taxonomic distribution of the sponge glycosides is discussed. Sponges (phylum Porifera) are an oldest living group of Metazoa. Their general organization was so adaptable that they survived during dramatic changes in environment. This group of aquatic organisms (marine and freshwater) are very diverse and includes more than 8500 valid species ,2. The uIt is known that many sponge metabolites may be synthesized by symbiotic microorganisms . HoweverTetracyclic steroid and triterpene glycosides are characteristic substances for many terrestrial higher plants. Over the past few decades, a large number of triterpene glycosides have been identified in animals, specifically sea cucumbers  from various orders, exhibiting taxonomic specificity ,13,14,15Melophlus sarasinorum  is very common in the Indo-West Pacific tropical region. This sponge also has such junior synonymic names as Asteropus sarasinorum, Melophlus isis and Stellettinopsis isis -Bottom Hunter labeled C5a to its receptor (IC50 > 10 μM). Eryloside E (31) reveals also immunosuppressive activity (EC50 of 1.3 μg/mL). Its immunosuppressive action was specific and independent from general cytotoxicity . Gl. Gl48), 49), a similar lanostane bioside, as a result of their investigation into E. formosus. The sponge was harvested near the Bahamas through scuba diving at a depth of 55 feet. It has the same aglycone, also containing arabinose residue as the first monosaccharide residue linked to C-3 of the aglycone and the second sugar  attached at the arabinose C-2 position  wer werLipas, A2 (2) ,24, H1 ( H2 (15) , J (18),d M (21) .73–77 are 14-nor-lanostane derivatives having a 23-ketone and a 24(25)-double bond in the side chain. The aglycone of 73 has an 8(9)-double bond whereas aglycones of 74 and 75 possess a 9(11)-double bond. Aglycone of 74 has additional 5α- and 8α-hydroxyls whereas aglycon of 75 possess 5β- and 8β-hydroxyls. The aglycone of 76 has an 8(14)-double bond and 9α-hydroxy and 8(9)-epoxy groups. The aglycone of 77 has 8α,9α-diol group and C-18 methyl shifted to 14β-position. It also has a unique 12(13)-double bond. Most of these glycosides feature a pentasaccharide chain that is very typical of sarasinosides. However, glycoside 73 is unique in that it is a trioside lacking two glucose residues, specifically the terminal and pre-terminal ones . Th. Th80), 80 was proposed along with biogenetical reasons. All the isolated triterpene glycosides were not active against two human solid cancer cell lines, Huh-7 (hepatocarcinoma) and A549 (lung carcinoma) . Al. AlPoecie A (96) . AdditioPandaros acanthifolium belonging to the family Microcionidae was harvested from canyon rock near Martinique Island. A series of new steroid glycosides, named pandarosides, were isolated as result of the investigation into this sponge. Pandarosides A–D (108–111) are glycosides with uncommon sterol aglycones, which have an unusual cis-C/D ring junction that is oxidized in ring D [D-glucose and D-glucuronic acids or D-glucuronic acid. The carbohydrate moiety is attached to C-3 of the aglycone (The sample of Caribbean sponge n ring D . Their caglycone . During 112–117) and their methyl esters 112a–117a, have been isolated from the same sponge [114) and its methyl ester 114a were inhibitors of the growth of Leishmania donovani  and Trypanosoma brucei rhodesiense  [In continuation, the next series of steroidal glycosides, pandarosides E–J  .118–120) were isolated as minor components from the same species [118a–120a were also obtained during the isolation procedure probably as artefacts  and its methyl ester (114a), which were inactive on T. b. rhodesiense [Finally, pandarosides K–M  and pandarosides G and L  have additional 8(9)- and 7(8)-double bonds, respectively. All pandarosides have a 23-keto group in their side chains. Differences were observed at C-24, which is either free or contains methyl, ethyl, methylene, or ethylene groups. Four types of carbohydrate chains were found in pandarosides, and all contain β-D-glucuronic acid as the first monosaccharide residue. The obtaining methyl esters of pandarosides, most likely, are artifacts formed during the MeOH extraction steps.Thus, all pandarosides have rare aglycones with 14β-H, a 16(17)-double bond, and 3β,16-diol and 15-keto groups. Pandarosides E and F (121–126)  . Acanthi121–126) are connected to either C-15 (in glycosides 121–123) or C-16 (in glycosides 124–126) of their steroid aglycones. Acanthifoliosides A–C (121–123) are mono-β-D-xylopyranosides, whereas acanthifoliosides D and E (124 and 125) are mono-α-L-rhamnopyranosides. However, acanthifolioside F (126) is a branched trioside (126a) was also isolated and probably formed during the extraction process as previously observed for pandarosides [121–126) and the methyl ester of acanthifolioside F (126a) [P. acanthifolium glycosides and starfish steroid glycosides, because of oxidation of D-ring in C-15 and C-16 positions [The carbohydrate residues of acanthifoliosides A–F  . There iositions ,18,19.P. acanthifolium was harvested through scuba diving near the coast of Marathon in the Florida Keys  and was investigated by a group of Canadian chemists and biochemists [127–130) E-double bond in the side chain. The aglycones of 129 and 130 are 5α-poriferastan and poriferast-5-en derivatives, respectively. The side chains of the both of aglycones possess a hydroxyl at C-23 and ethyl group at a 24-position, as well as in acanthifoliosides D–F [127) exhibited antioxidant and cytoprotective activities [The next sample of chemists . They ha127–130) . These gides D–F . The hydtivities .Mycale  by Russian scientists in 1981 [131) was isolated from the Caribbean sponge Mycale laxissima as individual substance [132–141) were reported [D-galactopyranose, one D-glucopyranose, and one D-arabinopyranose residues. In mycalosides D, F, and K  the glucopyranose residue has acetate group at C-6. One galactopyranosyl moiety attached to C-4 of glucopyranosyl residue by a β-glycosidic bond, another galactopyranosyl unit attached to C-2 of arabinopyranose by a α-glycosidic bond. Their aglycones are steroid derivatives, as they have oxidized functionalities in rings A, D, and side chains. All mycalosides, with the exception of mycaloside I (139), have an unusual oxidation at C-21, which is characteristic of steroids from ophiuroids [132) and C (133) are 27- and 28-norsteroid derivatives of the glycoside 131, respectively. Mycaloside D (134) differs from mycaloside A (131) only by the presence of an additional acetyl at C-6 of the first glucose in the carbohydrate moiety. Mycaloside E (135) is a 28-nor-4-deoxy-mycaloside A. Mycalosides F–H (136–138) have new 5(6)-unsaturated 3β,4β,21-trihydroxy-15-keto-steroidal aglycones and differ from each other by aglycone side chains. These glycosides have nonacetylated  or acetylated by C-6 of the first glucose residue (136) in the tetrasaccharide carbohydrate chains. Mycaloside I (139) contains a tetraoside with a novel aglycone featuring a 7,24(28)-diunsaturated-3β,15β,29-trihydroxystigmastane derivative. Mycaloside I (135) contains a new aglycone, which differs from the aglycone of 131 by the absence of C-4 hydroxyl. Mycaloside K (141) is C-24 epimer of mycaloside D (134) (131) as well as micaloside G (137) have an inhibition action on the fertilization of eggs by sperm of the sea urchin Strongylocentrotus nudus after preincubation [The steroid oligoglycosides, similar to starfish asterosaponins by containing several monosaccharide residues, were first found in a sponge of the genera  in 1981 . Unfortuubstance . Then, treported ,75  . The totcubation .Niphates olemda , (family Niphatidae) is characteristic for the Indo-Pacific region and was collected at a depth of 40 m in Micronesia. The found in this sponge hapaioside (142) has an uncommon aglycone with a polycyclic nucleus, resembling several steroid hormones. It has a 4-hydroxy-6-oxo-19-norpregnane skeleton. The glycoside is a monoside and its monosaccharide residue is 6-deoxy-β-L-altropyranose-4-acetate [The sponge -acetate   and B (144), as well as sulfated polyhydroxysteroids were also obtained. The specimen was collected from shallow waters in the Solomon Islands. , which has been harvested in various geographical locations ranging from Guam to the north-western coast of Australia. Sarasinoside A3 (3) has been isolated from M. sarasinorum harvested in shallow waters near the north-western coast of Australia, Guam Island, Palau Islands, and Sulawesi Island. Similarly, sarasinoside A2 (2) was found in specimens harvested from shallow waters near the north-western coast of Australia, Palau Island, and other regions. A very similar character of distribution was found for erylosides of Erylus formosus, from which formoside (47) and eryloside F (49) were isolated from the samples harvested near Puerto Morelos and Bahamas in the Gulf of Mexico. Discoveries of glycosides with identical structures in different species, even those within the same genus, are uncommon. However, an instance of such a finding is eryloside H (67), which has been identified in Erylus formosus harvested near Puerto Morelos and in samples of E. nobilis from the waters of Jaeju Island, Republic of Korea. Nevertheless, some sponge genera may be characterized by the occurrence of closely related glycosides. This may be exemplified by erylosides found in different species of Erylus, which was collected in the shallow waters of three oceans . These glycosides contain 14-carboxy- or 14-nor-methyl-lanostane aglycone. Some of them are additionally dealkylated at C-4 and alkylated in sidechains. General carbohydrate chains architectures for a series of glycosides isolated from Erylus spp. are frequently close, but, as a rule, exact structures are a little different.Frequently, the presence of occurring triterpene and steroid glycosides in sponges can result in highly complex mixtures, making their separation a challenging task. Consequently, the difficulty in isolating these glycosides may hinder their utilization as chemotaxonomic markers. Due to the complexity of glycoside fractions, many researchers may only isolate a portion of the components at random, whereas another group may isolate a different portion. Comparing such data may lead to inaccuracies. Furthermore, distinct structural series of triterpene and steroid glycosides are distributed among taxa that are different from each other and widely separated. The most frequently the tetracyclic triterpene and steroid glycosides are presented in two orders—Tetractinellida (suborder Astrophorina) and Poecilosclerida. In total, the glycosides were found in the sponges belonging to five orders  very similar to other glycosides of eryloside series [Petrosia sp. and Petrosia nigricans, the representative of the order Haplosclerida [Pachastrella scrobiculosa (family Pachastrellidae) and Poecillastra compressa (family Vulcanellidae), which were harvested near Miura Peninsula, Japan [As we already have noted, a Korean group recently reported on the finding of a series of sarasinosides from the sponge w waters . Anothere series . A seriesclerida ,48. Verya, Japan  and the a, Japan , respectThe discovery of similar occurrences suggests a parallel origin and evolution of terpenoid and steroid glycosides in Demospongiae.Erylus formosus harvested near Bahamas and its absence in the specimens collected in Floridian waters.Therefore, triterpene and steroid glycosides can be regarded as taxonomic markers that are specific to certain species and occasionally even to the level of genus or subgenus. However, the use of them for improvement of sponge taxonomy may be difficult because of the presence of very complicated mixtures of close related substances in glycosidic fractions, and seasonal and ecological variability in the content of some components of glycosidic fractions in the different collection of the same sponge. This was described, for example, by Kubanek et al. , who fouErylus formosus harvested in shallow waters near Bahamas and Southern Florida deters fish-predators Thalassoma bifasciatum. Formoside (47) and formoside B (48), which have four monosaccharide residues in their carbohydrate chains, were found to be less active compared to the non-separated hexaoside fraction, which contains penasterol as the aglycone. The total glycoside sum indicated more activity than any separate subfraction or individual glycoside. Thus, triterpene glycosides of sponges may be a defensive agent against fish-predators [Sponges are very important parts of coral reef ecological systems . Kubanekredators .Erylus formosus and Ectyoplasia ferox demonstrated the activities of the glycosides not only as fish deterrents, but also as antifouling agents against biofilm-forming bacteria, invertebrates, and algae [18) [114) [95) [1 (1) [The investigation on triterpene glycosides from two Caribbean sponges nd algae ,83. Morend algae ,83,84,85gae [18) , sokodosgae [18) , some ergae [18) ,31,41, a8) [114) , may als8) [114) ,26,41, i14) [95) , as well) [1 (1) .Danio rerio rejected an artificial diet containing the sponge triterpene glycosides. Transcripts from a zebra fish cDNA library were expressed in oocytes of Xenopus laevis and checked for activation of a chemoreceptor via electrophysiology and electrophysiological responses of ectyoplasides A, B , and formoside (47) were revealed [Ecological activities of sponge triterpene glycosides may be moderated even by minor structural differences. Some triterpene glycosides are not excreted in sea water, but their concentration on surface of sponge specimens was high. However, these sponges may deter their enemies and competitors by surface direct contacts with triterpene glycosides. This is more probable than by contacts via sea water . The disrevealed . Later, revealed ,85. It wBiological activities of sponge steroid glycosides are similar to such of terpenoid ones see . For exaAbout 144 sponge saponins were isolated by the end of 2022, including 94 tetracyclic triterpenoid glycosides and 50 steroid ones. The presence of triterpene and steroid glycosides is a characteristic feature of a few taxonomic groups . All of them were found within the class Demospongiae, but they are not characteristic for the class as a whole. The fact that terpenoid and steroid glycosides are distributed in a mosaic pattern within the class Demospongiae, and that glycosides with similar structures can be found in representatives of different orders, along with the data on their biological activities and roles, suggests that these substances may have evolved independently as defensive agents in several taxonomic groups of Demospongiae, and may have undergone parallel evolution.These glycosides are very diverse from a chemical point of view and quite different from the structures of the both aglycone and carbohydrate chains. They are more structurally diverse than other saponins from marine invertebrates or terrestrial plants. The structures of both steroid and triterpene aglycones are the result of intensive oxidative processes that allow high activity of the corresponding cytochrome P-450 family of enzymes in the sponges belonging to saponin-producing taxa. These glycosides may be not only mono- and biosides, but also oligoglycosides. All the monosaccharides are in pyranose form and most of them belong to D-series; however, L-sugars also may be found. The monosaccharides may be acetylated by C-6 or may be in uronic acid form ; however, they do not contain any OMe groups. Sponge triterpene glycosides demonstrate deterrent and ichthyotoxic properties, and many of them and their steroid analogs have cytotoxic and antiprotozoal activities that may be a cause of their defensive, antifouling, and allelopathic biological functions. It seems probable that new marine expeditionary research and the progress in development of separation and spectral technique may allow for the discovery of new, interesting structural versions of triterpene and steroid glycosides in sponges.


Here, we investigated the association of vgll3 and six6 in the domestication of gilthead seabream and European seabass, both undergoing selective breeding for growth‐related traits in the Mediterranean. We analysed two different sets of samples using two different genotyping approaches. The first dataset comprised farmed and wild populations from Greece, genotyped for SNPs within the two genes (‘gene‐level genotyping’). The second dataset examined 300–600 k SNPs located in the chromosomes of the two genes, derived from a meta‐analysis of a Pool‐Seq experiment involving farmed and wild populations distributed widely across the Mediterranean (‘chromosome‐level genotyping’). The gene‐level analysis revealed a statistically significant allele frequency differences between farmed and wild populations on both genes in each species. This finding was partially supported by the chromosome‐level analysis, identifying highly differentiated regions may be involved in the domestication process at varying distances from the candidate genes. Noteworthy genomic features were found, such as a CpG island in gilthead seabream and novel candidate genes in European seabass, warranting further investigation. These findings support a putative role of vgll3 and six6 in the maturation and growth of gilthead seabream and European seabass, emphasizing the need for further research on their conserved function.Gene function conservation is crucial in molecular ecology, especially for key traits like growth and maturation in teleost fish. The  vgll3 and six6 genes, known to influence age‐at‐maturity in Atlantic salmon, in gilthead seabream and European seabass, both undergoing selective breeding for growth‐related traits in the Mediterranean. Two distinct genotyping approaches, gene‐level and chromosome‐level, were employed, revealing significant domestication effects on both genes in each species, with noteworthy genomic features identified. These findings indirectly support a potential role of vgll3 and six6 in the maturation and growth of gilthead seabream and European seabass, highlighting the need for further research on their conserved function.The research investigates the impact of  By leveraging the accumulated knowledge and insights gained from often costly and labour‐intensive genetic and ecological experiments conducted in model species, researchers can apply these insights to other ecologically or commercially important species. This strategy is widely used in molecular ecology within the framework of the candidate gene approach, to address diverse research questions, most notably the genetic basis of various traits in a broad range of species , revealing evidence of functional conservation across large evolutionary distances. The vestigial‐like family member 3 (vgll3) and SIX homeobox 6 (six6) genes have emerged as strong predictors of age‐at‐maturity in both wild and farmed Atlantic salmon selected for growth  and Steelhead trout , but not in Chinook salmon  and Coho salmon  , vgll3 has been suggested to play a role in growth during early development  and the European seabass (Dicentrarchus labrax) are economically important fish species in the Mediterranean region, with unresolved genetic bases of maturation and somatic growth  and obtained the intron/exon information from the associated gff file (GCF_900880675.1_fSpaAur1.1_genomic.gff). For European seabass, we obtained gene locations and intron/exon information from the annotations deposited at Ensembl for the European seabass genes . We then mapped the gene sequences to the chromosome‐level assembly of the species, namely the European seabass_V1.0 assembly (GenBank accession: GCA_000689215.1), using blastn sequence similarity searches. To ensure evolutionary conservation of the binding site, we anchored all primers to exons. After designing the primer pairs, we tested them for successful PCR amplification and then Sanger‐sequenced the amplicons in subsets of farmed and wild‐collected samples from different origins to increase the likelihood of finding polymorphic SNPs in a cost‐efficient manner . We further characterized the coding SNPs as missense or synonymous using the AliView program  and 2.5 μL of 10x Reaction Buffer . We assessed the success of the PCR products by electrophoresis in 1.5% (w/v) agarose gels. We subjected the PCR products that amplified successfully to enzymatic cleanup using ExoI and rSAP (New England Biolabs) as per the manufacturer's protocol and outsourced the cleaned PCR products to the Genewiz company  for Sanger sequencing. To identify SNPs, we aligned the resulting sequences with the respective reference sequence of GenBank using the Geneious program , and the other half at an earlier period, between 2004 and 2007  , incorporating the ‘forward/backward’ strategy in R to identify the model that best fitted the data given the factors of ‘origin’ (farmed and wild populations) and ‘time’ (two periods of collection: 2004–2007 and 2012–2016) as described above ). To account for the proportions of the data, we used a binomial error with the logit function. The AIC is defined as −2*log‐likelihood + 2*npar, where likelihood is the likelihood corresponding in each model and npar represents the number of parameters in the fitted model. All statistical analyses and visualizations were conducted in R v.3.6.1  implemented with the 6 in gilthead seabream and 104 in European seabass to 100 individuals. These reductions are consistent with historical events documented to have occurred about 5–10 generations ago .To address the confounding factor of known marked reductions in effective population sizes in the farmed populations due to breeding programs, we simulated bottleneck scenarios with parameters matching the described characteristics of the farmed populations in the two species. Specifically, we developed custom Python code to simulate bottlenecks, reducing the effective population size from 102.2vgll3 and six6 genes in each species. Specifically, we focused on chromosomes 9 and 16 harbouring the vgll3 and six6 genes in gilthead seabream, as well as on linkage groups 15 and 12 containing the vgll3 and six6 genes in European seabass.We utilized whole‐genome sequencing data of pooled samples (Pool‐Seq) from 14 farmed and 10 wild populations of gilthead seabream, as well as from 12 farmed and 12 wild populations of European seabass. These populations originated from nine countries within the Mediterranean Sea region  using the bwa mem algorithm , Fisher's exact test  and the Cochran–Mantel–Haenszel (CMH) test  between farmed and wild populations for each SNP.The allele frequencies between farmed and wild populations were compared using two programs: PoPoolation2  from BioMart for each species (Sparus_aurata.fSpaAur1.1.108 and Dicentrarchus_labrax. seabass_V1.0.105).BayPass was executed in Pool‐Seq mode with a burn‐in of 10,000 iterations  and recorded 10,000 samples with thinning (i.e. the number of iterations between two recorded samples) set to the default value of 25. Consequently, the post‐burn‐in length of the MCMC chain was 250,000 iterations. Other parameters were maintained at their default values. BayPass was employed to calculate the In addition, the biallelic SNPs identified at the gene‐level approach were cross‐referenced with the Pool‐Seq data. The population frequencies of these SNPs were examined using a non‐parametric Wilcoxon test to assess the statistical significance of the genotype frequency differences between farmed and wild populations in the Pool‐Seq dataset.33.1vgll3 and six6 in gilthead seabream and European seabass using 16 pairs of primers , SNPsix6: 16:14406471 (henceforth SNP2) in gilthead seabream, and SNPvgll3: 15:9203633, SNPsix6: 12:11591053 (henceforth SNP1), SNPsix6: 12:11591093 (henceforth SNP2) in European seabass. The genomic locations are according to the GCA_900880675.2 and GCA_000689215.1 genome assemblies in gilthead seabream and European seabass respectively. The exonic SNP was synonymous in gilthead seabream, whereas it resulted in a missense mutation (Ser332Ala) in European seabass. These SNPs did not concern the same codon between the two species.We PCR‐amplified nearly the entire transcribed region of p = 5.60e‐3 [vgll3]; p = 4.00e‐4 [six6 – SNP1]; p = 3.70e‐3 [six6 – SNP2]; European seabass: p = 3.95e‐2 [vgll3]; p = 3.50e‐3 [six6 – SNP1]; p = 1.70e‐3 [six6 – SNP2]; Figures p = 5.61e‐2; farmed populations: p = 7.25e‐2). GLMs confirmed allele frequencies differed significantly between domesticated and wild populations on the datasets, especially for six6 in gilthead seabream, even after accounting for time‐of‐sampling effects  ranging from 11% to 20% in gilthead seabream, and from 11% to 13% in European seabass, at a 5% significance level. At a 1% significance level, FPR ranged from 1% to 3% for both species and the two candidate genes , and 585,183 biallelic SNPs on chromosome 16 containing the six6 gene (SNP density: 51.39 ± 192.53 bp) in gilthead seabream. Similarly, we analysed 343,064 biallelic SNPs on linkage group 15 containing the vgll3 gene (SNP density: 74.52 ± 127.18 bp), and 310,807 biallelic SNPs on linkage group 12 containing the six6 gene (SNP density: 74.75 ± 133.6 bp) in European seabass. The significant difference in the reported number of SNPs, approximately 600 K on each of the gilthead seabream chromosomes compared to approximately 300 K on each of the European seabass chromosomes, can be attributed to technical factors. The gilthead seabream samples were sequenced using the higher throughput HiSeq X Ten platform, whereas the European seabass pools were sequenced using the HiSeq 4000 platform  located at 2.5 Mbp upstream of the vgll3 gene in gilthead seabream  and with non‐parametric Wilcoxon tests . Overall, the Pool‐Seq data revealed 41 biallelic SNPs were found on the vgll3 gene of gilthead seabream , 12 SNPs on six6 gene of gilthead seabream , 34 SNPs on vgll3 of European seabass  and 21 SNPs on six6 of European seabass .The biallelic nature of the SNPs of the gene‐level approach was confirmed in the Pool‐Seq data. The two studied SNPs of 3.3vgll3 gene in gilthead seabream contained a CpG island. Table Through the analysis of genes within 100 kbp of the identified peaks of differentiation, we discovered a range of potential regulatory elements and additional candidate genes Table . Notably4vgll3 and six6 genes between farmed and wild populations and their potential role in the domestication process of two commercially significant fish species, namely the gilthead seabream and the European seabass. Our findings support their potential involvement in their domestication process, and thus in growth, given that domestication in these fish is closely linked to growth‐related traits. To ensure the robustness of our conclusions, we employed a comprehensive approach that involved the genotyping of two distinct sample collections.In this study, we investigated the genetic differentiation in the 4.1vgll3 and six6 genes revealed significant allele and genotype frequency differences between farmed and wild populations suggesting that they may be involved in the domestication process  to a hydrophobic one . More experiments need to be performed to clarify the degree, if any, to which vgll3 and six6 may directly influence growth and maturation in gilthead seabream and European seabass.It is known that the domestication of the two species, gilthead seabream and European seabass, has started in the 1980s . The gpr161 has been linked to developmental processes in various species, such as in zebrafish , which participates in the citric acid cycle. A locus of a malic enzyme (mMEP‐2*) located on the chromosome of vgll3 in Atlantic salmon, has been shown to affect early maturation ; formal analysis ; investigation ; methodology ; project administration (supporting); visualization (supporting); writing – original draft ; writing – review and editing . Theopisti Nikolaou: Methodology (supporting); writing – review and editing (supporting). Smaragda Sismanoglou: Methodology (supporting); writing – review and editing (supporting). Konstantinos Gkagkavouzis: Methodology (supporting); project administration ; visualization (supporting); writing – review and editing (supporting). Nikoleta Karaiskou: Methodology (supporting); project administration (supporting); writing – review and editing (supporting). Efthimia Antonopoulou: Investigation (supporting); methodology (supporting); supervision (supporting); writing – review and editing (supporting). Alexandros Triantafyllidis: Conceptualization (supporting); funding acquisition (supporting); investigation (supporting); methodology (supporting); project administration (supporting); supervision ; writing – review and editing (supporting). Spiros Papakostas: Conceptualization (lead); data curation (lead); formal analysis (lead); funding acquisition (lead); investigation ; methodology ; project administration ; resources (lead); software (lead); supervision ; validation ; visualization ; writing – original draft ; writing – review and editing (lead).https://github.com/spirospapakostas/MedFish_domestication.This article has earned Open Data and Open Materials badges. Data and materials are available at NCBI's Sequence Read Archive (SRA) accession number PRJEB40423 and