Systematic analyses for candidate genes of milk production traits in water buffalo (Bubalus Bubalis)

Water buffalo (Bubalus bubalis) is of great economic importance as a provider of milk and meat in many countries. However, the milk yield of buffalo is much lower than that of Holstein cows. Selection of candidate genes related to milk production traits can be applied to improve buffalo milk performance. A systematic review of studies of these candidate genes will be greatly beneficial for researchers to timely and efficiently understand the research development of molecular markers for buffalo milk production traits. Here, we identified and classified the candidate genes associated with buffalo milk production traits. A total of 517 candidate genes have been identified as being associated with milk performance in different buffalo breeds. Nineteen candidate genes containing 47 mutation sites have been identified using the candidate gene approach. In addition, 499 candidate genes have been identified in six genome‐wide association studies (GWASes) including two studies performed with the bovine SNP chip and four studies with the buffalo SNP chip. Genes CTNND2 (catenin delta 2), APOB (apolipoprotein B), FHIT (fragile histidine triad) and ESRRG (estrogen related receptor gamma) were identified in at least two GWASes. These four genes, especially APOB, deserve further study to explore regulatory roles in buffalo milk production. With growth in the number of buffalo genomic studies, more candidate genes associated with buffalo milk production traits will be identified. Therefore, future studies, such as those investigating gene location and functional analyses, are necessary to facilitate the exploitation of genetic potential and the improvement of buffalo milk performance.     

Systems genetics approach reveals candidate genes for parasite resistance from quantitative trait loci studies in agricultural species

A systems genetics approach combining pathway analysis of quantitative trait loci (QTL) and gene expression information has provided strong evidence for common pathways associated with genetic resistance to internal parasites. Gene data, collected from published QTL regions in sheep, cattle, mice, rats and humans, and microarray data from sheep, were converted to human Entrez Gene IDs and compared to the KEGG pathway database. Selection of pathways from QTL data was based on a selection index that ensured that the selected pathways were in all species and the majority of the projects overall and within species. Pathways with either up- and down-regulated genes, primarily up-regulated genes or primarily down-regulated genes, were selected from gene expression data. After comparing the data sets independently, the pathways from each data set were compared and the common set of pathways and genes was identified. Comparisons within data sets identified 21 pathways from QTL data and 66 pathways from gene expression data. Both selected sets were enriched with pathways involved in immune functions, disease and cell responses to signals. The analysis identified 14 pathways that were common between QTL and gene expression data, and four directly associated with IFNγ or MHCII, with 31 common genes, including three MHCII genes. In conclusion, a systems genetics approach combining data from multiple QTL and gene expression projects led to the discovery of common pathways associated with genetic resistance to internal parasites. This systems genetics approach may prove significant for the discovery of candidate genes for many other multifactorial, economically important traits.     

A functional genomics approach to evaluate candidate genes located in a QTL interval for milk production traits on BTA6

The potential genetic and economic advantage of marker-assisted selection for enhanced production in dairy cattle has provided an impetus to conduct numerous genome scans in order to identify associations between DNA markers and future productive potential. One area of focus has been a quantitative trait locus on bovine chromosome 6 (BTA6) found to be associated with milk yield, milk protein and fat percentage, which has been subsequently fine-mapped to six positional candidate genes. Subsequent investigations have yet to resolve which of the potential positional candidate genes is responsible for the observed associations with productive performance. In this study, we analysed candidate gene expression and the effects of gene knockdown on expression of β- and κ-casein mRNA in a small interfering RNA transfected bovine in vitro mammosphere model. From our expression studies in vivo , we observed that four of the six candidates ( ABCG2 , SPP1 , PKD2 and LAP3 ) exhibited differential expression in bovine mammary tissue over the lactation cycle, but in vitro functional studies indicate that inhibition of only one gene, SPP1 , had a significant impact on milk protein gene expression. These data suggest that the gene product of SPP1 (also known as osteopontin) has a significant role in the modulation of milk protein gene expression. While these findings do not exclude other positional candidates from influencing lactation, they support the hypothesis that the gene product of SPP1 is a significant lactational regulatory molecule.     

Genome-wide association mapping in Norwegian Red cattle identifies quantitative trait loci for fertility and milk production on BTA12

Reproductive performance is a critical trait in dairy cattle. Poor reproductive performance leads to prolonged calving intervals, higher culling rates and extra expenses related to multiple inseminations, veterinary treatments and replacements. Genetic gain for improved reproduction through traditional selection is often slow because of low heritability and negative correlations with production traits. Detection of DNA markers associated with improved reproductive performance through genome-wide association studies could lead to genetic gain that is more balanced between fertility and production. Norwegian Red cattle are well suited for such studies, as very large numbers of detailed reproduction records are available. We conducted a genome-wide association study for non-return rate, fertility treatments and retained placenta using almost 1 million records on these traits and 17 343 genome-wide single-nucleotide polymorphisms. Genotyping costs were minimized by genotyping the sires of the cows recorded and by using daughter averages as phenotypes. The genotyped sires were assigned to either a discovery or a validation population. Associations were only considered to be validated if they were significant in both groups. Strong associations were found and validated on chromosomes 1, 5, 8, 9, 11 and 12. Several of these were highly supported by findings in other studies. The most important result was an association for non-return rate in heifers in a region of BTA12 where several associations for milk production traits have previously been found. Subsequent fine-mapping verified the presence of a quantitative trait loci (QTL) having opposing effects on non-return rate and milk production at 18 Mb. The other reproduction QTL did not have pleiotropic effects on milk production, and these are therefore of considerable interest for use in marker-assisted selection.     

Differential expression of genes in milk of dairy cattle during lactation

The milk fat globule (MFG) is one of the most representative of mammary gland tissues and can be utilized to study gene expression of lactating cows during lactation. In this study, RNA‐seq technology was employed to detect differential expression of genes in MFGs at day 10 and day 70 after calving between two groups of cows with extremely high (H group) and low (L group) 305‐day milk yield, milk fat yield and milk protein yield. In total, 1232, 81, 429 and 178 significantly differentially expressed genes (false discovery rate q < 0.05) were detected between H10 and L10, H70 and L70, H10 and H70, and L10 and L70 respectively. Gene Ontology enrichment and pathway analysis revealed that these differentially expressed genes were enriched in biological processes involved in mammary gland development, protein and lipid metabolism process, signal transduction, cellular process, differentiation and immune function. Among these differentially expressed genes, 178 (H10 vs. L10), 4 (H70 vs. L70), 68 (H10 vs. H70) and 22 (L10 vs. L70) were found to be located within previously reported QTL regions for milk production traits. Based on these results, some promising candidate genes for milk production traits in dairy cattle were suggested.     

Integration of QTL and bioinformatic tools to identify candidate genes for triglycerides in mice

To identify genetic loci influencing lipid levels, we performed quantitative trait loci (QTL) analysis between inbred mouse strains MRL/MpJ and SM/J, measuring triglyceride levels at 8 weeks of age in F2 mice fed a chow diet. We identified one significant QTL on chromosome (Chr) 15 and three suggestive QTL on Chrs 2, 7, and 17. We also carried out microarray analysis on the livers of parental strains of 282 F2 mice and used these data to find cis-regulated expression QTL. We then narrowed the list of candidate genes under significant QTL using a "toolbox" of bioinformatic resources, including haplotype analysis; parental strain comparison for gene expression differences and nonsynonymous coding single nucleotide polymorphisms (SNP); cis-regulated eQTL in livers of F2 mice; correlation between gene expression and phenotype; and conditioning of expression on the phenotype. We suggest Slc25a7 as a candidate gene for the Chr 7 QTL and, based on expression differences, five genes (Polr3 h, Cyp2d22, Cyp2d26, Tspo, and Ttll12) as candidate genes for Chr 15 QTL. This study shows how bioinformatics can be used effectively to reduce candidate gene lists for QTL related to complex traits.     

Dissection of the genetic architecture of three seed‐quality traits and consequences for breeding in Brassica napus

Genome‐wide association studies (GWASs) combining high‐throughput genome resequencing and phenotyping can accelerate the dissection of genetic architecture and identification of genes for plant complex traits. In this study, we developed a rapeseed genomic variation map consisting of 4 542 011 SNPs and 628 666 INDELs. GWAS was performed for three seed‐quality traits, including erucic acid content (EAC), glucosinolate content (GSC) and seed oil content (SOC) using 3.82 million polymorphisms in an association panel. Six, 49 and 17 loci were detected to be associated with EAC, GSC and SOC in multiple environments, respectively. The mean total contribution of these loci in each environment was 94.1% for EAC and 87.9% for GSC, notably higher than that for SOC (40.1%). A high correlation was observed between phenotypic variance and number of favourable alleles for associated loci, which will contribute to breeding improvement by pyramiding these loci. Furthermore, candidate genes were detected underlying associated loci, based on functional polymorphisms in gene regions where sequence variation was found to correlate with phenotypic variation. Our approach was validated by detection of well‐characterized FAE1 genes at each of two major loci for EAC on chromosomes A8 and C3, along with MYB28 genes at each of three major loci for GSC on chromosomes A9, C2 and C9. Four novel candidate genes were detected by correlation between GSC and SOC and observed sequence variation, respectively. This study provides insights into the genetic architecture of three seed‐quality traits, which would be useful for genetic improvement of B. napus.     

Construction of a cucumber genetic linkage map with SRAP markers and location of the genes for lateral branch traits

Using SRAP (sequence-related amplified polymorphism) markers a genetic linkage map of cucumber was constructed with a population consisting of 138 F₂ individuals derived from a cross of the two cucumber lines, S06 and S52. In the survey of parental polymorphisms with 182 primer combinations, 64 polymorphism-revealing primer pairs were screened out, which generated totally 108 polymorphic bands with an average of 1.7 bands per primer pair and at most 6 bands from one primer pair. The constructed molecular linkage map included 92 loci, distributed in seven linkage groups and spanning 1164.2 cM in length with an average genetic distance of 12.6 cM between two neighboring loci. Based on this linkage map, the quantitative trait loci (QTL) for the lateral branch number (lbn) and the lateral branch average length (lbl) in cucumber were identified by QTLMapper1.6. A major QTL lbn1 located between ME11SA4B and ME5EM5 in LG2 could explain 10.63% of the total variation with its positively effecting allele from S06. A major QTL lbl1 located between DC1OD3 and DC1EM14 in LG2 could account for 10.38% of the total variation with its positively effecting allele from S₀₆.     

奶牛产奶性状与乳房炎相关基因CpG含量及分布特征的比较分析

DNA甲基化是表观遗传学的重要组成部分,基因启动子区及第一外显子区的CpG甲基化通常抑制该基因的表达,而去甲基化则促进基因表达。已有的研究发现荷斯坦牛的乳房炎指标SCC(Somatic cell count)与产奶量呈较强负相关。文章分析并比较了这两类性状的相关基因的启动子区、第一外显子、下游2 000 bp序列中CpG含量及分布特征。结果表明,乳房炎相关基因的启动子、第一外显子中CpG含量显著低于产奶性状相关基因,而两类性状基因下游2 000 bp序列中CpG含量无显著性差异。另外,文中提出了两个量化基因序列中CpG特征的指标,一个是CpG平均距离,用来衡量序列中的CpG分布;另一个是条件概率p(G|C),用以量化序列中二核苷酸CpG随碱基C出现的可能性,并对两类基因的启动子和第一外显子区域的这两个指标做了统计检验。研究结果对产奶性状与乳房炎相关基因的DNA甲基化调控研究奠定了基础。     

Whole-genome scan to detect QTL for milk production, conformation, fertility and functional traits in two US Holstein families

A genome scan was conducted in two US Holstein half-sib families to identify quantitative trait loci (QTL) affecting milk production and conformation traits using the granddaughter design. The sires of the two studied families were related as sire and son and had 96 and 212 sons respectively. A total of 221 microsatellite loci were scored in both families. Statistical analysis was performed using two different analytical methods; half-sib least squares regression and Bayesian Monte Carlo Markov Chain. Traits analysed included five traditional milk production traits, somatic cell count, daughter pregnancy rate, male fertility and 20 conformation traits. A total of 47 tests achieved at least genome-wise significance. However, results from the two methods of analysis were only concordant for QTL location and level of significance in eight instances.     

Detection of loci affecting milk production and health traits in an elite US Holstein population using microsatellite markers

Quantitative trait loci (QTL) affecting health and milk production traits were studied in seven large half-sib US Holstein families by using the granddaughter design. Genotyping for 16 markers was completed and marker allele differences within and pooled-across families were analysed. Potential QTL were identified for somatic cell score (SCS), fat yield, fat percentage, protein yield and protein percentage. Three markers (BM203, BM4505 and BM2078) were associated with significant effects for different traits and, after further analysis, may be useful in marker-assisted selection in specific families. Comparisons between these data and previously identified QTL support the location of a QTL for milk yield and protein yield on chromosome 21.     

A genome‐wide association study suggests new candidate genes for milk production traits in Chinese Holstein cattle

A genome‐wide association study (GWAS) was conducted on 15 milk production traits in Chinese Holstein. The experimental population consisted of 445 cattle, each genotyped by the GGP (GeneSeek genomic profiling)‐BovineLD V3 SNP chip, which had 26 151 public SNPs in its manifest file. After data cleaning, 20 326 SNPs were retained for the GWAS. The phenotypes were estimated breeding values of traits, provided by a public dairy herd improvement program center that had been collected once a month for 3 years. Two statistical models, a fixed‐effect linear regression model and a mixed‐effect linear model, were used to estimate the association effects of SNPs on each of the phenotypes. Genome‐wide significant and suggestive thresholds were set at 2.46E‐06 and 4.95E‐05 respectively. The two statistical models concurrently identified two genome‐wide significant (P < 0.05) SNPs on milk production traits in this Chinese Holstein population. The positional candidate genes, which were the ones closest to these two identified SNPs, were EEF2K (eukaryotic elongation factor 2 kinase) and KLHL1 (kelch like family member 1). These two genes could serve as new candidate genes for milk yield and lactation persistence, yet their roles need to be verified in further function studies.     

Reducing the interval of a growth QTL on chromosome 4 in laying hens

In our previous research, we identified a QTL with an interval of 3.4 Mb for growth on chicken chromosome (GGA) 4 in an advanced intercross population of an initial cross between the New Hampshire inbred line (NHI) and the White Leghorn inbred line (WL77). In the current study, an association analysis was performed in a population of purebred white layers (WLA) with White Leghorn origin. Genotypic data of 130 SNPs within the previously identified 3.4‐Mb region were obtained using a 60K SNP chip. In total, 24 significant SNPs (LOD ≥ 4.44) on GGA4 were detected for daily weigh gain from 8 to 14 weeks and two SNPs (LOD ≥ 4.80) for body weight at 14 weeks. The QTL interval was reduced by 1.9 Mb to an interval of 1.5 Mb (74.6–76.1 Mb) that harbors 15 genes. Furthermore, to identify additional loci for chicken growth, a genome‐wide association study (GWAS) was carried out in a WLA population. The GWAS identified an additional QTL on GGA6 for body weight at six weeks (19.8–21.2 Mb). Our findings showed that by using a WLA population we were able to further reduce the QTL confidence interval previously detected using a NHI × WL77 advanced intercross population.     

Nested‐association mapping (NAM)‐based genetic dissection uncovers candidate genes for seed and pod weights in peanut (Arachis hypogaea)

Multiparental genetic mapping populations such as nested‐association mapping (NAM) have great potential for investigating quantitative traits and associated genomic regions leading to rapid discovery of candidate genes and markers. To demonstrate the utility and power of this approach, two NAM populations, NAM_Tifrunner and NAM_Florida‐07, were used for dissecting genetic control of 100‐pod weight (PW) and 100‐seed weight (SW) in peanut. Two high‐density SNP‐based genetic maps were constructed with 3341 loci and 2668 loci for NAM_Tifrunner and NAM_Florida‐07, respectively. The quantitative trait locus (QTL) analysis identified 12 and 8 major effect QTLs for PW and SW, respectively, in NAM_Tifrunner, and 13 and 11 major effect QTLs for PW and SW, respectively, in NAM_Florida‐07. Most of the QTLs associated with PW and SW were mapped on the chromosomes A05, A06, B05 and B06. A genomewide association study (GWAS) analysis identified 19 and 28 highly significant SNP–trait associations (STAs) in NAM_Tifrunner and 11 and 17 STAs in NAM_Florida‐07 for PW and SW, respectively. These significant STAs were co‐localized, suggesting that PW and SW are co‐regulated by several candidate genes identified on chromosomes A05, A06, B05, and B06. This study demonstrates the utility of NAM population for genetic dissection of complex traits and performing high‐resolution trait mapping in peanut.     

Investigating the genetic regulation of the expression of 63 lipid metabolism genes in the pig skeletal muscle

A comprehensive and systematic view of the genetic regulation of lipid metabolism genes is still lacking in pigs. Herewith, we have investigated the genetic regulation of 63 porcine genes with crucial roles in the uptake, transport, synthesis and catabolism of lipids. With this aim, we have performed an expression QTL (eQTL) scan in 104 pigs with available genotypes for the Illumina Porcine SNP60 chip and microarray measurements of gene expression in the gluteus medius muscle. Analysis of the data with gemma software revealed 13 cis‐ and 18 trans‐eQTL modulating the expression of 19 loci. Genes regulated by eQTL participated in a wide array of lipid metabolism pathways such as the β‐oxidation of fatty acids, lipid biosynthesis and lipolysis, fatty acid activation and desaturation, lipoprotein uptake, apolipoprotein assembly and cholesterol trafficking. These data provide a first picture of the genetic regulation of loci involved in porcine lipid metabolism.     

Association analysis of adiponectin and somatostatin polymorphisms on BTA1 with growth and carcass traits in Angus cattle

This study tested positional candidate genes adiponectin (ADIPOQ) and somatostatin (SST) for effects on carcass traits in a commercially relevant cattle population. Both genes are located within a region of BTA1 previously reported to harbour quantitative trait loci (QTL) that affect marbling, quality grade, yield grade, ribeye area and weaning weight in Bos taurus x Bos indicus crosses. Except for the first intron of ADIPOQ, both genes, including over 2 kb upstream of the promoters, were sequenced in five registered Angus sires to identify polymorphisms. A variable copy duplication and three single nucleotide polymorphisms (SNPs) in ADIPOQ and one SNP in SST were genotyped and tested for association with 19 traits in a 14-generation pedigree of 1697 registered Angus artificial insemination sires representing all the major USA lineages of the breed. Linear models that parameterized predicted genetic merits in terms of allele substitution effects were fit by weighted least squares, and goodness-of-fit tests were employed to differentiate causal mutations or polymorphisms in strong linkage disequilibrium (LD) with causal mutations from markers in weak LD with QTL. We confirmed the presence of QTL affecting marbling, ribeye muscle area and fat thickness in the vicinity of SST and ADIPOQ on BTA1 in Angus; excluded SST as underlying the ribeye muscle area QTL; and excluded ADIPOQ as underlying the marbling score QTL. However, association analysis provides very limited information about QTL location and has little intrinsic value when performed in the absence of linkage or LD analysis using flanking marker data to localize the QTL effect relative to positional candidate genes.     

Construction of a cucumber genetic linkage map with SRAP markers and location of the genes for lateral branch traits

Cucumber (Cucumis sativus L. 2n = 2x = 14), thatbelongs to Cucurbitaceae family, is one of majorvegetables with a planting area second to that of to-mato in the world[1]. Due to its economical importanceplant breeders and geneticists have paid much atten-tion to the genetic study on this important vegetablecrop, but the research progress in cucumber is muchless than that in tomato. In 1990, Pierce[2] reviewed allthe reported genes of cucumber that had been geneti-cally analyzed since the 1930…     

Associations of functional candidate genes derived from gene-expression profiles of prenatal porcine muscle tissue with meat quality and muscle deposition

Ten genes (ANK1, bR10D1, CA3, EPOR, HMGA2, MYPN, NME1, PDGFRA, ERC1, TTN), whose candidacy for meat-quality and carcass traits arises from their differential expression in prenatal muscle development, were examined for association in 1700 performance-tested fattening pigs of commercial purebred and crossbred herds of Duroc, Pietrain, Pietrain x (Landrace x Large White), Duroc x (Landrace x Large White) as well as in an experimental F(2) population based on a reciprocal cross of Duroc and Pietrain. Comparative sequencing revealed polymorphic sites segregating across commercial breeds. Genetic mapping results corresponded to pre-existing assignments to porcine chromosomes or current human-porcine comparative maps. Nine of these genes showed association with meat-quality and carcass traits at a nominal P-value of < or = 0.05; PDGFRA revealed no association reaching the P < or = 0.05 threshold. In particular, HMGA2, CA3, EPOR, NME1 and TTN were associated with meat colour, pH and conductivity of loin 24 h postmortem; CA3 and MYPN exhibited association with ham weight and lean content (FOM) respectively at P-values of < 0.003 that correspond to false discovery rates of < 0.05. However, none of the genes showed significant associations for a particular trait across all populations. The study revealed statistical-genetic evidence for association of the functional candidate genes with traits related to meat quality and muscle deposition. The polymorphisms detected are not likely causal, but markers were identified that are in linkage disequilibrium with causal genetic variation within particular populations.     

Confirmation and refinement of a QTL on BTA5 affecting milk production traits in the Fleckvieh dual purpose cattle breed

We analysed a QTL affecting milk yield (MY), milk protein yield (PY) and milk fat yield (FY) in the dual purpose cattle breed Fleckvieh on BTA5. Twenty-six microsatellite markers covering 135 cM were selected to analyse nine half-sib families containing 605 sons in a granddaughter design. We thereby assigned two new markers to the public linkage map using the CRI-MAP program. Phenotypic records were daughter yield deviations (DYD) originating from the routinely performed genetic evaluations of breeding animals. To determine the position of the QTL, three different approaches were applied: interval mapping (IM), linkage analysis by variance component analysis (LAVC), and combined linkage disequilibrium (LD) and linkage (LDL) analysis. All three methods mapped the QTL in the same marker interval ( BM2830-ETH152 ) with the greatest test-statistic value at 118, 119.33 and 119.33 cM respectively. The positive QTL allele simultaneously increases DYD in the first lactation by 272 kg milk, 7.1 kg milk protein and 7.0 kg milk fat. Although the mapping accuracy and the significance of a QTL effect increased from IM over LAVC to LDL, the confidence interval was large (13, 20 and 24 cM for FY, MY and PY respectively) for the positional cloning of the causal gene. The estimated averages of pair wise marker LD with a distance <5 cM were low (0.107) and reflect the large effective population size of the Fleckvieh subpopulation analysed. This low level of LD suggests a need for increase in marker density in following fine mapping steps.