A 31-bp Indel in the 5' UTR region of the GNB1L gene is signicantly associated with chicken body weight and carcass traits

Background: G-protein subunit beta 1 like (GNB1L) can encode a G-protein beta-subunit-like polypeptide, the chicken GNB1L gene is up-regulated in the breast muscle of high-feed eciency chickens, and its expression is 1.52-fold that of low-feed eciency chickens. However, there are no reports describing the effects of GNB1L gene Indel on the chicken carcass and growth traits. Results: This study identied a 31-bp Indel in 5' UTR of the GNB1L gene and elucidated the effect of this gene mutation on the carcass and growth traits in chickens. The results showed that the 31-bp Indel was highly signicant associations with body weight at 8 different stages, and also signicantly correlated with daily gain of 0 to 4 weeks and 4 to 8 weeks. Similarly, the mutation was signicantly associated with small intestine length, breast width, breast deep and breast muscle weight in carcass traits. Moreover, DD and ID are inferior genotypes for the growth and carcass traits of chickens. Conclusions: In a word, these results show that the 31-bp Indel of GNB1L gene is signicantly affected chicken body weight and carcass traits, and can serve as a candidate molecular marker for chicken genetics and breeding programs.

growth traits of cattle [13]. A19-bp Indel mutation in the PLAGI gene intron region affects the growth traits of the Chinese cattle [14]. The 16-bp Indel in 5′ untranslated regions (UTR) of the ZNF132 gene was signi cantly affected the body length of the Hainan black goat [3]. Recent studies revealed that 11-bp Indel in the DNMT3B intron region was signi cant correlation with the litter size at rst parity of goat [15]; two Indels (P2-16bp and P14-15bp) of DSCAML1 were markedly related to sperm quality in male goat, and three Indels of DSCAML1 were signi cant correlation with the litter size at rst parity in female goat [16]. A study has shown that 13-bp Indel mutation in the DGAT2 gene 3' UTR affects its expression and fat deposition in porcine [17]. In poultry research, two novel Indels in the QPCTL gene signi cantly affected chicken carcass traits and body weight at 5 different weeks of age [18]; the Indel of the CDKN3 gene was signi cantly associated with chicken carcass and growth traits [19]; a 22-bp Indel of the ZNF764L gene was markedly related to chicken birth weight, body slanting length, chest breadth and subcutaneous fat weight [20]. A 65-bp Indel in the chicken GOLGB1 gene intron signi cantly affected body weight and carcass traits at 13 weeks [21]. The 80-bp Indel in the PRLR was signi cantly associated with chicken leg weight, body weight and shank length [22].
In the present study, we veri ed a 31-bp Indel in the GNB1L from 10x whole-genome resequencing data of ten XH and ten RW chickens (data unpublished) (EVA accession number: PRJEB36864). The chicken GNB1L gene is located on chromosome 15, comprising 15 exons. Furthermore, a total of 80 Indels were found in the GNB1L gene in the Ensembl database (http://asia.ensembl.org/Gallus_gallus/Gene/Variation_Gene/Table?db=core;g=ENSGALG0000 0001925; r=15:1232691-1273276;t=ENSGALT00000002979). However, there is no report and verify about the Indel of chicken GNB1L gene. The main purpose of this research is to verify the Indel mutation of the GNB1L gene, to clarify the effect of the GNB1L Indel on chicken economic traits, and to analyze the GNB1L expression in different tissues, leg muscles and chest muscle tissues at different embryonic development stages. In addition, we examined the distribution of 31-bp Indel in different populations. These results indicate that the 31-bp Indel mutation in the GNB1L gene can serve as a candidate molecular marker for chicken growth traits, and provide a reference for molecular breeding of chickens.

Polymorphism detection and genotyping
A novel 31-bp Indel polymorphism in the 5' UTR region of the GNB1L was observed by DNA sequencing ( Figure S1) (TSINGKE, Guangzhou, China). All PCR ampli cation products were detected using 3.0% agarose gel electrophoresis, we found three genotypes including the 301bp homozygous DD genotype, the heterozygous ID genotype (332 bp and 301bp) and 332bp homozygous II genotype ( Figure S2).
Genetic diversity of the 31-bp Indel in different populations The genetic parameters, allele frequencies and genotype frequencies of in seven different breeds and F2 population were analyzed ( Table 1). The results suggest that the D allele frequency was lower than that of I in all breeds, except for LS chicken. Meanwhile, we counted different genotype distribution among the dual-purpose chickens (ND, GX, WC, QY and LS), F2 population, commercial broilers (RW) and commercial layers (ISA). The percentage of the DD genotype was the lowest in all breeds ( Figure S3). The results of a c 2 test suggested that the genotype frequencies of F2, ND and RW were not in HWE (P < 0.05), and WC, QY, ISA and LS were in HWE (P > 0.05). The values of He is from 0.46 to 0.50, and the values of Ne is from 1.85-1.99. The smallest and largest values of PIC are 0.35 and 0.37, respectively. The results revealed that the 31-bp Indel of GNB1L represents intermediate polymorphism, and lack of high genetic diversity in all populations (Table 1).
Genetic differentiation of the 31-bp Indel Results of differential selection suggested that between the LS and QY, LS and GX with medium genetic differentiation (0.05 < Fst < 0.15). Moreover, we observed between the other breeds with little genetic differentiation (Fst < 0.05) (Table S1).
Correlation between the GNB1L 31-bp Indel and economic traits Mixed Model were used to analysis the correlation between genotypes and economic traits. As Table 2 shows, the three genotypes showed signi cant correlation with 11 chicken growth traits, and greatly signi cantly associated with 9 growth traits. Especially, different genotypes were very signi cantly related to body weight at 7, 14, 21, 28, 35, 42, 49 and 56 weeks, Daily gain of 0 to 4 weeks (P < 0.01), and were signi cantly related to Daily gain of 4 to 8 weeks and shank length of 49 weeks (P < 0.05) (Figure 1a, 1b) ( Table 2). Importantly, the DD and ID genotypes were greater than the II genotype in all related growth traits.
Notably, the 31-bp Indel displayed highly signi cant correlation with breast width, breast deep, breast muscle weight and small intestine length in carcass traits, and were signi cant correlation with fat cingula width (Table 3). Interestingly, the DD and ID genotypes were greater than the II genotype in all related carcass traits. In the association analysis of 31-bp Indel and meat quality traits, the different genotypes showed signi cant correlation with dry matter content of leg muscle, and critical correlation with crude fat content of leg muscle in Table S2.

GNB1L expression in chickens
The GNB1L gene expression in 12 tissues of 20 weeks QY spotted-brown chickens was detected by qPCR. Based on qPCR, GNB1L was relatively highly abundant in heart, breast muscle, leg muscle, kidney and ovary, and in small intestine, spleen, liver, lung, and abdominal fat had relatively low expression levels ( Figure 2). Furthermore, the GNB1L gene expression level increases rst and then decrease in breast muscle at different embryonic stages, and expression level decreases rst and then increases in leg muscle at different embryonic stages (Figure 3a, 3b).

Transcription Factor Prediction in the GNB1L 31-bp Indel
The transcriptional binding sites in the 31-bp Indel of GNB1L gene were analyzed by online prediction website, and the results revealed ve potential transcription factors (NF-1, SP1, T3R, RAR-α and GR) ( Figure S4).

Discussion
The allelic frequency of genes can re ect the genetic diversity between different groups, which means that new mutations are introduced to some extent [2,23]. In recent decades, the breeding of commercial broilers and layers focuses on growth and reproductive traits, respectively. In these commercial breeds, dominant genotypes for speci c traits may be selected for breeding. Moreover, manual selection also determines the distribution and amount of genetic variation during domestication [23]. In this study, I allele was the predominant allele in the ND, GX, WC, QY, F2 population, RW and ISA, except for LS chicken. The results show that the LS chickens may undergo different selection pressure during evolutionary processes than other chickens. Interestingly, LS chicken is the only breed that can produce blue eggs in these breeds [24].
Body weight of chickens is a heritable trait with about 0.24%-0.47% heritability during growth [25].
Compared with commercial broilers, Chinese domestic broilers have a relatively low growth rate and body weight. Therefore, we studied the correlation between the 31-bp Indel in the GNB1L 5′ UTR region and F2 population carcass and growth traits. As Table 2 shows, the 31-bp Indel highly signi cant correlation with body weight at 8 different stages. Moreover, the three different genotypes were also signi cantly correlated with daily gain of 0 to 4 weeks and 4 to 8 weeks, and shank length of 49 weeks ( Table 2).
Signi cantly, the DD and ID genotypes were greater than the II genotype in all related growth traits, the DD genotype has the greatest weight at 7, 14, 21, 28, 35, 42 and 49 weeks, except for 56 weeks. Interestingly, the DD is the dominant genotype in daily gain of 0 to 4 weeks, and the ID is the dominant genotype in daily gain of 4 to 8 weeks. We hypothesize that DD and ID genotypes may have a higher feed conversion ratio during chicken development. In summary, the II genotype is a disadvantaged genotype in all growth traits.
Chinese domestic chickens have a good carcass yield, with breast muscles accounting for about 30% of the carcass weight, and the weight of muscles accounts for about 40% of the weight of the carcass [26].
Therefore, individuals with larger breast width, breast depth and breast weight are also the breeding direction of local yellow-feathered broilers. As Table 3 shows, the mutation was signi cantly related to breast width, breast muscle weight, breast deep and small intestine length of carcass traits. Similarly, the DD and ID genotypes were greater than the II genotype in all related carcass traits. Growing evidence suggests that the small intestine mainly responsible for the e cient absorption and metabolic processing of nutrients, and the small intestinal villi are the main parts for absorbing nutrients [27]. Perhaps the longer length of the small intestine is helpful to improve the e ciency of animal absorption of food. We speculate that GNB1L 31-bp Indel may affect the conversion e ciency of feed by affecting the length of the small intestine, which ultimately leads to differences in carcass and growth traits of individuals with different genotypes. Previous research results also indicate that GNB1L is related to higher feed e ciency [9].
Studies have demonstrated that mutations in 5′ UTR of some genes can affect gene expression [28,29]. Furthermore, TFs are also essential factors that regulation gene expression, prediction results of TFs showed that there are ve potential TFs such as NF-1, SP1, T3R, RAR-α and GR in 31-bp of GNB1L. We guess these TFs may be involved in the transcription of GNB1L gene, which in turn lead to differences of phenotype in three genotypes. In this research, the expression of the GNB1L was relatively highly abundant in heart, leg muscle, breast muscle, kidney and ovary, and other tissues had relatively low expression levels. Moreover, the expression of GNB1L increases rst and then decreases in breast muscle at different embryonic stages, and decreases rst and then increases in leg muscle. These results showed that the GNB1L may be related to the embryonic muscle development.

Conclusion
In conclusion, we rst time found that the GNB1L, a candidate gene for high-feed e ciency, has a 31-bp Indel in its 5′ UTR that is signi cantly related to chicken carcass and growth traits. Moreover, DD and ID are inferior genotypes for the carcass and growth traits of chickens. In addition, our research once again proved that GNB1L gene may be a candidate gene for higher feed conversion rate. In summary, this study showed that the GNB1L may be involved in the chicken embryonic development and growth, and the 31bp Indel of GNB1L gene can serve as a candidate molecular marker for genetics and breeding programs of chicken.

Methods
Animal samples and trait measurement DNA samples of 766 chickens from eight populations, Lushi chickens (LS, n = 39, 6 weeks), Ningdu chickens (ND, n = 95, 12 weeks), Wenchang chickens (WC, n = 65, 7 weeks), Qingyuan Partridge chickens (QY, n = 70, 7 weeks), Recessive White Rock chickens (RW, n = 55, 7 weeks), ISA Brown laying hen (ISA, n = 54, 20 weeks), Guangxi chickens (GX, n = 71, 12 weeks) and F2 population (F2, n = 360, 13 weeks) were used. These DNA samples are all from the chicken breed resource library kept in our laboratory. In eight different breeds, LS, ND, WC, QY and GX are domestic chicken breeds in China, RW and ISA are commercial broilers and layer hens, respectively. And the F2 resource population is a hybrid strain of RW and Xinghua (XH) chickens, XH chickens represent a slow-growing Chinese domestic chicken. In our laboratory, 2 mL of 5% Pentobarbital was injected intraperitoneally into the chicken (No. 57-33-0 of Chinese Academy of Sciences, Beijing Siyuan Technology Co., Ltd.). After 2-3 minutes, the chicken was sacri ced by bleeding through the carotid artery. All F2 population had data records about economic traits, and detailed information on measuring methods is as previously described [30].
12 different tissues were obtained from four QY chickens. Moreover, breast muscle of six embryonic periods (E10-15) and leg muscle of four embryonic periods (E12-15) was used to detect the relative GNB1L expression.
cDNA synthesis and qRT-PCR RNA extraction using the TRIzol (Takara, Dalian, China) method, reverse transcription by the cDNA reverse transcription kit (Takara, Dalian, China) followed by PCR. Relative gene expression was calculated by the 2 -ΔΔCt method, and signi cance using ANOVA followed by Duncan's test. All reactions using three biological and technical repetitions. The relative expression of GNB1L in different tissues and embryo ages were analyzed by qRT-PCR. Primers of GNB1L qRT-PCR and internal control β-actin are listed in Table S3.

Indel detection and diversity analysis of different breeds
A 31-bp Indel in the GNB1L gene from whole-genome re-sequencing data of ten XH and ten RW chickens (unpublished data). Genotyping of GNB1L 31-bp Indel by PCR ampli cation and gel electrophoresis in eight diverse populations. Blood samples were used for the extraction of DNA, and the nal concentration of DNA used for ampli cation was diluted to 50 ng/µL. GNB1L PCR primers based on the genome is listed in Table S3. Each 15-µL PCR ampli cation volume contained 1µL DNA, 1.5 µL primer, 7.5 µL 2×Taq Master mix (TSINGKE, Beijing, China), and 5µL double-distilled water. PCR procedure included at 95°C for 3 min, 35 cycles at 95°C for 30 s, 60°C for 30 s, 72°C for 30 s, and a nal extension at 72°C for 10 min. The PCR products after ampli cation were separated by 3.0% gel electrophoresis.
The genotypes and allele frequencies of the mutation were calculated directly in different breeds. Hardy-Weinberg equilibrium (HWE) was analyzed using the SHEsis online website (http://analysis. biox.cn). Moreover, allele numbers (Ne), genetic indices of heterozygosity (He), effective polymorphism information content (PIC) and population differentiation were analyzed by PopGene software (Version 1.3.1) [31, 32].

Transcription Factor Prediction
The transcription factors (TFs) in the 31-bp Indel mutation of 5′ UTR regions of the GNB1L gene were predicted by online AliBaba software (Version 2.1) [24].

Statistics
Association analysis of F2 population by SPSS 22.0 software, and was used two different models in the analysis. All growth traits use Model (Yijkl=µ+ Gi + Sj + Hk + + eijkl), and all carcass traits use Model II (Yijkl=μ + Gi + Sj + Hk + + b (Wijkl -W(-)) + eijkl), and carcass weight serve as a concomitant variable of Model II. Yijkl represents the observed value, µ is the overall population mean, is the xed effect of family, Gi is the xed effect of genotype, Hk is the xed effect of hatch, Sj is the xed effect of sex, b is the regression coe cient for carcass weight, W(-) is average slaughter weight, Wijkl represents the individual slaughter weight, and eijkl represents the random error in two Models. Signi cance was set at P-value < 0.05, and the Bonferroni's test serve as multiple comparisons [18].

Declarations
Ethics approval and consent to participate We followed the guidelines of Institutional Animal Care and Use Committee for use and care of laboratory animals, and approved by the South China Agricultural University (approval ID: SCAU#0014). All efforts were made to minimize damage to the animal.

Consent for publication
Not applicable.

Availability of data and materials
All the data and materials supporting the conclusions of the study are included in the manuscript and Additional le 1.

Competing interests
The authors declare that this article has no con ict of interest.  PIC: polymorphism information content; P-value: P-value of Hardy-Weinberg equilibrium.   Figure 1 The daily gain of three different genotypes at different growth stages. * indicate P < 0.01.

Figure 2
Relative expression patterns of GNB1L in different tissues. The data represent Mean ± SD (n = 3).