Screening and identification of mutation sites of ST3GAL4 gene in chicken
The SNP site rs475471G>A were identified at ST3GAL4 gene based on previous GWAS results. (Unpublished data). To verify this SNP, and 9 mutation sites were found. The DNA samples of 468 Hubbard broilers were sequenced by PCR-SBT (Table S1) and sequencing map is shown in Fig 1, and the position of the mutation site in the gene structure is shown in Fig 2.
Linkage disequilibrium analysis of chicken ST3GAL4 gene mutation sites
Haploview software was used for linkage disequilibrium analysis. The D' value of 9 SNPs (rs475435C>T, rs475460C>T, rs475471G>A, rs475533C>T, rs475621A>G, rs475647C>A, rs475695C>T, rs475743A>G and rs475744A>G) in ST3GAL4 gene were calculated. The results showed that the D' value of pairing of the above SNPs were greater than 0.97 except for the constraint condition that the D' values of rs475435C > T and rs475743A > G were less than 0.8; The calculation of r2 value showed that except for rs475435C > T and rs475743A > G, the r2 value of pairing between rs475460C>T, rs475471G>A, rs475533C>T, rs475621A>G, rs475647C>A, rs475695C>T and rs475744A>G was greater than 0.9. When the restriction conditions of r2 value greater than 0.33 and D' value greater than 0.8 are satisfied between two polymorphic loci, they are considered to be in a strong linkage disequilibrium state. Through the analysis of the results, we found that the D' value and r2 value of rs475471G>A, rs475533C>T, rs475621A>G and rs475647C>A were all 1, which were completely linked. The results are shown in Fig 3 and Table 1.
Table 1 Analysis of pairwise linkage disequilibrium base on 9 SNPs of ST3GAL4 gene
Note:The lower triangle is r2, the upper triangle is D'.
Association analysis of genetic variation loci of ST3GAL4 gene with production traits and blood biochemical indexes of Hubbard broilers
The blood samples of 468 Hubbard broilers were sequenced by PCR. After statistical typing, 9 SNPs were associated with production traits of Hubbard broilers, all the 9 SNPs of ST3GAL4 gene were significantly related to ALP (P <0.01). The results were shown in Table 2. In addition, rs475471G>A, rs475533C>T, rs475621A>G and rs475647C>A were significantly associated with body weight and tibial length (P <0.05).
Table 2 Association analysis of 9 SNPs of ST3GAL4 with ALP of Hubbard broilers
SNP loci
|
ALP levels in all genotypes
|
P-value
|
475435C>T
|
CC=292
|
CT=176
|
|
|
47.89±2.26a
|
29.74±2.95c
|
|
2.65E-07
|
475460C>T
|
CC=133
|
CT=255
|
TT=80
|
|
23.81±3.18c
|
45.06±2.39 a
|
56.85±4.01 a
|
1.66E-08
|
475471G>A
|
GG=143
|
GA=250
|
AA=75
|
|
24.50±3.06 c
|
45.72±2.40 a
|
58.18±4.20 a
|
1.32E-08
|
475533C>T
|
CC=143
|
CT=250
|
TT=75
|
|
24.50±3.06 c
|
45.72±2.40 a
|
58.18±4.20 a
|
1.32E-08
|
475621A>G
|
AA=143
|
GA=250
|
GG=75
|
|
24.50±3.06 c
|
45.72±2.40 a
|
58.18±4.20 a
|
1.32E-08
|
475647C>A
|
CC=143
|
AC=250
|
AA=75
|
|
24.50±3.065
|
45.72±2.40 a
|
58.18±4.20 a
|
1.32E-08
|
475695C>T
|
CC=141
|
CT=251
|
TT=76
|
|
24.82±3.07 c
|
45.62±2.43 a
|
57.43±4.11 a
|
1.36E-08
|
475743A>G
|
AA=138
|
GA=314
|
GG=16
|
|
52.17±3.27 a
|
36.677±2.23 b
|
26.881±11.95 c
|
1.75E-06
|
475744A>G
|
AA=131
|
GA=256
|
GG=81
|
|
23.81±3.18 c
|
44.84±2.39 a
|
57.29±3.94 a
|
1.29E-08
|
The same letters within a row indicate no significant difference (P > 0.05), the difference between adjacent letters is significant (P < 0.05), and the difference between alternate letters is extremely significant (P < 0.01).
Association analysis of ST3GAL4 genotype and economic traits of F2 resource population
In order identify the association between the mutations of ST3GAL4 gene and production traits in chicken, especially with ALP. The rs475471G>A of four complete linkage sites was selected as tag SNP, and Hinfl restriction enzyme was used for natural enzyme digestion, which enzyme products were electrophoretic with 2% agarose gel. The typing map was shown in Fig 4, and different genotypes were identified according to agarose gel electrophoresis. The amplification product of GG genotype was a single band with the size of 589 bp, the amplification product of GA genotype was three bands with the size of 589bp, 451bp and 138bp, respectively, the amplification product of AA genotype was two bands with the size of 451 bp and 138 bp. Because of the 138 bp band was too weak, only the other two bands could be used for genotyping.
The results showed that there was a significant correlation between different genotypes and pH value of breast muscle and leg muscle (P <0.05). The pH value of chest muscle and leg muscle of AA genotype individuals were lower than that of GG genotype and GA genotype individuals. The different genotypes of the locus were significantly correlated with 4-week body weight, chest width, body slanting length, pelvis breadth and 12 week tibial circumference (P <0.05), the AA genotype and GA genotype had significantly higher percentage of full evisceration, half evisceration, head weight and leg weight than GG genotype (P <0.05), the most important result of this work was to find a significant differences in ALP levels among different genotypes (P <0.01). The results were shown in Table 3.
To analysis of the above results, the GG genotype had the lowest ALP and grew slowly. The ALP level of AA and AG genotypes were significantly higher than that of GG genotype, and exhibit better growth performance than those individuals with GG genotype. This is consistent with the analysis in Hubbard broilers. In summary, allele A is positively correlated with ALP content, and the genotype with high alkaline phosphatase content was the dominant genotype.
Table 3 Association analysis results of 475471G>A polymorphic loci of ST3GAL4 gene and economic traits of F2 resource population
Traits
|
GG=238
|
GA=407
|
AA=94
|
P值
|
CPH
|
6.10±0.02c
|
6.09±0.01b
|
5.97±0.03a
|
0.002
|
LPH
|
6.65±0.01c
|
6.67±0.01b
|
6.59±0.03a
|
0.044
|
BW4 (g)
|
314.35±6.93b
|
324.20±6.66b
|
326.31±7.87a
|
0.014
|
CW4 (cm)
|
4.03±0.03b
|
4.13±0.03a
|
4.06±0.05ab
|
0.025
|
BSL4 (cm)
|
11.30±0.09b
|
11.41±0.08ab
|
11.52±0.11a
|
0.043
|
PW4 (cm)
|
5.09±0.03b
|
5.18±0.03a
|
5.17±0.05a
|
0.027
|
SEW (g)
|
81.06±0.23b
|
81.53±0.21a
|
81.63±0.28a
|
0.011
|
EW (g)
|
67.71±0.33b
|
68.03±0.32ab
|
68.31±0.36a
|
0.026
|
LW (g)
|
146.21±3.65b
|
150.78±3.52a
|
152.83±4.13a
|
0.024
|
ALP(U/mL)
|
592.56±57.83c
|
776.94±54.23b
|
886.46±70.33a
|
2.65E-07
|
CPH, Chest muscle PH, LPH, leg muscle PH,BW4, Body Weight at 4 weeks,CW4, Chest width at 4 weeks,BSL4, Body slant length at 4 weeks, PW4, Pelvic width, SEW, Semi-evisceration weight, EW, Evisceration weight, LW, Leg weight, ALP, Alkaline phosphatase.
Genotypic and allelic frequencies and related genetic parameters for the chicken ST3GAL4 gene
To detect the variations among commercial broilers, egg-type chickens, dual-type chickens, we performed a PCR analysis on 4825 individuals from 18 populations were analyzed, and to investigate the genotype distribution and gene frequencies of ST3GAL4 in different populations in this study. The number of genotypes of each variety is shown in Table S2. As shown in Table 4, the genotype frequencies of CB, R308, B817, HBD broilers and F2 resource groups were significantly different from those of egg-type chickens and dual-type chickens. GG genotype mainly existed in egg-type chickens and dual-type chickens, while AA genotype mainly distributed in commercial broilers and F2 resource population.
PIC, He and Ne are not only indicators to measure the degree of allele polymorphism and gene mutation, but also indicators to reflect the genetic variation within the population. According to the standard polymorphism information, PIC < 0.25 represents low polymorphism, 0.25 < PIC <0.50 represents moderate polymorphism, while PIC >0.50 represents high polymorphism. The F2 resource population and commercial broilers all showed moderate polymorphism, which indicated that the F2 generation and commercial broilers exhibited greater genetic variation and selection potential than egg-type chickens and dual-type chickens. Allele A is positively correlated with ALP and mainly existed in commercial broilers and F2 generation, what's interesting is that the growth performance of individuals with high ALP level is better, which might be due to the result of artificial selection.
Table 4 Genotypic and allelic frequencies and related genetic parameters for the chicken ST3GAL4 gene
Breeds/n
|
Genotypic and allelic frequencies
|
He
|
Ne
|
PIC
|
|
GG
|
GA
|
AA
|
G
|
A
|
|
|
|
AA/597
|
0.035
|
0.628
|
0.337
|
0.349
|
0.651
|
0.455
|
1.833
|
0.351
|
CB/224
|
0.259
|
0.598
|
0.143
|
0.558
|
0.442
|
0.493
|
1.973
|
0.372
|
R308/179
|
0.274
|
0.570
|
0.156
|
0.559
|
0.441
|
0.493
|
1.973
|
0.372
|
B817/71
|
0.141
|
0.704
|
0.155
|
0.493
|
0.507
|
0.500
|
2.000
|
0.375
|
HBD/468
|
0.297
|
0.538
|
0.165
|
0.566
|
0.434
|
0.491
|
1.966
|
0.371
|
F2/739
|
0.322
|
0.551
|
0.127
|
0.597
|
0.403
|
0.481
|
1.927
|
0.365
|
YY/43
|
0.814
|
0.186
|
0.00
|
0.907
|
0.093
|
0.169
|
1.203
|
0.155
|
DX/186
|
0.758
|
0.226
|
0.016
|
0.871
|
0.129
|
0.225
|
1.290
|
0.200
|
GC/78
|
0.833
|
0.154
|
0.013
|
0.910
|
0.090
|
0.163
|
1.195
|
0.150
|
GS/177
|
0.655
|
0.316
|
0.028
|
0.814
|
0.186
|
0.303
|
1.435
|
0.257
|
HD/158
|
0.842
|
0.158
|
0.000
|
0.921
|
0.079
|
0.146
|
1.171
|
0.135
|
HL/307
|
0.57
|
0.417
|
0.013
|
0.779
|
0.221
|
0.345
|
1.526
|
0.285
|
LS/92
|
0.554
|
0.413
|
0.033
|
0.761
|
0.239
|
0.364
|
1.572
|
0.298
|
WH/288
|
0.729
|
0.236
|
0.035
|
0.847
|
0.153
|
0.259
|
1.349
|
0.225
|
XC/384
|
0.833
|
0.159
|
0.008
|
0.913
|
0.087
|
0.159
|
1.189
|
0.147
|
RW/221
|
0.778
|
0.213
|
0.009
|
0.885
|
0.115
|
0.204
|
1.257
|
0.183
|
CS/95
|
0.663
|
0.274
|
0.063
|
0.800
|
0.200
|
0.320
|
1.471
|
0.269
|
FB/113
|
0.584
|
0.398
|
0.018
|
0783
|
0.217
|
0.340
|
1.154
|
0.282
|
AA, Arbor Acres broiler, CB, Cobb broiler, R308, Ross308, B817, 817 broiler, HBD, Hubbard broiler, F2, F2 resource population, YY, Yunyang chicken, DX, Dongxiang chicken, GC, Henan gamecock, GS, Gushi chicken, HD, Houdan chicken, HL, Hy-line brown hen, LS, Lushi chicken, WH, Wuhei chicken, XC, Xichuan chicken, RW, Recessive white chicken, CS, Changshun chicken, FB, Field Broiler. He, expected heterozygosity; Ne, effective allele numbers; PIC, polymorphism information content.
Expression of different genotypes of ST3GAL4 gene in different tissues
By analyzing the expression of different genotypes of STCGAL4 gene in different tissues, we found that STCGAL4 is most highly expressed in the duodenum, while almost not expressed in the pectoral and leg muscle Fig.5. In addition, the expression level of GG and GA genotype were significantly higher than that of AA genotype in spleen (P<0.05), the GA and AA genotype were extremely significantly higher than that of GG genotype in duodenum (P<0.01).