Whole Genome Resequencing and Templated Assembly
Next-generation WGR data was generated for eight pools of 10 individual DNA samples representing two biological replicates for each gender for both AS resistant and susceptible birds from the unselected, Relaxed (REL) line. The REL is descended from an elite commercial line [36]. Each pool was sequenced to >66x coverage using paired-end (2 x 125-bp) Illumina sequencing. FASTQ reads for each pool were mapped onto the May 2018 GRCg6a reference genome. The average read counts for all pools was 534,902,802±6,325,923, with 93.4±0.2% reads successfully assembled onto the reference genome. Potential SNPs identified by the read mapping totaled 12,024,469 in male pools and 11,933,041 in female pools. SNP counts were recorded, and SNP densities plotted for each chromosome according to gender (Figure 1). The SNP density was observed to be higher for some of the microchromosomes in the assemblies for both genders. Whereas the average chromosomal SNP density is 1.36±0.61, SNP densities for microchromosomes 30, 31 and 33, are 2.3 to 3.6 SNPs per 100 bp. Most chromosomes have GC content of ≤ 47%. Chromosomes 30, 31 and 33 have a GC% of 58.7, 52.3, and 53.9, respectively. The current assembly of chromosome 30 is 1.82 Mbp, while chromosomes 31 and 33 are more complete at 6.15 and 7.82 Mbp, respectively. However, the assembly for chromosome 28 is 5.12 Mbp at 53% GC and the SNP density is 1.276/100 bp. This pattern of SNP density by chromosome is consistent with that observed for 3 commercial lines (manuscript in preparation) so it is not unique to the REL line. The SNP density is also not a function of read depth as read depth was roughly equivalent across all the chromosomes.
To identify potential QTLs for AS phenotype, the difference between the frequency of the SNP in resistant versus susceptible phenotypes were calculated (resistant SNP frequency - susceptible SNP frequency). The SNP frequency differences were visually inspected using the Integrative Genomics Viewer (IGViewer; [37]) according to chromosome and chromosomal location for both genders to visually identify clusters of SNPs with frequencies skewed with respect to phenotype for either or both genders. Specifically, the SNP frequencies were scanned for clusters of SNPs showing frequency differences of >20%. Twenty eight regions on 13 chromosomes were detected showing clusters of SNPs differentially represented in the different phenotypes (Table 1). The identified regions included 15 that showed association in both genders, while 8 appeared to be specific to males and 5 to females. The majority of regions showed a higher frequency of non-reference SNPs in the ascites resistant birds, with 8 associated with resistance in both genders, 5 male specific and 3 female specific. The non-reference SNPs were associated with susceptibility (higher frequency in susceptible) for 1 region in both genders, 3 male specific and 2 female specific. There were two regions where the non-reference SNPs were associated with resistance in males and with susceptibility in females, while there were four regions where the reverse was found. The 64 genes identified in these regions (Table 1), were used to search the NCBI Phenotype-Genotype Integrator, (PheGenI; https://www.ncbi.nlm.nih.gov/gap/phegeni) for phenotypes that had been identified in human GWAS as associated with traits possibly contributing to PHS or AS. The most frequent traits from the PheGenI output are presented in Figure 2. Traits that are of particular interest regarding AS in broilers include: platelet function tests, blood pressure, body mass index, echocardiography, mycocardial infarction, erythrocyte indices, and heart failure. Thus, the human GWAS data supports these 28 regions as potential candidate QTLs for AS.
Table 1. Potential QTL regions for ascites syndrome based on WGR in the REL line.
|
Mbp
|
Res-Sus SNP frequency
|
|
Chr
|
Start
|
Stop
|
Size
|
Male
|
Female
|
Genes within Region
|
1
|
48.18
|
48.41
|
0.23
|
30%
|
30%
|
APLD,GPRC5A,HEBP1,FAM234B,GSG1, EMP1,MIR6581
|
1
|
170.483
|
170.53
|
0.05
|
40%
|
0%
|
CAB39L
|
1
|
175.68
|
175.87
|
0.19
|
-25%
|
30%
|
PDS5B
|
1
|
182.31
|
182.46
|
0.15
|
40%
|
20%
|
AASDHPPT,KBTBD3,MSANTD4
|
1
|
183.65
|
183.95
|
0.30
|
35%
|
35%
|
DCUN1D5,MMP13,MMP10,MMP3,MMP7,BIRC2
|
2
|
22.86
|
23.03
|
0.17
|
50%
|
0%
|
SAMD9L,HEPACAM2,VP50
|
2
|
34.47
|
34.61
|
0.14
|
40%
|
-20%
|
PLCL2
|
2
|
91.85
|
91.92
|
0.07
|
50%
|
0%
|
CNDP2,FAM69C
|
2
|
95.14
|
95.22
|
0.08
|
-30%
|
0%
|
CDH19
|
2
|
122.75
|
122.83
|
0.08
|
45%
|
-25%
|
CA2
|
2
|
126.97
|
127.09
|
0.12
|
40%
|
20%
|
CPQ
|
3
|
37.27
|
37.36
|
0.09
|
-30%
|
40%
|
RYR2
|
3
|
48.98
|
49.00
|
0.02
|
-20%
|
30%
|
RMND1
|
3
|
50.41
|
50.44
|
0.03
|
40%
|
0%
|
SCAF8
|
3
|
100.70
|
101.10
|
0.40
|
0%
|
-30%
|
OSR1
|
4
|
36.72
|
36.90
|
0.18
|
40%
|
0%
|
GRID2
|
5
|
13.26
|
13.29
|
0.03
|
0%
|
50%
|
DDTNFR23, CARS
|
5
|
25.44
|
25.47
|
0.04
|
-50%
|
25%
|
SPTBN5
|
6
|
28.82
|
28.87
|
0.05
|
0%
|
40%
|
ABLIM1
|
10
|
6.49
|
6.54
|
0.05
|
40%
|
30%
|
TJP1
|
14
|
1.48
|
1.66
|
0.18
|
0%
|
30%
|
LMTK2,BHLHA15,TECPR1,BRI3,BAIAP2L1, NPTX2
|
20
|
9.06
|
9.10
|
0.04
|
0%
|
-40%
|
PXDNL,PCMTD2
|
22
|
4.40
|
4.48
|
0.08
|
60%
|
20%
|
LRRTM4
|
27
|
7.85
|
7.98
|
0.13
|
-30%
|
0%
|
RAMP2,WNK4,COA3,BECN1,PSME3,AOC3, G6PC,PTGES3L,RPL27,IFI35,VAT1,RND2
|
28
|
0.59
|
0.63
|
0.05
|
-25%
|
0%
|
TIMM44,HNRNPM
|
Z
|
18.60
|
18.73
|
0.13
|
-25%
|
-50%
|
PDE4D
|
Z
|
19.10
|
19.50
|
0.40
|
25%
|
50%
|
ZSWIM6,KIF2A
|
Z
|
33.87
|
33.90
|
0.03
|
25%
|
25%
|
SLC24A2
|
Regions are listed by Chromosome (Chr) and Megabase pair (Mbp) Start, Stop and Size, in the chicken genome GRCg6a assembly. Res-Sus SNP frequency is the difference in the approximate maximum frequency difference for the non-reference SNP between the phenotypes of the indicated genders. Positive Res-Sus SNP frequency difference indicates the non-reference SNPs are associated with resistance, negative values indicate association with susceptibility.**Gga2 region previously verified as a QTL [35].
LRRTM4 Genotyping in Association with Ascites Syndrome
The region on chromosome 2 for CPQ has already been extensively analyzed for association with ascites phenotype and confirmed for association of the non-reference SNPs with ascites resistance in males [35]. The region on chromosome 22 associated with the LRRTM4 gene was selected for further investigation because an association was found in both genders, with a larger frequency difference in males than females. This region contains 711 successive, high-quality SNPs spanning the 4.400-4.455 Mbp region on Gga22 (Figure 3). The frequency difference (resistant SNP frequency - susceptible SNP frequency) averages 27% in males and 7% in females. In males 641 SNPs have a positive SNP frequency difference while only 11 are negative. In females there were 261 positive, and 12 negative, for SNP frequency difference. Of these SNPs, 560 covered 45.1 kbp of the 3’end of the LRRTM4 gene which spans 211.6 kbp from 4,409,156 to 4,620,835 Mbp. The LRRTM4 gene encodes the leucine-rich repeat transmembrane neuronal protein 4 which has been suggested to play a role in the regulation of dendritic spine development in the nervous system [38, 39]. NCBI PheGenI associates the human LRRTM4 gene region with traits such as antihypertension, carotid artery disease, coronary heart disease, and pulmonary embolism, supporting a probable association with AS in broilers.
An exonuclease assay was developed to target two SNPs (4,405,679, and 4,405,681 Mbp) which gave SNP frequency differences of approximately 50% in males and 15% in females. These SNPs are in the intergenic region, 3.5 kbp from the 3’ end of the LRRTM4 gene. The exonuclease assay was used to genotype more than 600 archived DNA samples from REL birds previously phenotyped for AS susceptibility in the hypobaric chamber [30, 34, 35]. The observed genotype frequencies were in agreement with calculated genotype frequencies (computed from allele frequencies) consistent with Hardy Weinberg Equilibrium (HWE) which implies that the quantitative Polymerase Chain Reaction (qPCR) genotyping is valid, there are no significant issues with null alleles, and that the samples of DNA utilized was non-biased. These same DNA samples had been used in the genotype associations with AS for the CPQ gene on chromosome 2 [35]. Based on the WGR SNP frequency plots the expectation was an association of resistance for males homozygous with the non-reference SNPs. Instead there was a significant association of the homozygous non-reference SNPs in females (AG genotype in females in Table 2; adjusted P-value=0.047). There was also a significant adjusted P-value (P=0.0083) for all samples, combining male and female data (Table 2). In both cases the homozygous non-reference SNPs were associated with increased susceptibility (AG genotype higher frequency of susceptible than RR or GA genotypes). The homozygous non-reference increases susceptibility by 19% in the females and 12 % in the entire population.
Table 2. Genotype data shows an association of LRRTM4 to ascites syndrome in female REL line birds.
|
|
All
|
|
Male
|
|
Female
|
Genotype
|
n
|
Sus
|
Res
|
P-val.
|
n
|
Sus
|
Res
|
P-val.
|
n
|
Sus
|
Res
|
P-val.
|
AG
|
246
|
45.6
|
54.4
|
0.0083
|
101
|
41.6
|
58.4
|
0.24
|
113
|
47.8
|
52.2
|
0.047
|
RR
|
426
|
31.0
|
69.0
|
0.13
|
192
|
27.7
|
72.3
|
0.21
|
157
|
32.7
|
67.3
|
0.83
|
GA
|
197
|
34.5
|
65.5
|
1.76
|
86
|
36.5
|
63.5
|
1.65
|
81
|
29.6
|
70.4
|
0.52
|
Data is based on SNP genotypes using a qPCR assay. Genotype is the composite of the two SNPs on chromosome 28 assayed with P1 and P2 (Table 4) (bases: 4,405,679 and 4,405,681). For each genotype the percentage of each phenotype (Sus=susceptible; Res=resistant) is presented along with the adjusted P-value (P-val.) for All samples, and each gender. Discrepancies in totals result from missing gender data for some samples. Phenotypes are Sus (susceptible) or Res (resistant).
Male broilers with homozygous non-reference genotype for intron 6 SNPs in CPQ gene have shown approximately 20% higher overall resistance [35]. To test for potential interactions between CPQ and LRRTM4, the genotype data for LRRTM4 with the genotype data for the same samples for the CPQ gene [35] were combined. The combined genotype of heterozygous for LRRTM4 and homozygous non-reference for CPQ (RRCC) genotype was significantly associated with ascites resistance in males (P=0.033), contrary to the LRRTM4 genotype alone. The RRCC genotype was the most abundant genotype in the samples (n=131). Whereas, for most combined genotypes the males were nearly 50:50, 85% of the 61 RRCC males were resistant to AS (Table 3). For CPQ genotypes alone, 72% of male homozygous non-reference males were resistant, so the combination of the two loci may increase resistance over CPQ alone. Additionally, for homozygous reference LRRTM4 (GA), the addition of a heterozygous CPQ also seems to favor resistance, but the P-values are not significant. In summary, the CPQ non-reference homozygous genotype confers approximately 2:1 odds on ascites resistance in males, but the addition of one non-reference allele for LRRTM4 appears to improve the odds to approximately 6:1.
[Insert Table3 here]
Tissue-specific Expression Evaluation of LRRTM4
LRRTM4 gene expression for each of the homozygous genotypes was measured by reverse transcriptase qPCR (RT-qPCR) for heart, lung, liver, brain, and testis samples. TATA-binding protein (TBP) served as the reference, as TBP is recommended across multiple human tissues [40], recommended for chicken cardio-pulmonary gene expression assays [41], and we have found it to show little variation across multiple chicken tissues [42]. No expression was detected in lung, liver, brain, or testis RNA samples, but expression was observed in the heart. When comparing the level of expression of LRRTM4 between homozygous reference (ΔΔCt ± sd = 6.8 ± 0.8) and homozygous non-reference (ΔΔCt ± sd = 5.7 ± 1.0) genotypes there was no difference in expression between genotypes (P=0.188). The expression data indicates that LRRTM4 in broiler heart is expressed at about 0.016 (0.56) the level as TBP. The RNAseq summary in NCBI for human LRRTM4 (Gene- Full Report) suggests that LRRTM4 expression is primarily restricted to the brain. Human RNAseq RPKM values for LRRTM4 are brain 5.6±1.1, heart 0.05±0.03, liver 0.033±0.012, lung 0.24±0.14, and testis 0.024±0.011. Based on the RPKM values for human TBP and LRRTM4 for these tissues, and an average ΔCt of 15.2±1.5 for TBP in the RT-qPCR analyses from REL broilers, amplification for all these tissues for LRRTM4 was expected within the 25 qPCR cycles employed. The relative expression in human heart of LRRTM4 to TBP is 0.0154 which is in agreement with the ΔΔCt values in heart for chickens. Thus, LRRTM4 expression in lung, liver, brain, and testis, is apparently much lower than the expression observed in human. Further, the two alternative alleles of LRRTM4, distinguished based on SNP differences distal to the 3’ end of the gene, do not appear to significantly differ in expression levels. Therefore, the contribution of LRRTM4 to AS more likely result from differences in the polypeptide sequences, or post-translational processes.