Alternative Traits for Genetic Evaluation of Mastitis Based on Lifetime Merit.

Genetic selection has achieved little progress in reducing mastitis incidence. Mastitis traits are problematic due to the lack of sensitivity of the data and reliance on clinical diagnosis, often missing subclinical cases, and/or on monthly somatic cell count (SCC) measurements. The current measure for mastitis is the lactation average of the somatic cells score (LSCS). We studied two datasets: (1) 148 heifers divided into non-intramammary infected, sub-clinically infected and clinical mastitis groups; (2) data from 89,601 heifers from Israeli Holsteins through the same period divided into "udder healthy" (UH) and "non-healthy" (UNH) by a threshold of SCC 120,000 cells/mL in all nine monthly milk recordings. In study 1, non-infected heifers had significantly (p < 0.05) more partum, production days and overall lifetime milk production compared to clinical and sub-clinically infected. In study 2, UH heifers (20.3%) had significantly higher (p < 0.01) lifetime milk, production days, and lactations. Subdividing datasets by sires, the same analyses detected differences in percentages of UH daughters between the sire groups. Lifetime milk production correlated (r = +0.83, p < 0.001) with udder health status. SCC threshold of less than 120,000 cells/mL during all first lactation measurements indicated healthy udder, providing a valuable insight that this dichotomous trait is advantageous for calculating lifetime net-merit index (NM$) over LSCS.

Re-Evaluation of Genotyping Methodologies in Cattle: The Proficiency of Imputation.

In dairy cattle, identifying polymorphisms that contribute to complex economical traits such as residual feed intake (RFI) is challenging and demands accurate genotyping. In this study, we compared imputed genotypes (n = 192 cows) to those obtained using the TaqMan and high-resolution melting (HRM) methods (n = 114 cows), for mutations in the FABP4 gene that had been suggested to have a large effect on RFI. Combining the whole genome sequence (n = 19 bulls) and the cows' BovineHD BeadChip allowed imputing genotypes for these mutations that were verified by Sanger sequencing, whereas, an error rate of 11.6% and 10.7% were encountered for HRM and TaqMan, respectively. We show that this error rate seriously affected the linkage-disequilibrium analysis that supported this gene candidacy over other BTA14 gene candidates. Thus, imputation produced superior genotypes and should also be regarded as a method of choice to validate the reliability of the genotypes obtained by other methodologies that are prone to genotyping errors due to technical conditions. These results support the view that RFI is a complex trait and that searching for the causative sequence variation underlying cattle RFI should await the development of statistical methods suitable to handle additive and epistatic interactions.

Genetic and Genomic Analysis of Cow Mortality in the Israeli Holstein Population.

"Livability" was defined as the inverse of the probability of death. The objectives of this study were to estimate the heritability, genetic and phenotypic trends for the livability of Israeli Holstein cows; estimate the genetic and environmental correlations between livability and the nine traits included in the Israeli breeding index; estimate the effect of the inclusion of livability in the Israeli breeding index on expected genetic gains; and compute a genome-wide association study (GWAS) for livability. Seven data sets were analyzed. All data were derived from the database of the Israeli dairy cattle herd-book. The mean livability for the complete data set of 523,954 cows born from 2000 through 2016 was 89.6%. Pregnancy reduced livability by 15%. Livability generally increased with parity and days in milk within parity. Heritability of livability was 0.0082. Phenotypic and genetic trends over the 14-year period from 2000 through 2013 were -0.42% and -0.22% per year. If livability is included in the Israeli breeding index, accounting for 9% of the index, livability would increase by 1.3% and protein production would decrease by 11 kg over the next decade, as compared to the current index. A marker in proximity to the oxytocin-vasopressin locus had the greatest effect in the GWAS. Oxytocin activity in cattle affects calving-associated pathologies and maternal death. Inclusion of livability in the Israeli breeding index is not recommended.

Master-Key Regulators of Sex Determination in Fish and Other Vertebrates-A Review.

In vertebrates, mainly single genes with an allele ratio of 1:1 trigger sex-determination (SD), leading to initial equal sex-ratios. Such genes are designated master-key regulators (MKRs) and are frequently associated with DNA structural variations, such as copy-number variation and null-alleles. Most MKR knowledge comes from fish, especially cichlids, which serve as a genetic model for SD. We list 14 MKRs, of which dmrt1 has been identified in taxonomically distant species such as birds and fish. The identification of MKRs with known involvement in SD, such as amh and fshr, indicates that a common network drives SD. We illustrate a network that affects estrogen/androgen equilibrium, suggesting that structural variation may exert over-expression of the gene and thus form an MKR. However, the reason why certain factors constitute MKRs, whereas others do not is unclear. The limited number of conserved MKRs suggests that their heterologous sequences could be used as targets in future searches for MKRs of additional species. Sex-specific mortality, sex reversal, the role of temperature in SD, and multigenic SD are examined, claiming that these phenomena are often consequences of artificial hybridization. We discuss the essentiality of taxonomic authentication of species to validate purebred origin before MKR searches.

Absence of Figla-like Gene Is Concordant with Femaleness in Cichlids Harboring the LG1 Sex-Determination System.

Oreochromis niloticus has been used as a reference genome for studies of tilapia sex determination (SD) revealing segregating genetic loci on linkage groups (LGs) 1, 3, and 23. The master key regulator genes (MKR) underlying the SD regions on LGs 3 and 23 have been already found. To identify the MKR in fish that segregate for the LG1 XX/XY SD-system, we applied short variant discovery within the sequence reads of the genomic libraries of the Amherst hybrid stock, Coptodon zillii and Sarotherodon galilaeus, which were aligned to a 3-Mbp-region of the O. aureus genome. We obtained 66,372 variants of which six were concordant with the XX/XY model of SD and were conserved across these species, disclosing the male specific figla-like gene. We further validated this observation in O. mossambicus and in the Chitralada hybrid stock. Genome alignment of the 1252-bp transcript showed that the figla-like gene's size was 2664 bp, and that its three exons were capable of encoding 99 amino acids including a 45-amino-acid basic helix-loop-helix domain that is typical of the ovary development regulator-factor-in-the-germline-alpha (FIGLA). In Amherst gonads, the figla-like gene was exclusively expressed in testes. Thus, the figla-like genomic presence determines male fate by interrupting the female developmental program. This indicates that the figla-like gene is the long-sought SD MKR on LG1.

Genome-wide recombination map construction from single sperm sequencing in cattle.

BACKGROUND: Meiotic recombination is one of the important phenomena contributing to gamete genome diversity. However, except for human and a few model organisms, it is not well studied in livestock, including cattle. RESULTS: To investigate their distributions in the cattle sperm genome, we sequenced 143 single sperms from two Holstein bulls. We mapped meiotic recombination events at high resolution based on phased heterozygous single nucleotide polymorphism (SNP). In the absence of evolutionary selection pressure in fertilization and survival, recombination events in sperm are enriched near distal chromosomal ends, revealing that such a pattern is intrinsic to the molecular mechanism of meiosis. Furthermore, we further validated these findings in single sperms with results derived from sequencing its family trio of diploid genomes and our previous studies of recombination in cattle. CONCLUSIONS: To our knowledge, this is the first large-scale single sperm whole-genome sequencing effort in livestock, which provided useful information for future studies of recombination, genome instability, and male infertility.

Comparing BeadChip and WGS Genotyping: Non-Technical Failed Calling Is Attributable to Additional Variation within the Probe Target Sequence.

Microarray-based genomic selection is a central tool to increase the genetic gain of economically significant traits in dairy cattle. Yet, the effectivity of this tool is slightly limited, as estimates based on genotype data only partially explain the observed heritability. In the analysis of the genomes of 17 Israeli Holstein bulls, we compared genotyping accuracy between whole-genome sequencing (WGS) and microarray-based techniques. Using the standard GATK pipeline, the short-variant discovery within sequence reads mapped to the reference genome (ARS-UCD1.2) was compared to the genotypes from Illumina BovineSNP50 BeadChip and to an alternative method, which computationally mimics the hybridization procedure by mapping reads to 50 bp spanning the BeadChip source sequences. The number of mismatches between the BeadChip and WGS genotypes was low (0.2%). However, 17,197 (40% of the informative SNPs) had extra variation within 50 bp of the targeted SNP site, which might interfere with hybridization-based genotyping. Consequently, with respect to genotyping errors, BeadChip varied significantly and systematically from WGS genotyping, introducing null allele-like effects and Mendelian errors (<0.5%), whereas the GATK algorithm of local de novo assembly of haplotypes successfully resolved the genotypes in the extra-variable regions. These findings suggest that the microarray design should avoid polymorphic genomic regions that are prone to extra variation and that WGS data may be used to resolve erroneous genotyping, which may partially explain missing heritability.

Cross-species conservation of a transposase-linked element enables genetic sexing of commercial populations of Russian sturgeon (Acipenser gueldenstaedtii).

All-female culture of sturgeon is essential for efficient caviar production. However, Russian sturgeon (Acipenser gueldenstaedtii) does not exhibit external sexual dimorphism, and therefore, commercial farms apply gonadal endoscopy or ultrasound at the earliest age of 4-5 years to separate the sexes, with ~90% accuracy. Recently, a dominant genomic marker (AllWSEX2) has been found with association to femaleness in sturgeons. We developed a duplex PCR (dAllWSEX2) with the adjacent bmp7 gene as an internal control, to validate an effective PCR. Robust amplification of control fragments was observed for all samples of our commercial A. gueldenstaedtii stock (n = 337). The dAllWSEX2 assay was significantly associated with sex (n = 43, p < 1.6 × 10-8 ), yet four (18%) of the endoscopy-determined females were genetic males. To examine whether some females display a male genetic profile, we tested 96 egg-producing females, which were all verified as genetic females, indicating that the observed mismatches may be attributed to wrong sexing by endoscopy. Application of dAllWSEX2 on 100 7-month-old fish showed no sex-dependent differences in body weight, indicating that weighing is not an applicable tool for sorting females at a young age. Sanger sequencing of the bmp7 fragment revealed octaploidy and sex-independent variation, suggesting that the critical sex-determining region harboring AllWSEX2 is small. In keeping with a model of a single-ploidy encoding female determination, AllWSEX2 showed no variation despite being a transposase-linked repetitive element. Cross-species conservation of AllWSEX2, and absence of annotated sex-determination genes in this region suggests that, in sturgeons, the sex-determining mechanism is different from mechanisms identified in other fish.

Towards the detection of copy number variation from single sperm sequencing in cattle.

BACKGROUND: Copy number variation (CNV) has been routinely studied using bulk-cell sequencing. However, CNV is not well studied on the single-cell level except for humans and a few model organisms. RESULTS: We sequenced 143 single sperms of two Holstein bulls, from which we predicted CNV events using 14 single sperms with deep sequencing. We then compared the CNV results derived from single sperms with the bulk-cell sequencing of one bull's family trio of diploid genomes. As a known CNV hotspot, segmental duplications were also predicted using the bovine ARS-UCD1.2 genome. Although the trio CNVs validated only some single sperm CNVs, they still showed a distal chromosomal distribution pattern and significant associations with segmental duplications and satellite repeats. CONCLUSION: Our preliminary results pointed out future research directions and highlighted the importance of uniform whole genome amplification, deep sequence coverage, and dedicated software pipelines for CNV detection using single cell sequencing data.

Gene Variant of Barrier to Autointegration Factor 2 (Banf2w) Is Concordant with Female Determination in Cichlids.

Oreochromis fishes exhibit variability of sex-determination (SD) genes whose characterization contributes to understanding of the sex differentiation network, and to effective tilapia farming, which requires all-male culture. However, O. niloticus (On) amh is the only master-key regulator (MKR) of SD that has been mapped (XY/XX SD-system on LG23). In O. aureus (Oa), LG3 controls a WZ/ZZ SD-system that has recently been delimited to 9.2 Mbp, with an embedded interval rich with female-specific variation, harboring two paics genes and banf2. Developing genetic markers within this interval and using a hybrid Oa stock that demonstrates no recombination repression in LG3, we mapped the critical SD region to 235 Kbp on the orthologous On physical map (p < 1.5 × 10-26). DNA-seq assembly and peak-proportion analysis of variation based on Sanger chromatograms allowed the characterization of copy-number variation (CNV) of banf2. Oa males had three exons capable of encoding 90-amino-acid polypeptides, yet in Oa females, we found an extra copy with an 89-amino-acid polypeptide and three non-conservative amino acid substitutions, designated as banf2w. CNV analysis suggested the existence of two to five copies of banf2 in diploidic Cichlidae. Disrupting the Hardy-Weinberg equilibrium (p < 4.2 × 10-3), banf2w was concordant with female determination in Oa and in three cichlids with LG3 WZ/ZZ SD-systems (O. tanganicae, O. hornorum and Pelmatolapia mariae). Furthermore, exclusive RNA-seq expression in Oa females strengthened the candidacy of banf2w as the long-sought LG3 SD MKR. As banf genes mediate nuclear assembly, chromatin organization, gene expression and gonad development, banf2w may play a fundamental role inducing female nucleus formation that is essential for WZ/ZZ SD.

Estimating Copy-Number Proportions: The Comeback of Sanger Sequencing.

Determination of the relative copy numbers of mixed molecular species in nucleic acid samples is often the objective of biological experiments, including Single-Nucleotide Polymorphism (SNP), indel and gene copy-number characterization, and quantification of CRISPR-Cas9 base editing, cytosine methylation, and RNA editing. Standard dye-terminator chromatograms are a widely accessible, cost-effective information source from which copy-number proportions can be inferred. However, the rate of incorporation of dye terminators is dependent on the dye type, the adjacent sequence string, and the secondary structure of the sequenced strand. These variable rates complicate inferences and have driven scientists to resort to complex and costly quantification methods. Because these complex methods introduce their own biases, researchers are rethinking whether rectifying distortions in sequencing trace files and using direct sequencing for quantification will enable comparable accurate assessment. Indeed, recent developments in software tools (e.g., TIDE, ICE, EditR, BEEP and BEAT) indicate that quantification based on direct Sanger sequencing is gaining in scientific acceptance. This commentary reviews the common obstacles in quantification and the latest insights and developments relevant to estimating copy-number proportions based on direct Sanger sequencing, concluding that bidirectional sequencing and sophisticated base calling are the keys to identifying and avoiding sequence distortions.

A novel c.1759T>G variant in follicle-stimulating hormone-receptor gene is concordant with male determination in the flathead grey mullet (Mugil cephalus).

Various master key regulators (MKRs) that control a binary switch of sex determination (SD) have been found in fish; these provide an excellent model for the study of vertebrate genetic SD. The SD region in flathead grey mullet has been previously mapped to a 1 Mbp region harboring 27 genes, of which one is follicle-stimulating hormone receptor (fshr). Although this gene is involved in gonad differentiation and function, it has not been considered as an MKR of SD. We systematically investigated polymorphism in mullet fshr using DNA shotgun sequences, and compared them between males and females. Capable of encoding nonconservative amino acid substitutions, c.1732G>A and c.1759T>G exhibited association with sex on a population level (N = 83; P ≤ 6.7 × 10-19). Hence, 1732 A and 1759 G represent a male-specific haplotype of the gene, designated as "fshry." Additional flanking SNPs showed a weaker degree of association with sex, delimiting the SD critical region to 143 nucleotides on exon 14. Lack of homozygotes for fshry, and the resulting divergence from Hardy-Weinberg equilibrium (N = 170; P ≤ 3.9 × 10-5), were compatible with a male heterogametic model (XY/XX). Capable of replacing a phenylalanine with valine, c.1759T>G alters a conserved position across the sixth transmembrane domain of vertebrate FSHRs. Amino acid substitutions in this position in vertebrates are frequently associated with constant receptor activation and consequently with FSH/FSHR signaling alteration; thus, indicating a potential role of fshr as an MKR of SD.

Preferential Mapping of Sex-Biased Differentially-Expressed Genes of Larvae to the Sex-Determining Region of Flathead Grey Mullet (Mugil cephalus).

Flathead gray mullet (Mugil cephalus) is a cosmopolitan mugilid species popular in fishery and aquaculture with an economic preference for all-female population. However, it displays neither sexual dimorphisms nor heteromorphic sex chromosomes. We have previously presented a microsatellite-based linkage map for this species locating a single sex determination region (SDR) on linkage group 9 (LG9) with evidence for XX/XY sex determination (SD) mechanism. In this work, we refine the critical SDR on LG9, and propose positional- and functional- candidate genes for SD. To elucidate the genetic mechanism of SD, we assembled and compared male and female genomic sequences of 19 syntenic genes within the putative SDR on mullet's LG9, based on orthology to tilapia's LG8 (tLG8) physical map. A total of 25 sequence-based markers in 12 genes were developed. For all markers, we observed association with sex in at least one of the two analyzed M. cephalus full-sib families, but not in the wild-type population. Recombination events were inferred within families thus setting the SDR boundaries to a region orthologous to ∼0.9 Mbp with 27 genes on tLG8. As the sexual phenotype is evident only in adults, larvae were assigned into two putative sex-groups according to their paternal haplotypes, following a model of XY/XX SD-system. A total of 107 sex-biased differentially expressed genes in larvae were observed, of which 51 were mapped to tLG8 (48% enrichment), as compared to 5% in random control. Furthermore, 23 of the 107 genes displayed sex-specific expression; and 22 of these genes were positioned to tLG8, indicating 96% enrichment. Of the 27 SDR genes, BCCIP, DHX32A, DOCK1, and FSHR (GTH-RI) are suggested as positional and functional gene candidates for SD.

Basal Levels of CD18 Antigen Presenting Cells in Cow Milk Associate with Copy Number Variation of Fc Gamma Receptors.

Differentiation of cells by flow cytometry provides informative somatic cell counts (SCCs) that allow analyzing leukocyte population patterns in udder infections of different etiologies. Postulating that this approach also enhances the statistical power to detect genetic variants linked to cell levels in milk of healthy mammary glands, we used monoclonal antibodies anti-CD18, anti-CD4, anti--CD14, and anti-PMN to count cells presenting these surface antigens, and performed a genome-wide association study of these counts in 125 Israeli Holsteins genotyped using SNP BeadChips. We identified an informative haplotype of 15 SNPs in the centromeric end of BTA3 that was strongly associated with CD18 cells (p < 2.3 × 10-9). Within this region, examination of the network of genes interacting with ITGB2 (CD18) indicated an Fc-γ-receptor gene cluster, including FCGR2A (CD32). Sanger-sequence analysis of FCGR2s-linked exon 3 variation to CD18 counts. Meta-analysis of RNA-Seq data revealed a significant negative correlation (R = -0.51) between expression of CD32 and CD18 in milk. Assembly of DNA-Seq reads uncovered FCGR copy-number variation and a variant, designated V7, was abundant in dairy cattle, probably reflecting adaptation to selection pressure for low SCC in Holstein milk.

Avian Leptin: Bird's-Eye View of the Evolution of Vertebrate Energy-Balance Control.

Discovery of the satiety hormone leptin in 1994 and its characterization in mammals provided a key tool to deciphering the complex mechanism governing adipose tissue regulation of appetite and energy expenditure. Surprisingly, despite the perfectly logical notion of an energy-storing tissue announcing the amount of fat stores using leptin signaling, alternate mechanisms were chosen in bird evolution. This conclusion emerged based on the recent discovery and characterization of genuine avian leptin - after it had been assumed missing by some, and erroneously identified by others. Critical evaluation of the past and present indications of the role of leptin in Aves provides a new perspective on the evolution of energy-balance control in vertebrates; proposing a regulation strategy alternative to the adipostat mechanism.

Mapping of the Sex Determining Region on Linkage Group 12 of Guppy (Poecilia reticulata).

Poecilia reticulata is one of the most popular ornamental fish species with a higher demand for males due to their large colorful fins. The objectives of this study were mapping of the sex-determining (SD) region on linkage group 12 of guppy, and identification of a sex specific marker. We generated eight polymorphic microsatellite markers distributed along the distal 5.4 Mbp sequence of the previously identified SD region on linkage group (LG) 12. The markers were tested for association with sex in 156 individuals of the Red Blonde and Flame strains, and 126 progeny of four full-sibs Red Blonde families. A male-specific allele was found for microsatellite gu1066 at position of 25.3 Mbp on LG12 for both strains, and gu832 at position 24.4 Mbp for the Flame strain. Thus, a region of 0.9 Mbp between these markers, harboring 27 annotated genes, was selected for analysis. Based on association of copy number variation and sex determination we mapped a duplicated region between LGs 9 and 12, of 1.26 Mbp, containing 59 genes on LG12. The common interval between the segment bounded by gu1066 and gu832, and the duplicated region of 0.43 Mbp on LG12 harbors 11 genes of which 6 have multiple copies (54%). Growth arrest and DNA damage inducible gamma-like (GADD45G-like) is a plausible positional and functional candidate gene for its role in male fertility. We characterized the genomic structure of the gene in guppy, and found two isoforms; but no sex-biased differences were evident in genomic sequence and gene expression.

Avian Expression Patterns and Genomic Mapping Implicate Leptin in Digestion and TNF in Immunity, Suggesting That Their Interacting Adipokine Role Has Been Acquired Only in Mammals

In mammals, leptin and tumor-necrosis factor (TNF) are prominent interacting adipokines mediating appetite control and insulin sensitivity. While TNF pleiotropically functions in immune defense and cell survival, leptin is largely confined to signaling energy stores in adipocytes. Knowledge about the function of avian leptin and TNF is limited and they are absent or lowly expressed in adipose, respectively. Employing radiation-hybrid mapping and FISH-TSA, we mapped TNF and its syntenic genes to chicken chromosome 16 within the major histocompatibility complex (MHC) region. This mapping position suggests that avian TNF has a role in regulating immune response. To test its possible interaction with leptin within the immune system and beyond, we compared the transcription patterns of TNF, leptin and their cognate receptors obtained by meta-analysis of GenBank RNA-seq data. While expression of leptin and its receptor (LEPR) were detected in the brain and digestive tract, TNF and its receptor mRNAs were primarily found in viral-infected and LPS-treated leukocytes. We confirmed leptin expression in the duodenum by immunohistochemistry staining. Altogether, we suggest that whereas leptin and TNF interact as adipokines in mammals, in birds, they have distinct roles. Thus, the interaction between leptin and TNF may be unique to mammals.

Bos taurus-indicus hybridization correlates with intralocus sexual-conflict effects of PRDM9 on male and female fertility in Holstein cattle.

BACKGROUND: Crossover localization during meiotic recombination is mediated by the fast-evolving zinc-finger (ZnF) domain of gene PRDM9. To study its impact on dairy cattle performance, we compared its genetic variation between the relatively small Israeli (IL) Holsteins and the North American (US) Holsteins that count millions. RESULTS: Initially, we analyzed the major BTA1 haplotypes present in IL Holsteins based on the 10 most telomeric SNPs of the BovineSNP50 BeadChip. Sequencing of representative haplotype carriers indicated that for all frequent haplotypes (> 6%), the variable PRDM9 ZnF array consisted of seven tandem ZnF repeats. Two rare haplotypes (frequency < 4%) carried an indicine PRDM9, whereas all others were variants of the taurine type. These two haplotypes included the minor SNP allele, which was perfectly linked with a previously described PRDM9 allele known to induce unique localization of recombination hotspots. One of them had a significant (p = 0.03) negative effect on IL sire fertility. This haplotype combined the rare minor alleles of the only SNPs with significant (p < 0.05) negative substitution effects on US sire fertility (SCR). Analysis of telomeric SNPs indicated general agreement of allele frequencies (R = 0.95) and of the substitution effects on sire fertility (SCR, R = 0.6) between the US and IL samples. Surprisingly, the alleles that had a negative impact on male fertility had the most positive substitution effects on female fertility traits (DPR, CCR and HCR). CONCLUSIONS: A negative genetic correlation between male and female fertility is encoded within the BTA1 telomere. Cloning the taurine PRDM9 gene, which is the common form carried by Holsteins, we encountered the infiltration of an indicine PRDM9 variant into this population. During meiosis, in heterozygous males, the indicine PRDM9 variant may induce incompatibility of recombination hotspots and male infertility. However, this variant is associated with favorable female fertility, which would explain its survival and the general negative correlation (R = - 0.3) observed between male and female fertility in US Holsteins. Further research is needed to explain the mechanism underlying this positive effect and to devise a methodology to unlink it from the negative effect on male fertility during breeding.

Array CGH-based detection of CNV regions and their potential association with reproduction and other economic traits in Holsteins.

BACKGROUND: Copy number variations (CNVs) are structural variants consisting of large-scale insertions and deletions of genomic fragments. Exploring CNVs and estimating their effects on phenotypes are useful for genome selection but remain challenging in the livestock. RESULTS: We identified 1043 CNV regions (CNVRs) from array comparative genomic hybridization (CGH) data of 47 Holstein bulls. Using a probe-based CNV association approach, we detected 87 CNVRs significantly (Bonferroni-corrected P value < 0.05) associated with at least one out of 41 complex traits. Within them, 39 CNVRs were simultaneously associated with at least 2 complex traits. Notably, 24 CNVRs were markedly related to daughter pregnancy rate (DPR). For example, CNVR661 containing CYP4A11 and CNVR213 containing CTR9, respectively, were associated with DPR and other traits related to reproduction, production, and body conformation. CNVR758 was also significantly related to DPR, with a nearby gene CAPZA3, encoding one of F-actin-capping proteins which play a role in determining sperm architecture and male fertility. We corroborated these CNVRs by examining their overlapped quantitative trait loci and comparing with previously published CNV results. CONCLUSION: To our knowledge, this is one of the first genome-wide association studies based on CNVs called by array CGH in Holstein cattle. Our results contribute substantial information about the potential CNV impacts on reproduction, health, production, and body conformation traits, which lay the foundation for incorporating CNV into the future dairy cattle breeding program.

Quantitative trait loci on LGs 9 and 14 affect the reproductive interaction between two Oreochromis species, O. niloticus and O. aureus.

Effective farming of tilapia requires all-male culture, characterized by uniformity and high growth rate. Males of O. aureus (Oa) and females of O. niloticus (On) produce all-male offspring, but there is a behavioral reproductive barrier between the two species that prevents mass production. In crosses between Oa and On broodstocks, few hybrid females are attracted to the Oa male nests (denoted responders), and if they harbor the On alleles for the sex determination (SD) sites on linkage groups (LGs) 1, 3, and 23, all-male progeny are produced. Yet, without controlling for the alleles underlying SD, the parental stocks gradually lose their capability for all-male production. Hypothesizing that marker-assisted selection for female responders would allow production of sustainable broodstocks, we applied genotyping-by-sequencing to generate 4983 informative SNPs from 13 responding and 28 non-responding females from two full-sib families. Accounting for multiple comparisons in a genome-wide association study, seven SNPs met a false discovery rate of 0.061. Lowest nominal probabilities were on LGs 9 and 14, for which microsatellite DNA markers were designed within the candidate genes PTGDSL and CASRL, respectively. By increasing the sample size to 22 responders and 47 non-responders and by genotyping additional established microsatellites, we confirmed the association of these LGs with female responsiveness. The combined effects of microsatellites GM171 and CARSL-LOC100690618 on LGs 9 and 14 explained 37% of the phenotypic variance of reproductive interaction (p < 0.0001). Based on these findings, we propose a strategy for mass production of all-male tilapia hybrids through selection for genomic loci affecting SD and female responsiveness.