DOI: https://doi.org/10.21203/rs.3.rs-24064/v1
Background: Ras and Rab interactor 2 (RIN2) gene, encoding RAS and Rab interacting protein 2, can interact with GTP-bound Rab5 and participate in early endocytosis. Deletion of RIN2 may impair Rab5-related endosome signaling, leading to abnormal phenotypes. However, no research has been reported on the functions of RIN2 related to animal production.
Results: A 61-bp insertion/deletion (indel) in the RIN2 intron region in this study. The genotype analysis of mutation sites was performed on 550 individuals from 7 different chicken breeds, and it was found that the indel exists in each breed and the local breed chickens are mainly DD genotypes.
Correlation analysis of the indel with growth traits and carcass traits of the F2 population of Xinghua and White Recessive Rock chicken showed that the RIN2 61-bp deletion mutation site was significantly correlated with abdominal fat weight, fat width and hatching weight traits (P < 0.05). RIN2 mRNA was expressed in all test tissues, and the expression level of abdominal fat was higher than that in other tissues. In addition, it was further found that the expression level of type II RIN2 mRNA in abdominal fat was significantly different from that of ID type and DD type (P < 0.05).
Conclusion: The results showed that the mutation was closely related to the abdominal fat-related and hatching weight traits of chickens, which may have certain reference value for molecular marker-assisted selection of chickens.
Ras and Rab interactor 2 (RIN2), known as RAS and Rab interacting protein 2, functions as a guanine nucleotide exchange factor (GEF). RIN2 is shown to interact with Rab5, a small GTPase that participates in early endocytosis [1, 2]. Rab5 is necessary for transport of endocytic vesicles to early endosomes [2]. Deletion of RIN2 may impair Rab5-related endosome signaling, and may also damage secreted proteins from endoplasmic reticulum to Golgi or Golgi to plasma membrane, leading to collagen fiber structure and observed phenotypic abnormalities [3].
Previous research has shown that RIN2 syndrome in humans is also called MACS (macrocephaly, alopecia, cutis laxa and scoliosis) syndrome that is a rare hereditary skin disease caused by the loss of the 1-bp homozygote of RIN2 [3, 4]. A genome-wide selective scan of purebred horses showed that RIN2 plays similar roles in signal transmission, indicating that the RIN2 gene is under strong artificial selection in horse racing [5]. Comparative analysis of the genome-wide methylation and transcriptome of the longest muscle in the sheep showed that RIN2 may be a functional gene that affects meat quality traits [6]. However, no research has been reported on the functions of RIN2 related to animal production, and the exact functional mechanism in chickens is unclear.
Indel is another form of mutation, and there are a large number of indel polymorphisms in the genome of model organisms [7]. Indel plays an important role in genetic diversity and phenotypic differentiation [8, 9]. Compared with single nucleotide polymorphisms (SNP), indel variants have the advantages of convenient detection and significant effects, and have higher efficiency and wider application prospects [10]. In poultry, the repeat indel in the promoter region of the CDKN3 gene is significantly related to the economic traits of chickens [11]. The 86-bp indel in the MLNR gene downstream region and the two indel variants in the QPCTL gene promoter region are both related to chicken growth and carcass traits [12, 7]. An 80-bp indel in PRLR was associated with growth traits [13]. A new indel in the promoter region of the TGFB2 gene is related to body weight at almost all stages [14]. In cattle, a new 19-bp indel in PLAG1 is related to the growth traits of breed cattle, and a 12-bp indel in NPM1 was associated with growth traits [15, 16].
In this study, a 61-bp deletion in the intron region of the RIN2 gene was identified by DNA sequencing and polymerase chain reaction (PCR) analysis. A total of 550 individuals from F2 resource groups, white feather broilers and local breed chickens, were tested for genotype. The correlations between polymorphisms in F2 resource populations of Xinghua-White Recessive Rock chicken hybrids and meat quality, growth traits and carcass traits were analyzed. Differences in RIN2 mRNA levels were investigated in the abdominal fat and breast muscle. The purpose of this study is to clarify the relationship between RIN2 gene variation and chicken performance traits, and to explore whether RIN2 gene can be used as a molecular marker for selecting production traits.
Based on the F2 resource group (N = 304) with meat quality, growth and carcass traits recorded in this laboratory (Xinghua and White Recessive Rock chicken full sib hybrid F2 generation, of which Xinghua chicken is a Chinese local slow-growing breed, White Recessive Rock chicken is a fast-growing broiler), and the correlation analysis of RIN2 was performed. More information on the F2 population was provided by a previous study [17].
To confirm the distribution of this genotypic variation in chickens of other breeds, genomic DNA samples were collected from a total of 267 healthy individuals. The number of samples contributed by each group was as follows: White Recessive Rock chickens (WRR, n = 41), Wenchang chickens (WC, n = 48), Qingjiaoma Chicken (QJ, n = 48), Lushi chickens (CS, n = 40), Guangxi yellow chickens (GX, n = 46) and Gushi chickens (GS, n = 44).
In addition, in order to detect the expression of RIN2 mRNA in different tissues, a total of 12 tissues (hearts, liver, spleen, lung, kidney, breast muscle, leg muscle, abdomen fat, jejunum, duodenum, hypothalamus and ovary) were collected from 24-week-old Yellow chickens. Different genotypes of abdominal fat and breast muscle were taken from four-week-old Yellow chickens to detect the expression of RIN2 mRNA in different genotypes. All tissues were stored at -80 °C.
The genomic DNA in the blood was extracted using the DNA extraction kit (Omega, Norcross, America), and the quality of the genome was detected by a spectrophotometer. All primers were designed using online tools provided by NCBI (http://www.ncbi.nlm.nih.gov/tools/primer-blast/) and synthesized by Beijing TsingKe Company (Supplementary Appendix Table 1). PCR was performed in a total volume of 10 µL, including 1 µL of DNA, 0.2 µL of each primer, 5 µL of 2 × M5 PCR Mix (Yuexing, Guangzhou, China), and 3.6 µL of water. The cycle parameters are: 95 °C for 3 min, 95 °C for 25 s, 61 °C for 25 s, 72 °C for 15 s, 72 °C extension for 5 min, a total of 34 cycles, and then refrigerated at 4 °C. An aliquot (7 µL) of each reaction was electrophoresed on a 2% agarose gel to determine the genotype.
| Breeds | Number | Genotype and Gene frequency | He | Ne | PIC | P-value | ||||
|---|---|---|---|---|---|---|---|---|---|---|
| II | ID | DD | I | D | ||||||
| F2 | 283 | 0.110 | 0.350 | 0.541 | 0.331 | 0.735 | 0.219 | 1.280 | 0.195 | 1.000 |
| WRR | 41 | 0.415 | 0.390 | 0.195 | 0.644 | 0.442 | 0.053 | 1.056 | 0.051 | 0.811 |
| WC | 48 | 0.000 | 0.063 | 0.938 | 0.032 | 0.968 | 0.476 | 1.908 | 0.363 | 0.249 |
| QJ | 48 | 0.000 | 0.125 | 0.875 | 0.067 | 0.935 | 0.061 | 1.064 | 0.059 | 1.000 |
| LS | 40 | 0.050 | 0.200 | 0.750 | 0.224 | 0.866 | 0.117 | 1.133 | 0.110 | 0.644 |
| GX | 46 | 0.000 | 0.109 | 0.891 | 0.058 | 0.944 | 0.053 | 1.056 | 0.051 | 0.811 |
| GS | 44 | 0.000 | 0.250 | 0.750 | 0.144 | 0.866 | 0.103 | 1.115 | 0.098 | 0.697 |
| Note: He represents gene heterozygosity, Ne represents effective allele numbers, Polymorphism information content (PIC). F2 generation resource population(F2), White Recessive Rock chicken(WRR), Wenchang chicken(WC), Qingjiaoma Chicken(QJ), Lushi chickens(LC), Guangxi Yellow chicken(GX), Gushi chicken(GS), P-value of the Hardy-Weinberg equilibrium (P-value). | ||||||||||
Use Trizol® reagent to extract total RNA from tissues. Quantitative real-time polymerase chain reaction (qPCR) was used to detect the expression level of RIN2 mRNA in each tissue. qPCR was performed using the CFX96 system (Bio-Rad, Hercules, CA, USA). The β-actin gene was used as an internal control. qPCR conditions were as follows: 95 °C for 5 min, 95 °C for 30 s, 60 °C for 30 s,72 °C for 30 s and a total of 35 cycles. The results were analysed using the method [18].
Statistical analysis of all sequence variations and important economic traits related to the F2 resource group was performed using SPSS Statistics 24 software. The mixed linear models used in the analysis are:
where is the observed value, is the overall average, is the genotype fixed effect, is the family fixed effect, is the gender fixed effect, is the hatching fixed effect, is the carcass weight regression coefficient, is the average slaughter weight, for individual slaughter weight, is random error. P-value < 0.05 was considered significant, and a Bonferroni's test was performed to control multiple comparisons [19]. Model I is used to assess genotypes related to growth traits, meat quality, and blood biochemical indicators. Considering the effect of body weight on carcass traits, Model II uses carcass weight as a covariate to calculate carcass traits.
Through whole-genome resequencing, a new 61-bp deletion mutation was found downstream of the RIN2 gene. As shown in Fig. 1, the Indel polymorphism was analyzed by PCR amplification of the region and electrophoresis of the product in a 2.0% agarose gel. Named II (646 bp), ID (646 bp and 585 bp), and DD (585 bp), respectively. The PCR products were sequenced to pinpoint the insertion (Fig. 2).
The genotype and allele frequencies, and other genetic parameters associated with the RIN2 Indel locus were calculated for 550 individuals in the study (Table 1). All seven varieties had a RIN2 61-bp deletion mutation, and with the exception of WRR, the allele frequency of D was higher than that of I. The genotype distribution of different populations is shown in Fig. 3.
By convention, PIC > 0.5 is highly polymorphic, 0.25 ≤ PIC ≤ 0.5 is moderate polymorphism, and PIC < 0.25 is low polymorphism. Except for WC showing moderate polymorphism, the other groups showed low polymorphism. The degree of genetic heterozygosity is 0.053–0.476, and the number of effective alleles is 1.056–1.908.
In order to determine whether the differential selection of RIN2 61-bp insertion sequence occurred during domestication of chickens, the pairwise fixed index (Fst value) was used to analyze the differentiation between populations. The analysis showed that the RIN2 61-bp site deletion type had strong genetic differentiation between WC, QJ, LS, GX, GS, and WRR (0.2 < Fst < 0.5; Table 2), indicating that the deletion mutation was in WRR already selected. FST values are usually lower among other breeds.
| Breeds | WRR | WC | QL | LS | GX | GS | |
|---|---|---|---|---|---|---|---|
| WC | 0.3977 | ||||||
| QJ | 0.3451 | 0.0055 | |||||
| LS | 0.2236 | 0.0448 | 0.0207 | ||||
| GX | 0.3556 | 0.0033 | 0.0003 | 0.0256 | |||
| GS | 0.2550 | 0.0312 | 0.0116 | 0.0013 | 0.0154 | ||
| F2 | 0.0533 | 0.0427 | 0.0324 | 0.0100 | 0.0338 | 0.0153 | |
| Note: F2 generation resource population(F2), White Recessive Rock chicken (WRR), Wenchang chicken(WC), Qingjiaoma Chicken(QJ), Lushi chickens(LC), Guangxi Yellow chicken(GX), Gushi chicken(GS). | |||||||
In the F2 population, the 61-bp indel of the RIN2 gene was significantly associated with slaughter performance. Significant correlations were detected in abdominal fat weight and fat width traits (P = 0.046, P = 0.005; Table 3). Among them, abdominal fat weight and fat width traits of DD genotypes were greater than those in chickens with the ID and II genotypes. There was no significant difference between ID and II individuals. The 61-bp indel was not significantly associated with other carcass traits (Supplementary Appendix Table 2).
| Traits | Mean ± SE | P-value | ||
|---|---|---|---|---|
| II | ID | DD | ||
| FW(mm) | 10.262 ± 0.678a | 11.455 ± 0.439a | 12.539 ± 0.354b | 0.005 |
| AFW(g) | 21.070 ± 3.893a | 24.757 ± 2.876a | 29.795 ± 2.492b | 0.046 |
| SFT(mm) | 3.647 ± 0.243 | 4.090 ± 0.154 | 4.208 ± 0.123 | 0.100 |
| Note: fat width(FW), abdominal fat weight(AFW), subcutaneous fat thickness(SFT), Different lowercase letters of the means superscript show significant differences (P < 0.05), the same letters show no difference (P > 0.05). | ||||
In the F2 population, the 61-bp indel of the RIN2 gene was significantly related to hatching weight, with a correlation of P = 0.027 (Table 4). Among them, hatching weight traits of II genotypes were greater than those in chickens with the ID and DD genotypes. There was no significant difference between ID and DD individuals. Other growth traits were not significantly associated with the indel (Supplementary Appendix Table 3). The 61-bp indel in the RIN2 intron was not significantly associated with meat quality traits (Supplementary Appendix Table 4).
| Traits | Age week | Mean ± SE | P-value | ||
|---|---|---|---|---|---|
| II | ID | DD | |||
| 0 | 30.70 ± 0.56a | 29.82 ± 0.48b | 29.79 ± 0.46b | 0.027 | |
| 1 | 60.52 ± 1.73 | 58.77 ± 1.29 | 59.50 ± 1.13 | 0.403 | |
| Body weight | 2 | 125.12 ± 3.65 | 123.66 ± 2.70 | 122.84 ± 2.34 | 0.810 |
| (g) | 3 | 213.71 ± 6.57 | 209.35 ± 4.81 | 208.37 ± 4.11 | 0.685 |
| 4 | 311.02 ± 10.29 | 308.69 ± 7.52 | 307.17 ± 6.41 | 0.930 | |
| 5 | 455.47 ± 15.05 | 433.91 ± 10.41 | 430.24 ± 8.82 | 0.218 | |
| Note: Different lowercase letters of the means superscript show significant differences (P < 0.05), the same letters show no difference (P > 0.05). | |||||
Chicken RIN2 gene is on chromosome 3 (GenBank accession number NC_006090.5),It consists of 20 exons and encodes a protein of 836 amino acids. The expression of RIN2 mRNA in various tissues of 171-day-old Yellow chickens was studied (Fig. 4), and the results showed that RIN2 was expressed in all test tissues, with the highest expression level in abdominal fat. The expression levels in heart, liver, lung, kidney, and hypothalamus are moderate. In contrast, the expression levels were lower in spleen, breast muscle, leg muscle, jejunum, and duodenum. The high expression of RIN2 in abdominal fat suggested that RIN2 may play a role in the formation of abdominal fat.
In abdominal fat, 22-week-old Xinghua bantam chicken type II RIN2 mRNA expression level was significantly different from ID type and DD type (P < 0.05; Fig. 5a). This indicates that the 61-bp indel site affects the expression of RIN2 and may affect slaughter traits such as abdominal fat weight. Therefore, it is important to examine the relationship between 61-bp indel sites and growth and slaughter traits in a larger chicken population. There was no significant difference in the expression of RIN2 mRNA between different genotypes in breast muscle (Fig. 5b).
In this study, we speculate that RIN2 61-bp indel may have a positive effect on the growth traits of chickens and a negative effect on the abdominal fat weight of chickens. This Indel may be a potential molecular marker for auxiliary selection of good quality broilers.
In animal breeding, the discovery of key genes and molecular mechanisms that affect growth traits is an important step to improve breeding efficiency and speed up the breeding process [20, 21]. In order to improve the selection effect of main traits, traditional selection methods can be complemented by gene-assisted selection or molecular marker-assisted selection (MAS). MAS is an effective way to improve short-term traits [22, 23]. In this study, a new 61-bp deletion mutation was identified on the RIN2 gene through whole genome resequencing and PCR product sequencing. In the genetic analysis of 550 individuals of 7 varieties, it was found that there was a deletion mutation of RIN2 61-bp in all varieties. No functional studies on RIN2 gene in animal production have been reported.
Hatching weight is the main indicator of chick quality evaluation [24]. Previous studies have shown that there is a positive correlation between the hatching weight of broilers and the weight of slaughter. For every 1 g increase in hatching weight, the slaughter weight increases by 8–13 g [25, 26]. The economic value of high-hatching weight broilers is also generally higher than that of low- hatching weight broilers [27].
The abdomen is an important part of the fat deposition in chickens. Abdominal fat weight is highly related to the total body fat deposition in chickens and can be used as an index for selecting chicken fat deposition. Previous studies have found that excessive fat deposits in modern commercial broiler breeds can waste a lot of feed, while reducing slaughter rates and economic benefits [28, 29]. For consumers, eating broilers that accumulate too much fat may also cause human obesity or cause other diseases [30]. Therefore, the excessive deposition of abdominal fat in chickens has become one of the problems to be solved in the current broiler production.
In this study, we found that RIN2 gene was expressed in different tissues (Fig. 4), which is consistent with previous reports of widespread RIN2 expression [31]. In addition, RIN2 gene is highly expressed in abdominal fat, kidney, and heart, suggesting that it may be related to fat deposition and growth. The RIN2 61-bp deletion mutation site was significantly negatively correlated with chicken hatching weight (P < 0.05, Table 4). The hatching weight of type II individuals was greater than that of ID type and DD type. During the pre-growth period (1–7 weeks), the weight of the genotype II has always been the highest, while the weight of the DD genotype has generally been the lowest. Analysis of the gene frequency of 7 different chicken breeds revealed that the 61-bp deletion mutation has been highly selected in WRR (Table 1). We speculate that in terms of growth performance, RIN2 61-bp indel type II is the dominant genotype, and DD type is the inferior genotype. The 61-bp deletion of RIN2 may have a positive effect on chicken growth traits.
It is worth noting that the expression of RIN2 gene is highest in abdominal fat tissue. Quantitative analysis of RIN2 mRNA expression in abdominal fat tissue of different genotypes showed that the expression level of II was significantly higher than ID and DD (P < 0.05; Fig. 5a). Correlation analysis found that abdominal fat weight of type II individuals was significantly lower than that of ID and DD individuals (P < 0.05, Table 3). Based on the above results, it can be speculated that RIN2 61-bp indel is involved in the deposition of fat in the abdomen, which may have a negative effect on the fat traits of chickens, but the specific reason is unknown.
The results of this study indicate that the 61-bp intron of the RIN2 gene is associated with slaughter and abdominal fatness traits in chickens. RIN2 mRNA is expressed in all tissues, but the expression level in abdominal fat is higher. Identification of indel related to growth traits, carcass traits, and abdominal fat weight indicates that the indel may be a useful marker in poultry breeding and related QTL identification studies.
Ethics approval and consent to participate
All animal experiments performed in this study comply with the requirements of the Institutional Animal Protection and Utilization Committee of South China Agricultural University (approval ID: SCAU # 0014), and the care and use of animals are in compliance with local animal welfare laws.
Consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing interests.
Funding
This work was supported by the China Agriculture Research System (Grant No. CARS-41-G03), and the Science and Technology Program of Guangzhou, China (Grant No. 201804020088).
Acknowledgments
The authors thank Meixing Wu for editing the language of this manuscript.
Authors’ contributions
These studies were designed by WJL and THR. They conducted experimental analysis and prepared charts. WJL and XQZ analyzed the data and drafted the manuscript. WYL and JYL contributed to the revision of the manuscript. MJX, MQL, DLH, SDL and WL helped to explain the results and revised the final version of the manuscript. All authors read and approved the final manuscript for publication.