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Research article
Identification of a two-marker-haplotype on Bos taurus autosome 18 associated with somatic cell score in German Holstein cattle
Bodo Brand†, Christine Baes†, Manfred Mayer, Norbert Reinsch and Christa Kühn*
Corresponding author:
† Equal contributors
Research Unit Molecular Biology, Research Institute for the Biology of Farm Animals, 18196 Dummerstorf, Germany
Research Unit Genetics and Biometry, Research Institute for the Biology of Farm Animals, 18196 Dummerstorf, Germany
For all author emails, please .
BMC Genetics 2009, 10:50&
doi:10.56-10-50
The electronic version of this article is the complete one and can be found online at:
Received:15 June 2009
Accepted:2 September 2009
Published:2 September 2009
& 2009 B licensee BioMed Central Ltd.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background
The somatic cell score (SCS) is implemented in routine sire evaluations in many countries
as an indicator trait for udder health. Somatic cell score is highly correlated with
clinical mastitis, and in the German Holstein population quantitative trait loci (QTL)
for SCS have been repeatedly mapped on Bos taurus autosome 18 (BTA18). In the present study, we report a refined analysis of previously
detected QTL regions on BTA18 with the aim of identifying marker and marker haplotypes
in linkage disequilibrium with SCS. A combined linkage and linkage disequilibrium
approach was implemented, and association analyses of marker genotypes and maternally
inherited two-marker-haplotypes were conducted to identify marker and haplotypes in
linkage disequilibrium with a locus affecting SCS in the German Holstein population.
We detected a genome-wide significant QTL within marker interval 9 (HAMP_c.366+109G&A - BMS833) in the middle to telomeric region on BTA18 and a second putative QTL in marker interval
12-13 (BB710 - PVRL2_c.392G&A). Association analyses with genotypes of markers flanking the most likely QTL positions
revealed the microsatellite marker BMS833 (interval 9) to be associated with a locus affecting SCS within the families investigated.
A further analysis of maternally inherited two-marker haplotypes and effects of maternally
inherited two-marker-interval gametes indicated haplotype 249-G in marker interval 12-13 (BB710 - PVRL2_c.392G&A) to be associated with SCS in the German Holstein population.
Conclusion
Our results confirmed previous QTL mapping results for SCS and support the hypothesis
that more than one locus presumably affects udder health in the middle to telomeric
region of BTA18. However, a subsequent investigation of the reported QTL regions is
necessary to verify the two-QTL hypothesis and confirm the association of two-marker-haplotype
249-G in marker interval 12-13 (BB710 - PVRL2_c.392G&A) with SCS. For this purpose, higher marker density and multiple-trait and multiple-QTL
models are required to narrow down the position of the causal mutation or mutations
affecting SCS in German Holstein cattle.
Background
Udder health, somatic cell score and subclinical and clinical mastitis remain major
challenges for the economy of milk production in respect to milk production efficiency
and animal health and welfare. Several studies have attempted to identify chromosomal
regions, genes and polymorphisms that influence udder health in order to improve breeding
strategies. SCS has been used as an indicator of udder health and is implemented in
routine sire evaluations in many countries []. SCS has a low to medium heritability (h2 = 0.15; []) and a strong correlation to mastitis in the German Holstein population (rg = 0.84; []). However, selection on low SCS as well as on decreased mastitis incidence is hampered
by three aspects: first the low heritability of SCS and liability to mastitis, second
the difficulties in recording mastitis related data and third by potential population-wide
antagonisms between milk production traits (milk, fat and protein yield) and udder
health [,]. Recently, marker assisted selection (MAS) has been determined as a promising tool
to improve current selection strategies based on phenotypic data []. MAS implements genetic marker information of confirmed QTL regions to identify individuals
with favourable genetic background concerning the trait of interest. Thus, the confirmation
and fine mapping of known QTL regions and the estimation of QTL effects will advance
the use of MAS.
For clinical mastitis (CM) or SCS, QTL have been detected on nearly all autosomes
[] and several studies repeatedly detected QTL for SCS or CM in the telomeric region
of BTA18 [-]. In addition, Kühn et al. [] have shown in a proof-of-principle approach that information of 5 markers located
in the telomeric region of BTA18 indeed enabled successful MAS, which identified halfsib
heifers prior to first calving that exhibited significant differences in SCS after
parturition.
The intention of this study was to further analyse the middle to telomeric region
of BTA18 with the aim of identifying markers and marker haplotypes in linkage disequilibrium
(LD) with SCS in German Holstein cattle to improve MAS for udder health. Therefore,
we increased the marker density in the telomeric region on BTA18 and selected four
functional candidate genes within the QTL regions reported by [,,]. Polymorphisms detected within these candidate genes were used as additional markers
for fine mapping previously identified QTL regions and to analyse effects of candidate
gene polymorphisms on SCS in the German Holstein. In the present study, we detected
a genome-wide significant QTL in the middle to telomeric region on BTA18. Furthermore,
we analysed effects of maternally inherited marker haplotypes and identified a two-marker-haplotype
associated with SCS in German Holstein cattle.
Selection of candidate genes
Based on the positional information derived from the previously mentioned QTL regions
identified by [,,] and preliminary results from microarray experiments, an intensive literature research
of genes bearing potential function in innate immune defence, immune defence, mammary
gland development or udder morphology was performed. To enable the investigation of
positional candidate genes within the telomeric region on BTA18, a comparative map
for BTA18 and Homo sapiens autosome 19 (HSA19) was constructed to take advantage of the more advanced gene annotation
of the human genome (Additional file : marker table). Four candidate genes were selected: calmodulin 3 (phosphorylase kinase, delta) (CALM3), Hepcidin antimicrobial peptide (HAMP), cadherin 1, type 1, E-Cadherin (ephitelial) (CDH1), and poliovirus receptor-related 2 (herpesvirus entry mediator B) (PVRL2). Calmodulin 3 is a ubiquitously expressed Ca2+-binding protein that is involved in many Ca2+ modulated signal pathways. It was selected due to its influence on smooth muscle contraction
[,] and the potential effects on milk leaking and milk flow, which is a trait with a
substantial genetic correlation to SCS (rg = 0.4; []). HAMP was selected as an immunological candidate gene because of its antifungal and antibacterial
activity [], the toll-like receptor-4 dependent induction by bacterial pathogens in myeloid cells
[] and to a lesser extent due to its function as a key regulator of iron metabolism
[]. For CDH1 and PVRL2 initial results from own microarray experiments indicated their differential expression
in clinically unaffected heifers with different predisposition to udder infection.
Additionally, both genes are involved in cell-cell junctions that have a strong impact
on tissue development. E-Cadherin, a Ca2+-dependent cell-cell adhesion molecule, is involved in tissue and organ development
as part of the cadherin-catenin-complex [,], and poliovirus receptor-related 2, a Ca2+-independent immunoglobulin-like cell adhesion molecule, is involved in the organisation
of intercellular junctions as part of the nectin-afadin-complex [-]. Furthermore E-Cadherin can serve as a receptor for pathogens and is involved in
their internalization [,].
Additional file 1. Marker table. Summary of marker information including marker name, intervals, number of alleles,
polymorphism information content, position of markers in own linkage map and in published
linkage and RH maps and position in the bovine whole genome assemblies NCBI Build3.1
[GenBank: ] and Btau4.0 [GenBank: ] as well as comparative position in the human whole genome assembly HSA36.3. Markers
of intervals with marker spacing set to small values greater than zero are indicated
by *. For some markers no accession number was available, therefore references for
sequence information are given [,]. The polymorphism information content was calculated using the software PowerMarker
v3.25 []. Marker positions are in order to positions of 5'-nucleotides of upstream primers
for microsatellite markers or the direct position of the SNP. In Build3.1 several
discrepancies in the sequence assembly were discovered. Discrepancies to the refined
marker order are highlighted in yellow. Comparative positions in HSA36.3 were assigned
by BLAST search of marker sequences in the bovine whole genome assembly Btau4.0 to
identify the nearest gene locus on BTA18 and locating this gene locus in HSA36.3 using
NCBI Map Viewer [].
Format: XLS
Size: 31KB This file can be viewed with:
Screening for polymorphisms
Polymorphisms within the candidate genes were detected by comparative sequencing of
genomic DNA from heifers selected for QTL alleles associated with high or low SCS.
The heifers originate from most likely QTL heterozygous sires selected from the German
Holstein population based on marker information regarding a confirmed QTL for SCS
on BTA18. The selection strategy for the heifers and their phenotypes are described
by Kühn et al. [] in detail. For sequencing, we selected three pairs of halfsibs, where one daughter
inherited the SCS increasing paternal chromosomal region (q) and the other inherited
the SCS decreasing paternal chromosomal region (Q). In addition, two animals were
screened for variants which originated from a genetically divergent Charolais × Holstein
F2 cross background.
For each gene except CDH1 the entire gene (large introns excluded), 800 to 1500 bp of the promoter region and
up to 500 bp downstream of the transcripts were investigated. For CDH1, only the genomic sequence spanning exon 13 to 15 was analysed. Primer information
and the genomic position for each primer are given in additional file : primer table sequencing. Sequencing was performed by amplification of genomic DNA
and subsequent sequence analysis with the DYEnamic ET Terminator Cycle Sequencing
reaction and the MegaBACE(TM)1000 DNA Analysis System (GE Healthcare, Munich, Germany).
For evaluation of polymorphisms BioEdit 7.0.5.2 [] was used. Essentially, polymerase chain reaction (PCR) primers were used for sequencing.
Additional sequencing primers were used only for longer PCR fragments or PCR fragments
that were difficult to sequence.
Additional file 2. Primer table sequencing. Summary of primers used for sequencing, including primer sequence, position in Btau4.0
and polymorphisms detected in PCR fragments.
Format: XLS
Size: 38KB This file can be viewed with:
The pedigree material used for genotyping included a total of 1,054 animals originating
from six paternal halfsib families. Some of the animals are a subset of the granddaughter
designs previously described by [,]. Numbers of sons per grandsire ranged from 60 to 353, with an average family size
of 175 sons. The German genetic evaluation center (VIT) in Verden, Germany provided
additional pedigree information including non-genotyped ancestors of genotyped animals
(7,627 animals).
Phenotypes
The phenotype information for SCS was provided by the VIT as daughter yield deviations
(DYD) for the first lactation (Table ). SCS is the log2 transformed somatic cell count (log2 (somatic cell count/100000) + 3). DYD for SCS were calculated based on a random regression
test day model []. The reliabilities associated with the DYD were expressed as the number of effective
daughter contributions (EDC) as described by []. DYD and EDC for genotyped animals were obtained from the official release of the
April 2008 routine genetic evaluation.
Descriptive statistics for daughter yield deviations
Marker Set
The marker set included a total of 28 markers covering the telomeric region of BTA18
from CDH1 to DIK4013. Six of the 28 markers were already genotyped within previous QTL mapping studies
[,,] and include an erythrocyte antigen marker. The other 22 markers were newly selected
and genotyped. Fifteen new microsatellite and seven new single nucleotide polymorphism
(SNP) markers were chosen based on the information of the putative QTL positions reported
by [,,]. The fifteen new microsatellite markers were selected using the bovine linkage map
of the United States Department of Agriculture's Meat Animal Research Center (MARC
USDA) []. SNP markers were selected from all detected polymorphisms within candidate genes
based on allele frequencies, position (coding- or non-coding region), effect of the
SNP (synonymous or nonsynonymous) and whether they are in linkage disequilibrium to
each other.
Genotyping
Microsatellite markers were genotyped by PCR or Multiplex-PCR with fluorescence labelled
primers followed by a fragment length analysis using the MegaBACE(TM)1000 DNA Analysis
System and MegaBACE Fragment Profiler Version 1.2 software (GE Healthcare, Munich,
Germany) (Additional file : primer table genotyping). The genotyping methods used for detection of SNP were
PCR-restriction fragment length polymorphism (RFLP) assays for CDH1_c.2102C&T [NCBI dbSNP: rs], HAMP_c.366+109G&A [NCBI dbSNP: ss], PVRL2_c.-1268G&C [NCBI dbSNP: ss], PVRL2_c.392G&A [NCBI dbSNP: rs], CALM3_c.3+1795C&T [NCBI dbSNP: ss] and a multiplex pyrosequencing assay for HAMP_c.86+430G&A [NCBI dbSNP: ss] and CALM3_c.3+1678C&T [NCBI dbSNP: ss] (Table ). The enzymes used for detection of RFLPs were identified with the NEBcutter V2.0
webtool [,] and PCR primers were designed with primer analysis software Oligo 4.1 (National Bioscience
Inc., Plymouth, MN, USA). For the RFLP assays, a PCR specific for each SNP was used
to amplify genomic DNA, and PCR products were subsequently incubated for 8 h with
SNP specific restriction enzymes. For visualization of the RFLP a 2.5% agarose gel
Additional file 3. Primer table genotyping. Summary of primers used for genotyping microsatellites, including primer sequence
and position in Btau4.0.
Format: XLS
Size: 17KB This file can be viewed with:
Primer and enzymes used for genotyping of SNP
Primer design for pyrosequencing was performed with Pyrosequencing(TM) Assay Design Software
(Biotage AB, Uppsala, Sweden). A SNP specific PCR was used for amplification of genomic
DNA and the products of both PCR were merged and analysed with the PSQ(TM)HS 96A pyrosequencing
system (Biotage AB, Uppsala, Sweden) in a multiplex run.
Linkage Map
The genetic linkage map was calculated based on a refined marker order using CRIMAP
software []. The marker order on BTA18 used for the calculation was evaluated in two steps. First,
information from published linkage maps [,], RH-maps [], and human and bovine sequence-assemblies [] were merged and compared to own linkage-mapping results. Second, in a region including
the markers BMON117, DIK4672, BMS833, DIK4232 and BB710, no unequivocal marker order was obtained. Thus, the marker order in this region
was verified by RH-mapping using the 12000 rad whole-genome radiation hybrid panel
[] and RH-MAP3.0 software []. For some marker groups, a recombination rate of zero was calculated. To avoid technical
difficulties arising in the calculation of transmitting probabilities the marker spacing
was set to small values greater than zero (Additional file : marker table).
QTL Mapping
A combined linkage and linkage disequilibrium analysis (LALD) was performed using
the software system TIGER []. TIGER is a UNIX script linking several individual Fortran programmes to perform
combined linkage and linkage disequilibrium analysis. Six steps are implemented in
the script. First, allele frequencies and transmitting probabilities for each putative
QTL position are calculated using BIGMAP []. The putative QTL positions were considered as the midpoint of each marker interval,
resulting in a total of 27 putative QTL positions. Second, the identical by descent
(IBD) sub-matrices for each putative QTL position are computed based on the gene dropping
procedure described by [-] and third, the IBD sub-matrices are tested for positive definiteness and inverted.
The software program COBRA [] computes a condensed gametic relationship matrix and its inverse at each putative
QTL position for the calculation of gametic effects. Transmitting probabilities and
IBD sub-matrices are used for the set up of the condensed gametic relationship matrix.
Finally, an LALD analysis is performed analysing every putative QTL position with
restricted maximum likelihood (REML) methods applied in ASReml []. A detailed description of the QTL mapping procedure and the model applied in ASReml
is given by [].
Analyses were conducted using a likelihood ratio test, where the REML of the full
model was compared with the REML of the model missing the QTL effect. Chromosome-wide
and genome-wide significance thresholds were determined as restricted log likelihood
ratio (RLRT) equivalents of logarithm of odds (LOD) units [] where a LOD & 2 indicates chromosome-wide (RLRT = 9.2) and a LOD & 3 indicates genome-wide
significance (RLRT = 13.8) []. Confidence intervals were estimated using the LOD drop-off method described by [].
Association analysis
To investigate the association of candidate gene polymorphisms and markers flanking
interval 9 and interval 12-13 with SCS, a mixed model including a random polygenic
effect and the fixed effect of marker genotypes was applied in ASReml:
where y is a vector of phenotypic observations (DYD) for sires, μ is the overall mean, MGi is the fixed effect of the marker genotype i, aj is the random polygenic effect of animal j and eij is the random residual. The polygenic effect that accounts for the family structure
of the population was estimated using an extended pedigree of non-genotyped ancestors
of genotyped animals including a total of 7,627 animals. To account for multiple testing,
a 5% experiment-wise significance threshold was obtained by Bonferroni correction
of the nominal p-value assuming a 5% Type 1 error (pexp = 0.0057).
Analysis of maternally inherited two-marker haplotypes and two-marker-interval gametes
Due to the limited number of sires, paternally inherited chromosomes could have a
strong impact on genotype effects estimated in their offspring. To exclude these effects,
maternally inherited two-marker-intervals including flanking markers of most likely
QTL position were investigated. The most probable linkage phases of genotyped sires
were calculated to determine the maternally inherited haplotypes using BIGMAP. The
TIGER software system was then applied to estimate maternally inherited gametic effects
for the putative QTL positions in interval 9 and interval 12 based on IBD sub-matrices.
Finally, the estimated gametic effects of maternally inherited two-marker-interval
gametes were analysed and plotted using SAS software (SAS Institute, Cary, NC, USA).
To verify differences in estimated two-marker-interval gamete effects, we performed
an association analysis including a fixed maternally inherited two-marker-haplotype
effect independent of the IBD sub-matrices. For this purpose, the same model was used
as for marker genotypes (1), except that the fixed genotype effect was replaced by
the fixed maternally inherited two-marker-haplotype effect. Two-marker-haplotypes
with the highest and lowest mean for IBD gametic effects were tested against the total
of all other haplotypes within respective intervals. For interval 12-13 additionally
the most frequent two-marker-haplotype was investigated.
Screening for polymorphisms
Sequence analyses were based on the provisional reference sequences obtained from
NCBI []. For CALM3 [GenBank: ; GeneID: 520277], the entire gene (10728 bp) was resequenced including ~1.3 kb upstream
from the transcription start (assumedly promoter region). A total of eleven polymorphisms
were detected (Additional file : polymorphisms): ten SNP and one 12 bp deletion within the assumed promoter region.
Additional file 4. Polymorphisms. Summary of polymorphisms identified by comparative sequencing including polymorphism
name, accession number and sequence information for 200 nucleotides surrounding the
polymorphism.
Format: XLS
Size: 27KB This file can be viewed with:
For HAMP [GenBank: ; GeneID: 512301], in silico analyses revealed that this gene is not annotated in
Btau4.0 but has been annotated in NCBI Build3.1. The sequence of HAMP is still located on BTA18 in Btau4.0 [GenBank: : 476114 bp-477570 bp]. The whole gene (2777 bp) was resequenced, and three SNP were
detected (Additional file : polymorphisms), one SNP within intron 1 and two SNP within the first 110 nucleotides
downstream of HAMP transcript.
For CDH1, only the coding sequence was provided as provisional mRNA reference sequence [GenBank:
GeneID: 282637]. Due to discrepancies between the mRNA reference sequence and the
genome assembly sequence, only the genomic reference sequence was used for sequence
comparisons. The genomic sequence including exon 13 to 15 (1673 bp) was investigated
and six SNP were detected (Additional file : polymorphisms) two of them were located in exon 13 and cause an amino acid substitution.
For PVRL2 [GenBank: ; GeneID: 505580], the whole gene, excluding 13181 bp of intron 2 and 670 bp of intron
4, was resequenced (11766 bp). A total of 17 SNP and two length polymorphisms were
identified (Additional file : polymorphisms). The examination of the promoter region (1 kb upstream of transcription
start) of PVRL2 revealed a gap of 367 bp with missing sequence information in the genome assembly
Btau4.0. By a BLAST search [] a whole genome shotgun sequence (WGS)-Trace [Trace Archive: ] that overlaps
the gap was identified within the NCBI Trace Archives []. This WGS-Trace was incorporated in the reference sequence and sequence information
was confirmed by resequencing. Coordinates of polymorphisms upstream of the translation
initiation codon ATG are indicated according to the updated sequence [GenBank: ].
Markers and Map
The genetic marker map covered 50 cM of the telomeric region of BTA18 (Additional
file : marker table). Marker intervals ranged from 0.05 cM to 7.9 cM with an average marker
interval of 1.85 cM. The number of alleles for each marker ranged from two for SNP
to 34 for the erythrocyte antigen marker. The marker order is in good agreement with
previously published linkage- and RH-maps [-] and no discrepancies to the bovine sequence assembly Btau4.0 [] were observed (Additional file : marker table).
QTL Mapping
The restricted Log Likelihood Ratio profile of the combined LALD analysis is shown
in Figure . Two peaks exceeding the genome-wide significance level can be observed. The maximum
of the first peak is located in marker interval 9 at position 71.775 cM with HAMP_c.366+109G&A and BMS833 as flanking markers. The maximum of the second peak is located in marker interval
12 at position 77.6 cM with the flanking markers BB710 and PVRL2_c.-1268G&C. The LOD drop-off method was used to estimate confidence intervals for each of the
peaks. An approximate 96% confidence interval included marker intervals 3 to 9 for
the first peak and the marker intervals 2 to 15 for the second peak.
LALD Profile. Restricted Log Likelihood Ratio (RLRT) profile of a combined linkage and linkage
disequilibrium analysis testing for a putative QTL affecting SCS on BTA18. The profile
is plotted for putative QTL positions located at the midpoint of each marker interval.
QTL positions are indicated by black dots. Thick black lines indicate confidence interval
for the first maximum at interval 9 (upper line) and the second maximum at interval
12 (lower line). Dashed lines indicate genome- and chromosome-wide significance thresholds.
Association analysis
An association analysis between the candidate gene polymorphisms and SCS was performed
to evaluate the influence of the selected candidate genes on the variation in SCS
in German Holstein cattle. The markers BMS833 and BB710 were also included in the association analysis, as they are flanking markers of the
maxima observed in the LALD test statistic. The association analyses (Table ) indicated a significant effect of the BMS833 genotype (p = 0.004) on SCS within the 5% experiment-wise significance threshold (pexp = 0.0057). PVRL2_c.392G&A (p = 0.017) and CALM3_c.3+1678C&T (p = 0.055) approached nominal significance, but were not significant at the 5% experiment-wise
significance level. For the genotypes of SNP markers CDH1_c.2102C&T, HAMP_c.366+109G&A, HAMP_c.86+430G&A, PVRL2_c.-1268G&C and CALM3_c.3+1795C&T no significant effects on SCS were observed.
Association analyses of marker genotypes with SCS in six German Holstein halfsib families
Analysis of maternally inherited two-marker haplotypes and two-marker-interval gametes
To exclude any specific sire gamete effects that occur in a paternal halfsib design,
maternally inherited gametic effects for two-marker-interval gametes were analysed
and plotted for the putative QTL positions in interval 9 and interval 12-13. In interval
9 (HAMP_c.366+109G&A - BMS833), six maternally inherited two-marker-allele combinations occurred. Two-marker-interval
gametes including BMS833 alleles 115 and 119 were excluded, because they both occurred only once (T Figure ). Gametes carrying the BMS833 allele 117 in interval 9 have a mean estimated maternally inherited gametic effect of 0.022 (±
0.003) and 0.028 (± 0.003), respectively, whereas gametes carrying allele 113 of marker BMS833 have mean effects of - 0.0006 (± 0.0014) and - 0.0059 (± 0.0022), respectively. Investigating
the gametic effect of each flanking marker of interval 9 alone (Figure ), revealed that the main differences in gametic effects in the two-marker-interval
results from the discrimination by microsatellite marker alleles. The difference in
the mean effects for two-marker-interval gametes carrying the two alleles of HAMP_c.366+109G&A is 0.009, whereas the difference between gametes carrying alleles 113 and 117 of marker BMS833 is 0.0268.
Estimated effects on SCS for maternally inherited two-marker-gametes in interval 9
and interval 12-13
Box-whisker plot for estimated effects of maternally inherited two-marker-interval
gametes in interval 9. Two-locus-interval (A) and single locus (B) gametes of the two-marker-interval flanking
the putative QTL in interval 9 are written on the X-axis. The boxes contain 50% of
all values, where (+) represents the mean, and the horizontal line within the box
(-) represents the median. The first and third quartiles are represented by the lower
and upper edge of the box and the whiskers extend to the highest and lowest values.
For interval 12-13 (BB710 - PVRL2_c.392G&A) we selected PVRL2_c.392G&A (interval 13) as a flanking marker. Both polymorphisms, PVRL2_c.-1268G&C (interval 12) and PVRL2_c.392G&A (interval 13) are located in close vicinity within the PVRL2 gene and showed a high linkage disequilibrium (r2 = 0.68). In interval 12-13 (BB710 - PVRL2_c.392G&A), two-marker-interval gametes with twelve different two-marker-allele combinations
were observed. Two-marker-interval gametes carrying the two BB710 alleles 243 and 245 were excluded, because they both only occurred once. In addition, the frequencies
for allele combinations 253-G and 257-G were smaller than 1% and therefore gametes carrying these two allele combinations
are not included in Figure
(Table ). Analogous to the situation in interval 9, the SNP alleles of PVRL2_c.392G&A seem to have only a small influence on discriminating estimated two-marker-interval
gamete effects (Figure ), because the difference in the mean effects discriminated by the alleles is 0.0078.
For microsatellite marker alleles (Figure ), two-marker-interval gametes carrying the allele 249 have mean effects of 0.0378 (± 0.0018) and 0.0176 (± 0.0009) and gametes carrying
allele 253 have mean effects of - 0.0150 (± 0.0021) and - 0.0253 (± 0.0012). The biggest differences
in the mean estimated gametic effects was observed for the two two-marker-interval
gametes 249-G (0.0378 (± 0.0018)) and 253-A (-0.0253 (± 0.0012)). Thus, the difference in the mean maternally inherited gametic
effects equals 0.0631, which is equivalent to 0.16 phenotypic standard deviations.
Box-whisker plot for estimated effects of maternally inherited two-marker-interval
gametes in interval 12-13. Two-locus-interval (A) and single locus (B) gametes of the two-marker-interval BB710 (interval 12) and PVRL2_c.392G&A (interval 13) are written on the X-axis. Estimates are for the putative QTL position
in interval 12. The boxes contain 50% of all values where (+) represents the mean
and the horizontal line within the box (-) represents the median. The first and third
quartiles are represented by the lower and upper edge of the box and the whiskers
extend to the highest and lowest values.
To validate the differences observed between maternally inherited two-marker-interval
gametes, we performed a direct association analysis for maternally inherited two-marker-haplotypes
without considering IBD coefficients in interval 9 (HAMP_c.366+109G&A - BMS833) and interval 12-13 (BB710 - PVRL2_c.392G&A) (Table ). Only the two-marker-haplotypes with the highest and lowest mean for IBD gametic
effects were tested against the total of all other haplotypes. For interval 12-13,
we also tested 255-A, because it was the most frequent haplotype (frequency at 40%) in the data set. In
interval 9, no significant association of the maternally inherited haplotypes G-113 and G-117 were observed, whereas haplotype 249-G (p = 0.027) in interval 12-13 seems to be associated with SCS in the German Holstein
population.
Association analyses of maternally inherited two-marker-haplotypes with SCS
Discussion
In an approach to identify marker and marker haplotypes affecting SCS in the German
Holstein population, we analysed association of marker genotypes and maternally inherited
two-marker-intervals flanking putative QTL positions detected in an LALD analysis
for SCS on BTA18, and showed that the two-marker-haplotype 249-G of interval 12-13 (BB710 - PVRL2_c.392G&A) is in LD with SCS in the German Holstein population.
Initially, we detected a genome-wide significant QTL for SCS on BTA18 (Figure ). The maxima of the QTL test statistic were in interval 9 (HAMP_c.366+109G&A - BMS833) and in interval 12 (BB710 - PVRL2_c.-1268G&C). In the same region telomeric on BTA18, several studies reported QTL for SCS. Kühn
et al. [], Brink [] and Xu et al. [] all identified a QTL for SCS in the German Holstein population at the telomeric end
of BTA18 near marker TGLA227. Schulman et al. [] reported a QTL for SCS as well as for mastitis at the telomeric end of BTA18 in Finnish
Ayrshire cattle, and Ashwell et al. [] localized a QTL for SCS in the middle to telomeric region on BTA18 at marker BM2078
in US Holstein, whereas Lund et al. [] reported a QTL for SCS in Finnish Ayrshire, Swedish Red and White, and Danish Red
with the maximum of the test statistic in the middle of BTA18 between the markers
ILSTS002 and BMS2639. Interestingly Lund et al. [] as well as Xu et al. [] indicated that there might be more than one QTL for SCS on BTA18, but in both studies
no significant evidence could be provided for a second QTL. Due to the lower marker
density in previous studies and different approaches to detect QTL, a direct comparison
between our results and previously mentioned studies is impeded. Nevertheless, the
confidence intervals of the maxima observed in our LALD analysis did not include marker
TGLA227 at the telomeric end of BTA18, and the QTL position reported by Lund et al. [] as well as the assumption of a second QTL by Xu et al. [] further in the middle of BTA18, indicated that we identified a second QTL for SCS
in German Holstein cattle and possibly discovered a third QTL on BTA18 in our studies.
Further indications for a third QTL on BTA18 in German Holstein cattle arise from
association and haplotype analyses but no formal proof for a third QTL is given in
our studies.
Subsequent to QTL mapping, we performed an association analysis of marker genotypes
to verify the association of candidate gene polymorphisms and flanking markers of
the most likely QTL positions observed in LALD analysis with SCS. Our results indicated
that the microsatellite marker BMS833 (p = 0.004) is associated with SCS and PVRL2_c.392G&A (p = 0.017) and CALM3_c.3+1678C&T (p = 0.055) showed a respective tendency of association. Corresponding to the results
of our LALD analysis, these results confirmed the position of one QTL for SCS in interval
9, as BMS833 is one of the flanking markers of this interval. PVRL2_c.392G&A and CALM3_c.3+1678C&T are flanking markers of the intervals 13 (PVRL2_c.-1268G&C - PVRL2_c.392G&A), 14 (PVRL2_c.392G&A - DIK3014) and intervals 16 (DIK4234 - CALM3_c.3+1678C&T) and 17 (CALM3_c.3+1678C&T - CALM3_c.3+1795C&T). The weak association observed for these markers is also in accordance to our LALD
analysis, because interval 13 and 14 are within the genome-wide significance threshold
in LALD analysis. Additionally, the second maximum observed in our LALD analysis in
interval 12 is in an immediately adjacent interval and the putative QTL position in
interval 12 is approximately 1 MB upstream of PVRL2_c.392G&A. For CALM3_c.3+1678C&T, only interval 16 (DIK4234 - CALM3_c.3+1678C&T) is within the chromosome-wide significance level in our LALD test statistic confirming
the weaker association with SCS observed. To further test the results of our LALD
analysis and the association of BMS833 genotypes with SCS on a population wide level, we investigated maternally inherited
two-marker-intervals for interval 9 (HAMP_c.366+109G&A - BMS833) as well as for interval 12-13 (BB710 - PVRL2_c.392G&A). First we analysed effects of maternally inherited two-marker-interval gametes estimated
based on IBD coefficients and second we performed a direct association analysis for
maternally inherited two-marker-haplotypes of interval 9 and interval 12-13 without
considering IBD coefficients.
For interval 9 (HAMP_c.366+109G&A - BMS833), maternally inherited two-marker-interval gametes showed differences in estimated
effects for SCS (Figure ). The variance of estimated gametic effects was higher for two-marker-interval gametes
in interval 9 compared to two-marker-interval gametes in interval 12-13 and single
marker analyses revealed that the microsatellite marker alleles are the main force
in discriminating the effects of maternally inherited two-marker-interval gametes
(Figure , Figure ). Association analysis for maternally inherited two-marker-haplotypes of interval
9 showed that none of the maternally inherited two-marker-haplotypes in interval 9
(HAMP_c.366+109G&A - BMS833) are in LD with SCS in the German Holstein population. However, association analysis
of BMS833 genotypes showed an association with SCS in our half-sib design indicating that the
association is due to linkage but not linkage disequilibrium of the BMS833 locus with
the causal mutation affecting SCS.
For interval 12-13 (BB710 - PVRL2_c.392G&A), the biggest difference in gametic effects estimated for maternally inherited two-marker-interval
gametes was observed between 249-G (0.0378 (± 0.0018)) and 253-A (- 0.0253 (± 0.0012)), where a positive mean of estimates indicates an unfavourable
effect on SCS (high number of cells) and a negative mean of estimates indicates a
favourable effect on SCS (low number of cells). Association analyses for the maternally
inherited two-marker-haplotypes of interval 12-13 showed that 249-G (p = 0.027) is associated with SCS at the nominal 5% significance level in the German
Holstein population. The weak association of PVRL2_c.392G&A genotype with SCS within the families we investigated and the association of maternally
inherited two-marker-haplotype 249-G with SCS indicates that PVRL2_c.392G&A is not the causal mutation affecting SCS in German Holstein cattle, but the causal
mutation has to be located near or within marker interval 12-13 (BB710 - PVRL2_c.392G&A). Combining results obtained for markers BMS833 and PVRL2_c.392G&A it still remains unclear whether there are two mutations (two QTL), one located near
marker BMS833 (interval 9) and one near or within interval 12-13, or only one mutation, presumably
in interval 12-13, affecting SCS in the middle part of BTA18.
The candidate gene polymorphisms we investigated were not the causal mutations affecting
SCS in German Holstein cattle. However, the results of association analyses for single
marker genotypes and maternally inherited two-marker-haplotypes indicated that HAMP and PVRL2 were selected within the region harbouring at least one QTL for SCS. Particularly
PVRL2 still remains interesting. On the one hand, the association of the PVRL2_c.392G&A genotype and that of the 249-G haplotype suggests that another polymorphism within PVRL2 is the causal mutation affecting SCS in the German Holstein population. On the other
hand, PVRL2 was selected as a gene with possible impact on mammary gland development or udder
morphology and several studies have detected QTL for udder conformation on BTA18 [,]. Therefore, it is also possible that PVRL2 does not directly affect SCS but affects udder conformation traits like udder depth
or fore udder attachment that are correlated with SCS [].
Conclusion
In summary, our results suggest that the chromosomal region including interval 9 (HAMP_c.366+109G&A - BMS833) and interval 12-13 (BB710 - PVRL2_c.392G&A), in the middle to telomeric region on BTA18 has a strong impact on SCS in the German
Holstein population. The analyses of maternally inherited two-marker-interval gamete
effects and the association of the maternally inherited two-marker-haplotype 249-G of interval 12-13 (BB710 - PVRL2_c.392G&A) with SCS indicates that micosatellite marker BB710 could be a suitable candidate-marker for MAS, but association of microsatellite marker
BB710 with SCS has to be verified. To confirm the association of the two-marker-haplotype
249-G with SCS and approve the hypothesis of two QTL in this region a further investigation
is necessary. Thus, a mapping of udder conformation traits including a multiple-trait
and multiple-QTL model might be useful to verify the existence of two QTL, and whether
they are both directly affecting SCS or one is affecting a correlated trait. Likewise,
a higher marker density within this region has to be achieved and families segregating
for different BB710 alleles have to be identified. Hence, it might be useful to cover the region including
interval 9 and interval 12-13 with equally distributed SNP to narrow down the position
of the casual mutation or mutations affecting SCS in the German Holstein population
by a further fine mapping approach.
Authors' contributions
BB carried out the genotyping work, the polymorphism screening, the linkage map construction,
participated in the statistical analyses and drafted the manuscript. CB facilitated
the statistical analyses by the development of software packages, participated in
the statistical analyses and helped drafting the manuscript. MM participated in the
development of software packages and the statistical analyses. NR participated in
design and coordination of the study. CK devised the design of the study, coordinated
the study, and participated in the statistical analyses and in drafting the manuscript.
All authors read and approved the final manuscript.
Acknowledgements
The authors would like to thank M. Schwerin and A. Hartmann for information on unpublished
microarry data and the colleagues within the FUGATO M.A.S.net project for fruitful
discussions. The financial support of the German Federal Ministry of Education and
Research (BMBF) (Projekt FUGATO M.A.S.net, FKZ 0313390A) and the Development Association
for Biotechnology Research (FBF) e.V., Bonn, is gratefully acknowledged. In addition,
we thank F. Reinhardt, Z. Liu and the Vereinigte Informationssysteme Verden w.V. (VIT)
for assistance with the selection of animals and for providing data.
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