Identification of protein-damaging mutations in 10 swine taste receptors and 191 appetite-reward ...

17 Pages·2016·0.85 MB·English

by  Alex Clop

Clopetal.BMCGenomics (2016) 17:685
DOI10.1186/s12864-016-2972-z
RESEARCH ARTICLE Open Access
Identification of protein-damaging
mutations in 10 swine taste receptors
and 191 appetite-reward genes
Alex Clop1*, Abdoallah Sharaf1,2, Anna Castelló1, Sebastián Ramos-Onsins1, Susanna Cirera3, Anna Mercadé1,
Sophia Derdak4,5, Sergi Beltran4,5, Abe Huisman6, Merete Fredholm3, Pieter van As7 and Armand Sánchez1,8*
Abstract
Background: Taste receptors (TASRs) are essential for thebody’srecognitionof chemical compounds. Inthe
tongue, TASRs sense the sweet and umami and the toxin-related bitter taste thus promoting a particular eating
behaviour. Moreover, theirrelevance inother organs is now becoming evident. Inthe intestine, theyregulate
nutrient absorption and gut motility. Upon ligand binding, TASRs activate theappetite-reward circuitry to signal
thenervous system and keep body homeostasis. With the aim to identify genetic variation intheswine TASRs
and in thegenes from theappetiteand thereward pathways, we have sequenced theexons of 201TASRsand
appetite-reward genes from 304 pigs belonging to ten breeds, wild boars and to two phenotypically extreme
groups from
a F resource with data on growth and fat deposition.
2
Results: We identified 2,766codingvariants 395 ofwhich were predictedto have a strong impact onprotein
sequence and function. 334 variants were present inonly one breed and atpredicted alternativeallele frequency
(pAAF)≥0.1. The Asian pigs and thewild boars showed thelargest proportion of breed specific variants. We
also compared thepAAF ofthe two F groups and found thatvariants inTAS2R39 and CD36 display significant
2
differences suggesting that these genes could influence growthand fat deposition.We developed a 128-variant
genotyping assay and confirmed 57 of thesevariants.
Conclusions: We have identified thousandsof variants affecting TASRs as well as genes involved inthe appetite
and the reward mechanisms. Some ofthesegenes have been already associated to taste preferences, appetite or
behaviour inhumans and mouse. We have also detected indications of a potential relationship of some of these
genes withgrowth and fat deposition, which could have been caused by changes intaste preferences, appetite
or reward and ultimately impact on food intake. A genotypingarray with57 variantsin 31 of thesegenes is now
available for genotyping and start elucidating the impact of genetic variation in thesegenes on pig biology and
breeding.
Keywords: Taste receptors, Appetite-rewardpathways,Coding genetic variation, Genotyping array, Future
association studies
*Correspondence:[email protected];[email protected]
1CentreforResearchinAgriculturalGenomics(CRAG)CSIC-IRTA-UAB-UB,
CampusUAB,08193CerdanyoladelValles,Catalonia,Spain
Fulllistofauthorinformationisavailableattheendofthearticle
©2016TheAuthor(s).OpenAccessThisarticleisdistributedunderthetermsoftheCreativeCommonsAttribution4.0
InternationalLicense(http://creativecommons.org/licenses/by/4.0/),whichpermitsunrestricteduse,distribution,and
reproductioninanymedium,providedyougiveappropriatecredittotheoriginalauthor(s)andthesource,providealinkto
theCreativeCommonslicense,andindicateifchangesweremade.TheCreativeCommonsPublicDomainDedicationwaiver
(http://creativecommons.org/publicdomain/zero/1.0/)appliestothedatamadeavailableinthisarticle,unlessotherwisestated.
Clopetal.BMCGenomics (2016) 17:685 Page2of17
Background during fasting also promotes glutamate release. This
There are five canonical tastes that are sensed in the neurotransmitter, via a large catalogue of receptors, will
taste buds of the tongue, which include salty, sour, also excite NPY, AgRP and inhibit POMC neurons and
sweet, umami and bitter. While salty and sour are de- boost appetite. The catabolic product of glutamate,
tected by ion channels, the other three are sensed by a gamma aminobutyric acid (GABA) also boosts appetite
group of G-protein coupled receptors called taste recep- butusingdifferentneuronalmechanisms[10].Moreover,
tors (TASRs). Sweet and umami are appetizing tastes glutamate is also able to excite dopamine-secreting neu-
that characterize energy-rich food sources, namely sugar ronsinappetite-relevantareasofthebraintherebyindir-
and amino acid molecules, respectively, and are sensed ectly promoting the feeling of hunger [9]. In contrast,
by the TAS1R sub-type TAS1R1, TAS1R2 and TAS1R3 GABA can inhibit the same neurons and indirectly pro-
[1].Ontheotherhand,the–unpleasant-bittertastein- mote satiety [9]. Serotonin, a neurotransmitter that is
dicating the presence of toxic molecules, is sensed by mostly present in the gastrointestinal tract but also at
TAS2Rs, also known as bitter taste receptors [1], which much lower levels in the central nervous system, modu-
include a variable list of highly polymorphic genes with lates gastrointestinal motility, mood and appetite [11].
many species-specific orthologs. The annotation of the Serotonin inhibits appetite by stimulating its receptors
pig genome contains ten TAS2Rs according to the HTR2C and HTR1B, which in turn, activate the well-
Ensembl database (www.ensembl.org). In the recent known appetite-inhibitors POMC and MC4R and inhibit
years, it has become obvious that TASRs are expressed the appetite-promoter genes NPY and AGRP. Epineph-
in many other tissues and have additional chemo- rine/norepinephrine are two additional neurotransmit-
sensing functions. For example, they are present in the ters that seem to be key in food intake and in keeping
respiratory system where they regulate innate immunity energy balance and have been shown to respond to star-
and infection [2], and in sperm they have been linked to vation and low glucose levels in blood by activating the
motility and acrosomal reaction [3]. In the gastro- secretion ofghrelin[12].
intestinal tract, TASRs detect the molecules that are on In humans, the recent advent of whole genome [13]
transit and stimulate the appetite and reward (AR) cir- and exome [14] sequencing has shown that mutations
cuitries to promote the appropriate feeding behaviour, severely impacting on protein sequence are more abun-
thus keeping energy balance and body homeostasis [4, dant than previously thought, although due to purifying
5]. The AR mechanisms are highly interconnected and selection, they tend to have very low allele frequencies.
involve complex networks containing nutrients, neuro- Thus, protein-damaging polymorphisms in TASRs and
peptides, neurotransmitters, hormones and their related AR genes are likely to have an important impact on a
receptors and enzymes. These pathways engage the broad range of traits including feed intake, immune
gastrointestinal tract, pancreas, liver, muscle, adipose tis- function, behaviour or fertility both in livestock and
sue and brain. Appetite-related genes such as leptin humans. Consequently, understanding how these vari-
(LEP), leptin receptor (LEPR), cholecystokinin (CCK), ants affect phenotypes in farm animals may both help
Ghrelin (GHRL), Agouti-related protein (AgRP), neuro- improving the sustainability of the animal breeding sec-
peptide Y (NPY), proopiomelanocortin (POMC) and tor as well as benefit bio-medical research. Scientific
melanocortin 4 receptor (MC4R) encode for products interest in this gene family in the pig is now emerging
that inhibit or excite the dopamine, epinephrine, nor- and it has recently been shown that TASRs are
epinephrine, serotonin, and glutamate receptor pathways expressed in multiple porcine systems (immune, gastro
[6, 7] and modulate food intake and energy balance. In a intestinal, spermatogenic, etc. [15, 16]). The recent pub-
nutshell, ghrelin and LEP are two hormones with oppos- lication of the swine genome sequence and annotation
ite excitatory and inhibitory effects on the same neurons [17] and the development of genome capture assays
secreting appetite inducing NPY and AgRP or the feed- open unprecedented possibilities to identify deleterious
ing inhibitor POMC. These neuropeptides in turn, in- genetic variation. For instance, 295 coding variants in
activate or excite MC4R, which is a hunger repressor. swine TASRs have recently been identified after analyz-
Ghrelin is secreted by the stomach when it is empty and ing the low coverage whole genome sequences of 79 do-
LEP is released by adipocytes as a response to high en- mesticandwildpigsfromEuropeandAsia[18].
ergy stores [6]. CCK is a hormone secreted in the duo- Motivated by their functional relevance in the pig, we
denum as a response to luminal fat and protein and is a have sequenced ten porcine canonical TASRs and 191
strong inhibitor of food intake probably by decreasing AR genes in 304 pigs from multiple breeds in 16 DNA
gastric emptying and stimulating the vagus nerve [8]. pools with the aim to identify coding polymorphisms
LEP and ghrelin also inhibit and excite dopamine secre- and to provide a catalogue of potentially deleterious mu-
tion, respectively [9]. Dopamine signalling in certain tations likely to affect the function of these genes. More-
parts of the brain promotes appetite. Ghrelin secretion over, we have also detected indications that some of
Clopetal.BMCGenomics (2016) 17:685 Page3of17
these variants might be associated with growth and fat- and one novel splice-site donor. These variants affect
ness giving new insights in regard to the potential four TASRs with a clear over-representation in TAS1R1
phenotypic relevance of polymorphisms within these (Table 1). Three of these variants affecting TAS1R1 and
genes. TAS1R3 had a predicted minor allele frequency
(pMAF)≤0.01 (Tables 2 and 3). In addition, we identi-
Results fied 125 non-synonymous-coding variants and one
Sequencingstatistics codon-deletion, which are classified by snpEff as having
We initially selected 459 kb of target genomic DNA a moderate (M) impact. Thirty-four of the non-
(gDNA) covering the exons from the 12 TASRs and 201 synonymous changes were predicted to be deleterious
AR genes. After genome capturing, sequencing and read (Mdel) bySIFT[19](Table1).TheremainingMvariants
mapping, we successfully covered 372 kb of target were either SIFTpredicted astolerated (Mtol)ordidnot
sequence with a read depth at each nucleotide position yield any prediction. Hence, we have identified 44 vari-
(DP) above 1,000 in the 16 libraries as a whole. This ants (10 H and 34 Mdel) that are likely to have an im-
corresponds to 81 % of the initial target size and 201 portant effect on swine TASR function. Remarkably, all
genes fully or partially sequenced to a DP>1,000. The TASRs showed H or Mdel variants (Additional file 2).
poorly sequenced genes include TAS2R3, ENSSSCG Finally, 81 variants were predicted to be synonymous
000000029894 (a swine ortholog of human TAS2R16), changes with no apparent impact (L) on the subjacent
and 10 AR genes. The list of successfully sequenced proteins (Table 1). On average, the variants with strong
genes is shown in Additional file 1. After genome cap- impact on protein sequence (H and Mdel) were pre-
ture, sequencing and read mapping, 162,848,637 reads dictedtoberarerinthespeciesthanthose havingamild
mapped tothetargetgDNAregions. impact(MtolandL)(Table2)according topMAF.
Likewise, we sequenced 357,844 (80.1 % of the initial
VariantidentificationinTASRandARgenes selection) bp of exonic sequence from AR genes at a
We successfully sequenced (DP≥1,000) 14,598 bp DP>1,000, and identified 2,570 variant positions in AR
(94.5 % of the initial selection) of TASR exons and iden- exons, most of which were bi-allelic SNVs. 1,092
tified 219 coding variants in TASR exons, 113 of which (42.4 %) variants were not annotated in dbSNP
(52.1 %) do not have a dbSNP identifier and are thus (Additional file 2). Four of these positions were multi-
considered novel (Additional file 2). Two TASR variants allelic with each allele predicted to have a different effect
had the alternative allele fixed in the 16 pools and are onproteinsequence.Ofthe2,574assignedvarianteffects,
thus likely to be either errors or private variants in the 25 had the alternative allele fixed in the 16 pools. From
Duroc animal used to generate the reference sequence. the remaining 2,549 variants, 350 were classified as dele-
We excluded them from our list of putative polymor- terious(HandMdel)andaffected123genes(Table1and
phisms. Ten of the 217 remaining variants were Additional file 2). As for TASRs, H and Mdel as a whole
classified by snpEff to have a high impact (H) on the tended to have lower pMAF than Mtol and L (Table 2).
coding sequence and consequently, on the function of Weidentified74HvariantsinARgenes,29ofwhichhad
the gene according to the gene annotation in the swine pMAFs≤0.01 and are thus considered rare. These H rare
genome (Table 1). These include four single nucleotide variantsaffected21genes(Table3).
variants (SNVs) and six short indels, which cause three We also plotted the distribution of the AR gene vari-
stop-codon gains, one stop-codon loss, five frame-shift, ants predicted to have a strong impact on protein (H
Table1NumberofvariantsacrosstheTASRandARgenegroupspereachimpactclass
Highimpact Moderateimpact Lowimpact
Gene Splice Stopgained Stoplost Startloss Frameshift Mdel Mtol/SIFT Silent Startgained %strong Total
unknown impact
TotalTASRs 1 3 1 0 5 34 92 80 1 20.3% 217
TAS1R1(umami) 1 1 1 0 3 4 9 12 0 32.2% 31
TAS1R3(sweetandumami) 0 1 0 0 0 1 0 11 1 14.3% 14
TAS2Rs(bitter) 0 1 0 0 2 29 83 57 0 18.5% 172
AR 37 16 0 4 17 277 615 1,559 24 14.5% 2,549
Totalsegregating 38 19 1 4 22 311 707 1,639 25 2,766
Alternativeallelefixed 4 0 0 0 4 0 9 9 1 27
Total 42 19 1 4 26 311 716 1,648 26 2,793
Splice:variantspredictedtoaltereitherdonororacceptorsplicesites;%strongimpact:percentageofH+Mdelvariants
Clopetal.BMCGenomics (2016) 17:685 Page4of17
Table2VariantdistributionpereffectandpAAFwithineachgenegroup
Variantfrequencyclass Strongimpact Mildimpact
TASR Veryrare(pMAF<0.01) 28(63.6%) 51(29.5%)
Rare(pMAF=[0.010–0.019]) 6(13.6%) 28(16.2%)
Common(pMAF=[0.020–0.979]) 10(22.7%) 94(54.3%)
Totalnumber 44 173
pMAFmin-max(average) 0.0016–0.1910(0.028) 0.0016–0.4920(0.091)
AR Veryrare(pMAF<0.01) 184(52.6%) 560(25.5%)
Rare(pMAF=[0.010-0.019]) 41(11.7%) 235(10.7%)
Common(pMAF=[0.020–0.979]) 125(35.7%) 1,403(63.8%)
Totalnumber 350 2,198
pMAFmin-max(average) 0.0017–0.4800(0.029) 0.0016–0.4980(0.0992)
Thepercentagesareforthetotalnumberofvariantswithinthegroups:TASRstrongimpact,TASRmildimpact,ARstrongimpactandARmildimpact
and Mdel) and those with a mild impact (Mtol and L) observed (p-val=0.07) and the Duroc and Landrace
along the protein sequence divided in 10 consecutive were at the top (12.7 %) and bottom (9.4 %) ends, re-
position bins of equal amino acid length. We noted that spectively(Table4).
the strong impact variants tended to be more abundant Altogether, 26 TASR variants were present in a single
at the end of the protein (bin9+bin10), and that these breed at pAAF≥0.1 and might thus be breed-specific.
alsotendedtohave,onaverage,largerpMAF.Thistrend Not surprisingly, the Asian pool, which involved 15
was not observed in the set of mild impact variants Chinese Meishan and 7 Vietnamese pigs, showed the
(Fig. 1). highest proportion of pool-specific variants, with 12 be-
ing present only in this group (Additional file 3). The
Perbreedvariantdistribution wild boar also displayed several allelic particularities,
We compared the ten purebred pools and observed that, with nine unique variants. Noteworthy, seven of these
as expected, larger pools contained more variants (both wild boar variants, all with similar pAAF, mapped to
in TASR and AR genes). Nonetheless, the two Duroc TAS2R1 (Additional file 3). Overall, five (one H and four
pools together, with 45 samples, displayed less genetic Mdel) breed-specific variants were predicted to have a
diversity (1,042 variants), than the Large White, Land- strong impact on protein sequence (Additional file 3).
race and the Pietrain, which had a similar number of an- TheHvariant isa stop gaininTAS1R1thatispresent in
imals and were above 1,300 variants each (Table 4). The 17 % of the Mangalitza genomes, respectively. We also
Asian pool harboured the largest number of variants identified two variants, a synonymous (L) and a non-
(1,589 variants with a pool size=22) while the Iberian synonymous tolerated (Mtol) SNVs that affect TAS2R1,
pool was ranked as the most homogenous (746 and pool that whilst being present at very high frequencies or
size=13). Overall, we observed that the most ancient even fixed in all breeds (pAAF≥0.5), were absent in the
breeds, Mangalitza, Iberian, Majorcan Black were less Asianpool(Additionalfile3).
variableinoursetofgenes(Table4).Giventhatahigher We detected 306 AR coding variants that were
number of samples seem to be correlated to a higher uniquely present or absent in one breed (Additional file
number of variants, we also compared the genetic vari- 3). Of these, 35 variants involving 32 genes were pre-
ability using the Watterson estimate [20] at the neutrally dicted to be of functional importance (Additional file 3).
evolving synonymous sites (the third nucleotide position As in TASRs, the breed specific H and Mdel variants in
within each codon), which corrects the number of the AR genes were more abundant in the Asian and the
mutated sites by the number of animals included in the wild boar with 20 and five unique features, respectively.
analysis. This value (Bazna: 0.00118, Mangalitza: 0.00101, It was noteworthy that two of these H and Mdel vari-
Majorcan Black: 0.00103, Iberian: 0.00095, Duroc: 0.00090, ants, both in the Asian pools, were close to fixation
Pietrain:0.00131,Landrace:0.00130,LargeWhite:0.00130, (pAAF≥0.9).Thesevariantsmaptotheserotoninrecep-
wild boar:0.00109 and Asian: 0.00182) showed that all the tor, HTR3C, and to the cytochrome P450 gene, CYP2A6
populationshadsimilarvariabilitywiththeexceptionofthe (Additional file3).
Asian pool, which genetic variability doubled that of the We performed hierarchical clustering using an Un-
otherbreeds. weighted Pair Group Method with Arithmetic Mean
We also compared the percentage of variants that (UPGMA) based on the 2,523 (217 TASR and 2,306 AR)
were H or Mdel per breed. No obvious differences were variants that were present in at least one of the ten
Clopetal.BMCGenomics (2016) 17:685 Page5of17
Table3ListofrareHvariants
VariantID Effect Gene pMAF
chr12_15398873_C_T STOP_GAINED ACE 0.0017
chr12_62594159_A_G START_LOST ALDH3A2 0.0086
chr7_103136276_A_G START_LOST ALDH6A1 0.0017
chr7_103129739_C_G SPLICE_SITE_ACCEPTOR ALDH6A1 0.0034
chr9_110061864_G_A STOP_GAINED CD36 0.0017
chr7_28072405_A_C SPLICE_SITE_DONOR NOTCH4 0.0017
JH118674.1_43285_C_T SPLICE_SITE_DONOR GRIA1ortholog(ENSSSCG00000024560) 0.0049
chr16_66618439_A_T START_LOST GABRG2 0.0091
chr16_66618438_C_A,T START_LOST GABRG2 0.0017
chr1_64002416_A_G SPLICE_SITE_DONOR GABRR1 0.0034
GL896494.1_7864_G_T STOP_GAINED GPR179 0.0052
chr13_203412188_T_A SPLICE_SITE_ACCEPTOR GRIK1 0.0058
chr1_77441762_G_A SPLICE_SITE_ACCEPTOR GRIK2 0.0034
chr6_49600987_C_T SPLICE_SITE_DONOR GRIN2D 0.0034
chr6_49600986_A_G SPLICE_SITE_DONOR GRIN2D 0.0034
chr13_37136736_G_T SPLICE_SITE_ACCEPTOR GRM2 0.0017
chr13_37136747_T_G SPLICE_SITE_DONOR GRM2 0.0034
chr7_34927184_A_C SPLICE_SITE_DONOR GRM4 0.0017
chr7_34893335_T_G SPLICE_SITE_ACCEPTOR GRM4 0.0017
chr18_3214922_AC_A FRAME_SHIFT HTR5A_human_ortholog(ENSSSCG00000030573) 0.0030
chr18_3151609_AG_A FRAME_SHIFT HTR5A_human_ortholog(ENSSSCG00000023549) 0.0020
chr3_55922763_C_T SPLICE_SITE_ACCEPTOR NPAS2 0.0020
chr1_14768986_T_G SPLICE_SITE_ACCEPTOR OPRM1 0.0017
chr5_85218678_C_A STOP_GAINED PAHortholog(ENSSSCG00000000856) 0.0017
chr5_85218708_C_A STOP_GAINED PAHortholog(ENSSSCG00000000856) 0.0017
chr5_85218747_G_T SPLICE_SITE_DONOR PAHortholog(ENSSSCG00000000856) 0.0017
chr3_23446168_C_T STOP_GAINED SCNN1G 0.0017
chr16_85870363_G_C SPLICE_SITE_DONOR SLC6A3 0.0017
chr16_85870364_T_A SPLICE_SITE_DONOR SLC6A3 0.0017
chr6_58114941_G_A STOP_GAINED TAS1R3 0.0016
chr6_62357984_C_T STOP_GAINED TAS1R1 0.0065
chr6_62363203_G_C STOP_LOST TAS1R1 0.0065
Thevariantidentifier(ID)containsinformationonchromosome_position_referenceallele_alternativeallele
breeds and with pAAF information available in all these pAAFandphenotyperelationshipsintheF groups
2
populations. One-hundred variants were excluded from We also wanted to see whether we were able to detect
the purebred comparison since they were unique to the an indication of an effect of the variants on production
F animals. The resulting phylogenetic dendrogram is in traits. This was investigated by comparing the pAAFs of
2
line with what has been shown in other studies (Fig. 2). two F pools from the same experimental population,
2
Briefly, the Western (European and USA) breeds cluster each belonging to one of the tails of the phenotypic dis-
together and the Asian pool form a separate branch. tribution, for average daily gain and retroperitoneal fat
Within the Western cluster, the Duroc is the only mem- content. Out of the 217 TASR coding variants, 97 segre-
ber of a distant branch whilst the European commercial gated in the F resource, and within these, eight
2
breeds (Large White, Landrace and Pietrain) belong to displayed significantly different pAAFs in five TASRs
another sub-group and the more ancient breeds Iberian, (Table 5 and Additional file 4). Remarkably, four
Majorcan Black, and Mangalitza cluster together with TAS2R4 variants showed significant differences between
thewildboar. the pools. Likewise, we could compare 1,280 variants in
Clopetal.BMCGenomics (2016) 17:685 Page6of17
Fig.1DistributionandpMAFofstrong(H+Mdel)andmild(Mtol+L)impactvariantsalongtheproteinbodyofARgenes.Thebarplotsfor(a)
thestrongimpacteffectvariantsweremadewith310prematurestop,frame-shiftandMdel.Thebarplotforthemildimpacteffectvariants(b)
weremadewith2,174Mtol,in-frameindelsandsynonymousvariants.ThedotsindicatetheaveragepMAFineachofthe10positionpercentile
bins.Percentilebinsdividetheproteinbodyofeachgeneintenportionsofequalsize
AR genes segregating in this population and identified pigs. We could not genotype the remaining 67 pigsasall
56 significant differences (p-val≤0.05) involving 25 their DNA was used for the sequencing step. 9 % of the
genes (Table 5 and Additional file 4). After correcting assays failed to either amplify or clearly differentiate the
for multiple testing (p-val≤0.05/(97+1,280)=0.00003), genotype clusters, and 46 % did not show the alternative
one M variant in TAS2R39 and three variants in CD36 allele in any sample. 57 variants identified in our variant
remainedsignificant (Table5). calling pipeline were validated bytheTaqManassay. The
57 polymorphisms (3 TASR H, 12 AR H, 31 TASR M
Selectionandgenotype-basedvalidationofasetof and 11 AR M) affect eight TASRs and 23 AR genes
protein-damagingvariants (Additional file 6). All genotyped variants showed to be
With the double aim to validate the subset of variants in Hardy-Weinberg Equilibrium across breeds (data not
with more likely damaging impact and, to evaluate by shown). Although none of the four genotyped rare poly-
genetic association, their impact in pig breeding, we de- morphisms (all H) showed a homozygote state for the
signedaTaqMangenotypingassaytargeting128putative minor allele, one H SNV (chr16_45548628_G_A),
polymorphisms. We originally aimed at including all the unique to the Vietnamese pigs and with a MAF slightly
H variants found in the study and to fill the remaining above the rare variant threshold (MAF=0.013), was
of the assay with several M variants to cover all TASRs found in the homozygous state in two animals. This
and several AR genes. However, the assay had design re- variant was predicted to cause a stop codon gain in the
quirements that not all the variants fulfilled. Hence, we very last amino acid of the canonical HTR1A protein.
could only include nine H variants in TASR, 69 H vari- Another introduction of a stop codon
antsinAR genes,33MvariantsinTASRsand17Mvar- (chr8_38038074_G_A) was found in the middle of the
iants in AR genes. To validate the selected variants, we Gamma Aminobutyric Acid receptor GABRG1, and was
genotyped 237 (Additional file 5) of the 304 sequenced present only in Meishan pigs showing a MAF=0.022.
C
lo
p
e
t
a
l.
B
M
C
G
e
n
o
m
ic
s
(2
0
1
6
) 1
7
Table4Numberofvariantsandnumberofuniquevariantswithineachbreedandperimpactclass :6
8
5
Duroc Pietrain Largewhite Landrace Bazna Mangalitza Iberian Majorcanblack Wildboar Asian
Varianteffect nr(%) nr nr(%) nr nr(%) nr nr(%) nr nr(%) nr nr(%) nr nr(%) nr nr(%) nr nr(%) nr nr(%) nr
unique unique unique unique unique unique unique unique unique unique
Strongimpact 133 1 144 1 140 1 124 0 99 2 87 4 92 2 90 1 123 6 163 26
(12.7) (10.9) (10.5) (9.4) (10.2) (11.3) (12.3) (10.4) (12.4) (10.2)
High 44 1 46 0 49 0 33 0 28 0 29 1 30 1 30 0 35 0 48 9
(4.2) (3.5) (3.7) (2.5) (2.9) (3.8) (4.0) (3.5) (3.5) (3.0)
Moderate_Deleterious 89 0 98 1 91 1 91 0 71 2 58 3 62 1 60 1 88 6 115 17
(8.5) (7.4) (6.8) (6.9) (7.3) (7.5) (8.3) (6.9) (8.9) (7.2)
Mildimpact 909 5 1177 5 1194 5 1195 3 868 11 685 8 654 9 778 21 870 32 1426 195
(87.3) (89.1) (89.5) (90.6) (89.8) (88.7) (87.7) (89.6) (87.6) (89.7)
279 4 342 1 332 2 342 1 257 2 192 2 187 5 223 9 252 15 389 50
Moderate_Tolerated (26.8) (25.9) (24.9) (25.9) (26.6) (24.9) (25.1) (25.7) (25.4) (24.5)
Low 630 1 835 4 862 3 853 2 611 9 493 6 467 4 555 12 618 17 1037 145
(60.5) (63.2) (64.6) (64.7) (63.2) (63.8) (62.6) (63.9) (62.2) (65.3)
Total 1042 6 1321 6 1334 6 1319 3 967 13 772 12 746 11 868 22 993 38 1589 221
(0.6%)a (0.5%)a (0.4%)a (0.2%)a (1.3%)a (1.6%)a (1.5%)a (2.5%)a (3.8%)a (13.9%)a
Poolsize 45 41 39 40 15 12 13 17 22 22
Thistablewasdoneusingthe2,574variantsinTASRandARgenesincludingthemulti-allelicwithdifferenteffectsandthesewiththealternativeallelefixedinallthepopulations
aPercentageofvariantsthatarebreed-specific
P
a
g
e
7
o
f
1
7
Clopetal.BMCGenomics (2016) 17:685 Page8of17
Fig.2Phylogeneticdendrogramwiththe10breeds.Thenumbersindicatethesupportforeachnodeaccordingto1,000bootstrapiterations
We next compared the predicted and the matched chemical molecules that could be both sources of energy
genotype-observed frequency of the minor allele and or threatening toxins and promote an adequate re-
detected a strong correlation (r2=0.93) between the sponse. With the aim to characterize the coding genetic
two (Fig. 3). variation affecting swine TASRs and the AR circuitry
We also carried a genetic association analysis with the genes, we sequenced 16 gDNA pools corresponding to
genotypes and phenotypic values for retroperitoneal fat 304 pigs from ten breeds and European wild boar and
and daily gain in the F . 36 of the 57 variants segregated from two pools of an experimental F population with
2 2
in the F animals. The array contains two of the variants records on growth and retroperitoneal fat content. We
2
(chr2_3566521_A_C and chr18_7019623_G_A) that have mapped thousands of coding region genetic vari-
showed significant pAAF differences between the F ants, hundreds of which are expected to have a strong
2
groups with the Fisher test. For daily gain, only the impact on protein sequence, some of which are breed
marker chr2_3566521_A_Cwasnominallysignificant (p- specific. By comparing the pAAFs of these variants in
val=0.025) (Additional file 7) but it did not reach sig- two F pools divergent for growth and fat deposition, we
2
nificance after Bonferroni correction for multiple testing also identified many genotype - phenotype relationships.
(p-val≤0.05/36=0.0013). For retroperitoneal fat, two Our data provides detailed information of the gen-
markers, chr9_46397500_A_G and chr18_7019387_A_T, etic variation present in TASRs and AR genes. We
were nominally significant (p-val=0.028 and 0.044, also developed an assay to genotype 128 of the most
respectively) but again, none reached statistical sig- functionally relevant variants which is available to
nificance after Bonferroni correction (Additional file perform association studies with relevant traits in
7). Chr18_7019623_G_A, which was significant at pig populations.
the pAAF comparison between both F groups, did
2
not reach significance for any of the two traits (p- Technicalconsiderations
val=0.09 and 0.1 for daily gain and retroperitoneal DifferencesinDNAextractionmethodsandinaccuracies
fat, respectively). with respect to quantification might have a negative im-
pact onthe even distribution of sequencingreads among
Discussion the genomes within a pool thereby reducing the accur-
TASRs and the components of the AR circuitry are key acyofthepAAF/pMAFsasaproxyoftherealallelicfre-
genes in keeping body homeostasis as they recognize quency. Nonetheless,the comparison of predicted versus
Clopetal.BMCGenomics (2016) 17:685 Page9of17
Table5GeneswithvariantsshowingsignificantlydifferentpAAFbetweenF2_FandF2_L
Gene Totalnumber Numberandtypeofvariantswithsignificantdifferences FisherTestvaluerange
ispAAF (min-max)
ADRB1 1 1synonymous 0.0467
ALDH1B1 3 3synonymous 0.0253
ALDH2 1 1non-synoymoustolerated 0.0358
ALDH3B2 7 1non-synoymousdeleterious;6synonymous 0.0003–0.0137
ALDH6A1 1 1synonymous 0.0253
CD36 3 1frame-shift;1non-synoymousdeleterious;1synonymous 1.5xE-05a-2.58xE-05a
DISC1 3 3synonymous 0.0280–0.0357
TVPR1humanortholog(ENSSSCG00000017863) 5 1non-synoymousdeleterious;4synonymous 0.0111–0.0315
ALDH8A1ortholog(ENSSSCG00000023457) 1 1synonymous 0.0387
FOS 2 1non-synoymoustolerated;1synonymous 0.0284
GABRA3 1 1splice-sitedonor/acceptor 0.0253
GABRA6 1 1synonymous 0.0047
GPR179 9 2non-synoymousdeleterious;4non-synoymoustolerated; 0.0137–0.0324
3synonymous
GPRC5B 1 1non-synoymousdeleterious 0.0253
GPRC5C 1 1non-synoymoustolerated 0.0258
GRM1 3 3synonymous 0.0047–0.0383
GRM8 1 1synonymous 0.0178
HTR1B 1 1synonymous 0.0253
HTR3C 1 1synonymous 0.0357
LEPR 1 1synonymous 0.0253
MCHR2 2 2synonymous 0.0115–0.0253
MTNR1B 1 1non-synoymoustolerated 0.0357
P2RX2 2 2synonymous 0.0094–0.0324
P2X7 3 3synonymous 0.0006–0.0324
SIM1 1 1synonymous 0.0115
TAS2R16ortholog(ENSSSCG00000016433) 1 1synonymous 0.0315
TAS2R39 1 1codonchange 6.16xE-08a
TAS2R4 4 2non-synoymoustolerated;2synonymous 0.0002–0.0158
TAS2R41 1 1non-synoymoustolerated 0.0207
TAS2R60 1 1synonymous 0.0351
Total 64
aVariantswithFishertestvaluesremainingsignificantafterBonferronicorrectionforthemultipletestingfor1,377variants(p-val≤0.05/(1,377))
observed allele frequencies in the 57 genotyped variants depth of sequencing of 72× for each of the haploid ge-
showed that pAAF/pMAFs were very good predictors nomes. This read depth allowed us to detect all variants
and supply additional confidence of the quality of our present in the pools regardless of their frequency. Given
results(Fig.3). that we sequenced 304 animals harbouring in total 608
To the best of our knowledge, this is the first pub- chromosome sets (2 alleles each), we were able to detect
lished study from a targeted genome enrichment experi- rare variants with MAF≥0.0016. No previous high
ment in swine. As this approach reduces the sequencing throughput sequencing study in pigs reached this sensi-
throughput requirements, we were able to sequence the tivity to detect rare variants. We identified a large num-
target sub-genome (the exome of 201 TASRs and AR ber of variant events, most mapping to exon flanking
genes) in the largest number of pigs (n=304) reported regions including promoters, introns, and upstream and
to date, in a single experiment. By mapping nearly 163 downstream segments. Although some of these non-
million reads on the target exons, we reached an average coding variants could be functional, their regulatory
Clopetal.BMCGenomics (2016) 17:685 Page10of17
Fig.3LinearregressionandcorrelationbetweenpMAFandthegenotypeobservedMAFinthe57genotypedpolymorphisms
relevance is difficult to predict and was not the subject and Tamworth). 395 of the 2,766 “segregating” variants
of our study, which focused mainly, on the detection of arepredicted tohaveahigh impact(Hand Mdel) onthe
protein-damaging coding variation. proteinsequence of133 genes andare thus very likelyto
disrupt or strongly alter their function (Table 1 and
Variantidentification Additional file 2). Some of these variants showed allelic
Overall, we have identified 2,793 coding variants but 27 frequency relationship with daily gain and retro-
oftheseare fixed inoursampleset and arethus likelyto peritoneal fat deposition in the F resource with pheno-
2
be either errors in or private to the reference sequence. typic records (Table 5 and Additional file 4). In keeping
We annotated 217 variants segregating in the 10 canon- with previous findings in human [13], we also detected
ical swineTASRs in our samples. Da Silva et al. [18] de- that H and M variants tend to be more abundant and
scribed 279 coding variants in their list of 21 taste and have higher pAAFs in TASRs than in non-sensory genes
nutrient receptor genes after sequencing 79 pigs. How- (Tables 1 and 2) which indicates that TASRs are subject
ever, Da Silva and co-authors included 8 genes that we to balancing selection. TASRs are comparable to the
did not study. These are 5 likely-paralogs of canonical major histocompatibility complex genes (HLA in
TASRs and 3 genes that are not canonical TASRs but humans and SLA in pigs) as both are expressed at the
that are related to tasting fat (GPR41, and GPR84) and surface of cells to detect particular molecules that could
amino acids (GPRC6A). On the contrary, we included 2 be hazardous for the body. Whilst HLA and SLA detect
canonical TASRs (TAS2R4 and TAS2R40) not studied by antigens to promote an immune reaction, TASRs sense
Da Silva et al. Moreover, 4 we classified as AR genes chemical compounds to stimulate appropriate responses
(GPR40, GPR120, CASR, GRM1 and GRM4) that are (reward, acrosomal reaction, smooth muscle contraction
shared in both studies as they are not canonical TASRs or immune function among others) depending on the
or are receptors belonging to the glutamate pathway. cell type that is involved. Thus, a healthy animal popula-
138 of the 279 variants, 71 in canonical TASR and 67 in tion needs to be highly polymorphic in these genes in
the other shared genes, are common to both studies. order to maximize its adaptability and survival to vari-
The difference in the catalogue of identified variants is able environments facing multiple threads. As expected,
likely to be mostly due to the fact that Da Silva et al. H and Mdel variants had on average, lower pMAFs than
studied a different set of animals, some belonging to Mtol or L variants (Table 2), as the former are more
breeds we did not target (Hampshire, creole, Brazilian likely to be deleterious and consequently, subject to