A polymorphism within the equine CRISP3 gene is associated with stallion fertility in Hanoverian warmblood horses

Fertility of stallions is of high economic importance, especially for large breeding organisations and studs. Breeding schemes with respect to fertility traits and selection of stallions at an early stage may be improved by including molecular genetic markers associated with traits. The genes coding for equine cysteine-rich secretory proteins (CRISPs) are promising candidate genes because previous studies have shown that CRISPs play a role in the fertilising ability of male animals. We have previously characterised the three equine CRISP genes and identified a non-synonymous polymorphism in the CRISP1 gene. In this study, we report one non-synonymous polymorphism in the CRISP2 gene and four non-synonymous polymorphisms in the CRISP3 gene. All six CRISP polymorphisms were genotyped in 107 Hanoverian breeding stallions. Insemination records of stallions were used to analyse the association between CRISP polymorphisms and fertility traits. Three statistical models were used to evaluate the influence of single mutations, genotypes and haplotypes of the polymorphisms. The CRISP3 AJ459965:c.+622G>A SNP leading to the amino acid substitution E208K was significantly associated with the fertility of stallions. Stallions heterozygous for the CRISP3 c.+622G>A SNP had lower fertility than homozygous stallions (P = 0.0234). The pregnancy rate per cycle in these stallions was estimated to be approximately 7% lower than in stallions homozygous at this position.     

Evaluation of SPATA1-associated markers for stallion fertility

Stallion fertility is an economically important trait because the use of artificial insemination is increasing in the horse industry and superior sires are used more intensely. Molecular genetic markers may be useful as early indicators for a stallion's fertility and genetic improvement programmes. The testis-specific SPATA1 protein is involved in shaping the sperm head during spermatogenesis. Thus, the spermatogenesis associated 1 ( SPATA1 ) gene was chosen as candidate for stallion fertility, and we analysed intragenic single nucleotide polymorphisms (SNPs) as genetic markers for the least square means (LSM) of the pregnancy rate per oestrus of stallions and breeding values (BV) for the paternal and embryonic component of the pregnancy rate per oestrus. We sequenced the cDNA of SPATA1 to verify the annotated mRNA sequence. One SPATA1 -associated intronic SNP ( BIEC2-968854 ) showed a significant association with the embryonic component of BVs of stallions for the pregnancy rate per oestrus. The embryonic component of BVs was positively associated with homozygous C/C stallions. Both the additive and dominance effects were significant with values of −5.8% ( P  = 0.01) and −6.4% ( P  = 0.02) for the embryonic component of BVs. For the same SNP, a suggestive association was found for the LSM of the pregnancy rate per oestrus of stallions. Heterozygous stallions had higher pregnancy rates per oestrus than homozygous stallions. The dominance effect was 4.1% with a nominal P -value of 0.02. The SNP BIEC2-968854 can change an SP1 binding site and thus we assume that gene regulation may be influenced through this intronic mutation. This is the first report on SPATA1 being associated with the pregnancy rate per oestrus for stallions.     

Evaluation of ACE, SP17, and FSHB as candidates for stallion fertility in Hanoverian warmblood horses

The research of fertility in humans and other mammals has strongly advanced in the recent years. The examination of molecular mechanisms influencing horse fertility is relatively recent. We chose the angiotensin converting enzyme (ACE), the sperm autoantigenic protein 17 (SP17) and the follicle stimulating hormone (FSHB) as candidates for determining stallion fertility and to analyze associations of intragenic single nucleotide polymorphisms (SNPs), flanking microsatellites and candidate-gene linked haplotypes with the pregnancy rate per oestrus (PRO) in 179 Hanoverian stallions. Fertility traits analyzed were the least square means of PRO for stallions (LSMs) and the paternal and embryonic component of breeding values for PRO (BVs). We detected nine SNPs and two flanking microsatellites in ACE, eight SNPs and two flanking microsatellites in SP17 and four SNPs and one flanking microsatellite in FSHB. Three SP17-associated SNPs and the two flanking microsatellites showed significant association with the embryonic component of BVs and one SP17-associated microsatellite was also significantly associated with the paternal component of BVs. Two ACE-associated SNPs were significantly associated with the embryonic component of BVs. Significantly associated haplotypes were shown for all three candidate genes and the tested fertility parameters. The final regression analysis model indicated that haplotypes of all three candidate genes significantly contributed to the paternal and embryonic fertility components of PRO. This is the first report of associations of ACE, SP17 and FSHB with fertility traits of stallions.     

Refinement of quantitative trait loci on equine chromosome 10 for radiological signs of navicular disease in Hanoverian warmblood horses

Navicular disease is characterized by a progressive degenerative alteration of the equine podotrochlea. In this study, we refined a previously identified quantitative trait locus (QTL) on horse chromosome 10 for the abnormal development of canales sesamoidales (DCS) of the navicular bone in Hanoverian warmblood horses. Genotyping was done in 192 Hanoverian warmblood horses from 17 paternal half-sib groups. The whole marker set comprised 45 markers including seven newly developed microsatellites and 13 single nucleotide polymorphisms (SNPs) within positional candidate genes. Chromosome-wide significant QTL were confirmed and refined for DCS on horse chromosome (ECA) 10 at 0.16-2.70 Mb and at 14.45-36.37 Mb. Nine microsatellites and three SNP markers reached the highest multipoint Zmeans and LOD scores at 19.34-20.38 Mb and at 23.17-30.73 Mb with genome-wide error probabilities of P<0.05. In addition, a significant association of a SNP within VSTM1 and a significant haplotype-trait association within IRF3 could be shown. These results support a possible role of the candidate genes VSTM1 and IRF3 within the QTL on ECA10 for DCS. This study is a further step towards the identification of the genes responsible for navicular disease in Hanoverian warmblood horses.     

Evaluation of the presence of equine viral herpesvirus 1 (EHV-1) and equine viral herpesvirus 4 (EHV-4) DNA in stallion semen using polymerase chain reaction (PCR)

In the horse, the risk of excretion of two major equine pathogens (equine herpesvirus types 1 (EHV-1) and 4 (EHV-4)) in semen is unknown. The objective of our study was to assess the possible risks for the horizontal transmission of equine rhinopneumonitis herpesviruses via the semen and the effect of the viruses on stallion fertility.Samples of stallion semen (n = 390) were gathered from several different sources. Examination of the semen involved the detection of viral DNA using specific PCR. The mean fertility of the stallions whose sperm tested positive for viral DNA and the mean fertility of stallions whose sperm did not contain viral DNA, were compared using the Student's t-test.EHV-4 viral DNA was not detected in any of the semen samples. EHV-1 DNA was identified in 51 of the 390 samples, (13%). One hundred and eighty-two samples came from 6 studs and there was significant difference (p < 0.05) among the proportion of stallions whose semen tested positive for viral DNA from 0 to 55% between the studs.There was a significant difference (p < 0.014) between the fertility of stallions whose semen tested positive for viral DNA and those whose semen was free from viral DNA. The stallions that excreted the EHV-1 virus in their semen appeared to be more fertile than the non-excretors, but this difference was in fact related to the breeding technique since higher proportion of excretors were found among those whose semen was used fresh rather than preserved by cooling or freezing.In conclusion, this study suggests that the EHV-1 virus may be transmitted via the semen at mating or by artificial insemination as demonstrated with other herpes viruses in other species.     

A genome-wide association study of mare fertility in the Pura Raza Español horse

Despite the economic importance of fertility for the horse industry, few efforts have been made to achieve a better understanding of the genetic mechanisms underlying its control. This is probably due to the difficulty of obtaining reliable phenotypes and the complexity of modelling the environmental and management factors. This work is novel in that we propose to use reproductive efficiency (RE) as an indicator of mare fertility. To achieve this, we performed a genome-wide association study in the Pura Raza Español horse aimed at identifying genomic variants, regions, and candidate genes associated with fertility in mares. The dataset included 819 animals genotyped with the Affymetrix Axiom? Equine 670 K single-nucleotide polymorphisms (SNPs) Genotyping Array and the deregressed breeding values for RE trait, obtained using a ssBLUP model, employed as pseudo-phenotypic data. Our results showed 28 SNPs potentially associated with RE, which explained 87.19% of the genetic variance and 6.61% of the phenotypic variance. Those results were further validated in BayesB, showing a correlation between observed and predicted RE of 0.57. In addition, 15 candidate genes (HTRA3, SPIRE1, APOE, ERCC1, FOXA3, NECTIN-2, KLC3, RSPH6A, PDPK1, MEIOB, PAQR4, NM3, PKD1, PRSS21, IFT140) previously related to fertility in mammals were associated with the markers and genomic regions significantly associated with RE. To our knowledge, this is the first genome-wide association study performed on mare fertility.     

Polymorphism identification within 50 equine gene-specific sequence tagged sites

The continued discovery of polymorphisms in the equine genome will be important for future studies using genomic screens and fine mapping for the identification of disease genes. Segments of 50 equine genes were examined for variability in 10 different horse breeds using a pool-and-sequence method. We identified 11 single nucleotide polymorphisms (SNPs) in 9380 bp of sequenced exon, and 25 SNPs, six microsatellites, and one insertion/deletion in 16961 bp of sequenced intron. Of all genes studied 52% contained at least one polymorphism, and polymorphisms were found at an overall rate of 1/613 bp. Several of the putative SNPs were tested and verified by restriction enzyme analysis using natural restriction sites or ones created by primer mutagenesis. The lowest allele frequency for a SNP detected in pooled samples was 10%. Three of the SNPs verified in the diverse horse pool were further tested in six breed-specific horse pools and were found to be reasonably variable within breeds. The pool-and-sequence method allows identification of polymorphisms in horse populations and will be a valuable tool for future disease gene and comparative mapping in horses.     

A survey of the fertility of Icelandic stallions

Very limited information is available on the breeding performance of Icelandic stallions, let alone the effect that management practices may have had on such performance. As an extensively kept, largely genetically isolated breed of horse it provides a good model for the study of factors that affect reproductive performance without the additional complication of selective breeding, infectious infertility and breed effect. A survey was conducted using 27 Icelandic stallions covering 1590 mares within the normal Icelandic breeding system (May to September). During the season, stallions cover mares within three periods of time, each period being of a similar length (average 35.5 days). During period 1, mares are covered in hand and at pasture. During periods 2 and 3, all mares are covered at pasture. The overall fertility rate for Icelandic stallions was calculated. The effect of a range of variables on fertility was investigated statistically using a number of models in an attempt to minimise the effect of confounding factors. An overall adjusted fertility rate for Icelandic stallions of 67.7% was obtained. The following factors were shown to have a significant effect on fertility: age of mare (P<0.001), training level of stallion (P<0.05) and method of breeding (P<0.05). For some individual stallions reproductive status of the mare also had a significant (P<0.001) effect. Many of these factors have been observed to effect FR in other more intensively managed equine populations. However, the less dramatic detrimental effect of age and the lack of a significant effect of mare reproductive status in most stallions suggests that infertility problems are less evident in Icelandic mares, possibly due to less emphasis on selection for athletic performance and the accepted culling of subfertile stock.     

SNP-based pool genotyping and haplotype analysis accelerate fine-mapping of the wheat genomic region containing stripe rust resistance gene <Emphasis Type="Italic">Yr26</Emphasis>

Key message

NGS-assisted super pooling emerging as powerful tool to accelerate gene mapping and haplotype association analysis within target region uncovering specific linkage SNPs or alleles for marker-assisted gene pyramiding.

Abstract

Conventional gene mapping methods to identify genes associated with important agronomic traits require significant amounts of financial support and time. Here, a single nucleotide polymorphism (SNP)-based mapping approach, RNA-Seq and SNP array assisted super pooling analysis, was used for rapid mining of a candidate genomic region for stripe rust resistance gene Yr26 that has been widely used in wheat breeding programs in China. Large DNA and RNA super-pools were genotyped by Wheat SNP Array and sequenced by Illumina HiSeq, respectively. Hundreds of thousands of SNPs were identified and then filtered by multiple filtering criteria. Among selected SNPs, over 900 were found within an overlapping interval of less than 30 Mb as the Yr26 candidate genomic region in the centromeric region of chromosome arm 1BL. The 235 chromosome-specific SNPs were converted into KASP assays to validate the Yr26 interval in different genetic populations. Using a high-resolution mapping population (>?30,000 gametes), we confined Yr26 to a 0.003-cM interval. The Yr26 target region was anchored to the common wheat IWGSC RefSeq v1.0 and wild emmer WEWSeq v.1.0 sequences, from which 488 and 454 kb fragments were obtained. Several candidate genes were identified in the target genomic region, but there was no typical resistance gene in either genome region. Haplotype analysis identified specific SNPs linked to Yr26 and developed robust and breeder-friendly KASP markers. This integration strategy can be applied to accelerate generating many markers closely linked to target genes/QTL for a trait of interest in wheat and other polyploid species.

     

A genome-wide association study for quantitative trait loci of show-jumping in Hanoverian warmblood horses

Show-jumping is an economically important breeding goal in Hanoverian warmblood horses. The aim of this study was a genome-wide association study (GWAS) for quantitative trait loci (QTL) for show-jumping in Hanoverian warmblood horses, employing the Illumina equine SNP50 Beadchip. For our analyses, we genotyped 115 stallions of the National State stud of Lower Saxony. The show-jumping talent of a horse includes style and ability in free-jumping. To control spurious associations based on population stratification, two different mixed linear animal model (MLM) approaches were employed, besides linear models with fixed effects only and adaptive permutations for correcting multiple testing. Population stratification was explained best in the MLM considering Hanoverian, Thoroughbred, Trakehner and Holsteiner genes and the marker identity-by-state relationship matrix. We identified six QTL for show-jumping on horse chromosomes (ECA) 1, 8, 9 and 26 (-log(10) P-value >5) and further putative QTL with -log(10) P-values of 3-5 on ECA1, 3, 11, 17 and 21. Within six QTL regions, we identified human performance-related genes including PAPSS2 on ECA1, MYL2 on ECA8, TRHR on ECA9 and GABPA on ECA26 and within the putative QTL regions NRAP on ECA1, and TBX4 on ECA11. The results of our GWAS suggest that genes involved in muscle structure, development and metabolism are crucial for elite show-jumping performance. Further studies are required to validate these QTL in larger data sets and further horse populations.     

Two-dimensional polyacrylamide gel electrophoresis of equine seminal plasma proteins and their correlation with fertility

The objectives of this study were to 1) identify proteins found in stallion seminal plasma utilizing two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) in conjunction with Western blot analysis; and 2) to determine if any of these individual proteins were correlated with stallion fertility utilizing regression analysis. Fertility was quantified by assigning a breeding score for each stallion. Each score was calculated by dividing the number of conceptions by the number of breedings for each stallion for four successive breeding seasons (1992-1995). Ejaculates from stallions of known fertility (n = 6) were collected with a Missouri-style artificial vagina. Immediately after collection, the semen sample was filtered and the gel fraction removed. The resultant sperm-rich fraction was centrifuged in a Beckman Microfuge E at 10,000 x g and the seminal plasma aspirated from the pelleted sperm cells. Two-dimensional PAGE of the seminal plasma was performed under denaturing conditions which revealed that 14 proteins were common in all stallions in the research population. Four of these proteins (SP-1, SP-2, SP-3, and SP-4) were found to be significantly (P < 0.05) correlated with the breeding score assigned for each stallion. Regression analysis of protein optical densities with breeding score indicated that SP-1 (72 kDa, pI 5.6) was positively correlated with fertility (P < 0.05, r2 = 0.706), while SP-2 (75 kDa, pI 6.0), SP-3 (18 kDa, pI 4.3), and SP-4 (16 kDa, pI 6.5) were found to be negatively correlated (P < 0.05, r2 = 0.762, 0.730, 0.775 respectively) with fertility. Western blot analysis of SP-1 indicated there was an antigenic homology with a bovine 55 kDa fertility-associated seminal plasma protein identified in a study by Killian et al. (19). This suggests that the two proteins may have a similar physiological role and therefore common biological properties. These results indicate that analysis of stallion seminal plasma proteins can be used as an indicator of fertilizing capacity. Identification of such proteins in stallion seminal plasma could lead to better insight into the nature of subfertility or infertility in the horse, as well as to indicate better cryopreservation strategies.     

Profiling Deleterious Non-synonymous SNPs of Smoker's Gene CYP1A1

CYP1A1 gene belongs to the cytochrome P450 family and is known better as smokers’ gene due to its hyperactivation as a consequence of long term smoking. The expression of CYP1A1 induces polycyclic aromatic hydrocarbon production in the lungs, which when over expressed, is known to cause smoking related diseases, such as cardiovascular pathologies, cancer, and diabetes. Single nucleotide polymorphisms (SNPs) are the simplest form of genetic variations that occur at a higher frequency, and are denoted as synonymous and non-synonymous SNPs on the basis of their effects on the amino acids. This study adopts a systematic in silico approach to predict the deleterious SNPs that are associated with disease conditions. It is inferred that four SNPs are highly deleterious, among which the SNP with rs17861094 is commonly predicted to be harmful by all tools. Hydrophobic (isoleucine) to hydrophilic (serine) amino acid variation was observed in the candidate gene. Hence, this investigation aims to characterize a candidate gene from 159 SNPs of CYP1A1.     

Fine mapping of a quantitative trait locus for osteochondrosis on horse chromosome 2

In this study, we refine a quantitative trait locus for equine osteochondrosis (OC) on horse chromosome (ECA) 2 to a genome-wide significant interval at 20.08-30.94 Mb. The marker set contained 27 newly developed microsatellites equidistantly distributed over ECA2 and 44 nucleotide polymorphisms, located in 16 positional candidate genes for OC. Genotyping was performed in 211 Hanoverian horses from 14 paternal half-sib groups. A NCDN-associated SNP and haplotype were significantly associated with OC in fetlock and/or hock joints. This study is a further step towards the identification of genes responsible for OC in horses.     

Assignment of foal paternity when a mare is bred to two stallions

Accuracy of assigning foal paternity to the second serving stallion for foals whose dams had been bred to two stallions within an interval of 1 to 45 days was investigated using breeding dates from 108 equine paternity cases solved by blood typing. One hundred two of the foals were sired by the second stallion but six foals (5.5%) were excluded as being by the second sires. Thus, breeding dates are not reliable as a sole source of information to assign equine paternity accurately.