Indian Journal of Animal Research

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Indian Journal of Animal Research, volume 55 issue 6 (june 2021) : 624-628

Association Analysis between Polymorphism of GnRH-1 Genes and Carcass Traits of Quail (Coturnix coturnix)

Junyan Bai1,2,*, Zhihao Dong1, Zhengyu Fan1, Xinle Wang1, Ying Lei1,2, Youzhi Pang1,2, Shujuan Zhao1,2, Yu Chen1, Jingyun Li1
1College of Animal Science and Technology, Henan University of Science and Technology, Luoyang-471023, China.
2Key Laboratory of Animal Genetics and Breeding, Luoyang-471023, China.
Cite article:- Bai Junyan, Dong Zhihao, Fan Zhengyu, Wang Xinle, Lei Ying, Pang Youzhi, Zhao Shujuan, Chen Yu, Li Jingyun (2020). Association Analysis between Polymorphism of GnRH-1 Genes and Carcass Traits of Quail (Coturnix coturnix) . Indian Journal of Animal Research. 55(6): 624-628. doi: 10.18805/IJAR.B-1284.
Background: GnRH-1 mainly exists in hypothalamus and it is vital to gonad development and sexual maturity of animals. This study mainly analyzed the association between GnRH-1 polymorphism and carcass traits of quails, Research conclusions can provide references for breeding of new species of quail.

Methods: From 2018 to 2019, 31 female samples of Chinese Yellow quail, beijingbai quail and Korean quail were collected in Luolong District, Luoyang City, Henan Province, China.
SNP mutation sites of GnRH-1 gene were investigated in China yellow quail, Beijing white quail and Korean quail through PCR amplification and DNA sequencing technologys. Moreover, correlation analysis between polymorphism sites of GnRH-1 gene and carcass traits of quail was performed. 

Result: According to research results, a total of 14 SNP mutation sites of GnRH-1 were detected in China yellow quail, Beijing white quail and Korean quail, which were C71T, C108T, C168T, C178T, A184G, C206T, A209C, C215T, A252G, A279T, C281T, C293G, C339T and C458T. For A209C and C281T, only 2 genotypes were detected in China yellow quail and Beijing white quail, while for the remaining 12 SNP mutation sites, 3 genotypes were detected in three quail species. Correlation analysis showed that A209C, A279T, C281T and C339T were significantly correlated with liver weight (P<0.05), A209C, C281T and C339T were significantly correlated with dressing percentage (P<0.05), C215T and A279T were significantly correlated with breast muscle weight(whole) (P<0.05), A209C was significantly correlated with body weight, dressed carcass weight and whole net carcass weight (P<0.05).
With the characteristics of small investment, small scale, short growth period and short earning cycle, quail is highly appreciated by farmers and enjoys a promising market development prospect (Bai et al., 2020, 2017, 2016). According to comparative analysis of nutrients per 100g quail meat and chicken meat by Beijing Food Research Institute, it could be found that quail meat contained 22.2% proteins, which was higher than that chicken contained (21.5%). Besides, quail meat contained 20.4mg Ca, 277.1mg P, 6.2mg Fe which were significantly higher than those in chicken (11.0mg, 190.0mg and 1.5mg, respectively). Recently, many achievements have been made in the study of quail production performance and gene polymorphism (Li et al., 2019; Bai et al., 2020b, 2020c, 2020d).
 
Gonadotrophin releasing hormone (GnRH) is a decapeptide firstly obtained from hypothalamus of pig by Schally’s research team. GnRH mainly includes three types, namely, GnRH-1, GnRH-2 and GnRH-3. Among them, GnRH-1 mainly exists in hypothalamus and it is vital to gonad development and sexual maturity of animals. In addition, studied expression laws of GnRH genes in different development stages of Sichuan white goose, Xupu goose, small tailed han sheep and Microtus brandti (Zhang et al., 2016, Han et al., 2017, Ding et al., 2018, Liu et al., 2017). These studies all elaborated the close relationship between GnRH and production performance of animals. It can be used as an important candidate gene in the process of animal production. Therefore, this study analyzed polymorphism of GnRH-1 gene through sequencing technique and investigated carcass traits of quail, aiming to gain molecular markers related with carcass traits by association analysis method. Research conclusions can provide references for breeding and marker-assisted selection of new species of egg quail.
Genomic DNA extraction of quail
 
31 female samples of China yellow quail, Beijing white quail and Korean quail were collected, respectively. All samples were cultured in single cage which had been sterilized strictly before the experiment. Spraying sterilization was performed regularly to all cages during the experiment. All cages are piled up completely into four layers. The culture room was provided with 24h lights and quail were allowed to drink water and eat freely throughout the experiment. Fodders were supplemented artificially on the morning and evening every day. The culture temperature and humidity were determined according to culture management requirements. The feeding experiment ended at the age of 20 weeks, at the end of experiment, 5mL blood was collected from heart of quail. Besides, genotype DNA was extracted by poultry whole blood DNA extraction kit.
 
Measurement of carcass traits
 
At the end of culture experiment, quail samples were killed by jugular vein bleeding, removed hairs and then dissected. Weights of all parts were measured by an electronic scale. The slaughter traits measured included body weight, dressed carcass weight, whole net carcass weight, heart weight, liver weight, breast muscle weight (whole) and leg muscle weight (single). Whole net carcass rate (whole net carcass weight/live weight ×100%).
 
PCR amplification of GnRH-1 gene
 
In this experiment, GnRH-1 primers were: F: TCTTGG TTGGTGTTCTCCT and R: ATTGCTCAGCCTGGGAT (Pu, 2016), the segment size was 906bp. The total volume of PCR amplification reaction system was 15 μL, which was composed of 3.5μL deionized water, 1μL upstream primer, 1μL downstream primer, 2μL DNA and 7.5 μL2×Taq PCR Mix. The PCR amplification program consisted of 30 cycles of 5 min pre degeneration under 94°C, 30s degeneration under 94°C, 30s annealing under 59°C and 30s extension under 72°C, as well as 7min extension under 72°C. The reaction system was stored under 4°C.
 
Data analysis
 
PCR products of GnRH-1 gene were sent to Beijing Qingke biological Co., Ltd. for sequencing. Software Chromas was used to determine the genotype of the sequencing results. SPSS17.0 statistical software was used to analyze the association between different genotypes and carcass traits. The correlation analysis model between GnRH-1 gene and carcass traits is as follows: Analytical model:
 
                                Yijk =  μ + Bi + Mj + eijk

Yijk is the phenotype value of traits, μ is the total mean value, Bi is the effect of the i th variety (i = 1, 2, 3), Mj is the effect of the j th genotype effect, eijk is the residual effect.
SNP detection of GnRH-1 gene
 
PCR amplification products of GnRH-1 gene in China yellow quail, Beijing white quail and Korean quail were shown in Fig 1. Clearly, the amplification fragments of GnRH-1 gene gave high-definition bright bands near 900bp, which conformed to the target size of 906bp. Totally 14 SNP mutation sites of GnRH-1 gene were detected in three egg quail species, which were C71T, C108T, C168T, C178T, A184G, C206T, A209C, C215T, A252G, A279T, C281T, C293G, C339T and C458T respectively (Fig 2).
 

Fig 1: Agarose electrophoresis results of GnRH gene.


 

Fig 2: SNP detection of GnRH gene in quail.


 
GnRH-1 polymorphism of quail, they detected 6 mutation sites, including C108T, C168T, C178T, A184G, C206T and C215T, only C108T was silent mutation without causing change to corresponding amino acid  (Pu, 2016). In this study, 8 additional mutation sites were detected, which were C71T, A209C, A252G, A279T, C281T, C293G, C339T and C458T, demonstrating that GnRH-1 gene had rich polymorphism in 3 egg quail species.
 
Association analysis between GnRH gene and carcass traits
 
It could be seen from Table 1 that body weight, dressed carcass weight, whole net carcass weight and liver weight of AA and AC genotypes of A209C were significantly higher than those of CC genotype (P<0.05), while no significant differences were observed between AA and AC genotypes in term of the indexes above (P>0.05). Moreover, dressing percentage of AA genotype of A209C was significantly higher than that of AC and CC genotype (P<0.05), but no significant difference was observed between AC and CC genotypes (P>0.05). For C215T, breast muscle weight (whole) of CT genotype was far higher than that of TT genotype (P<0.05), but breast muscle weights (whole) of CT and TT genotypes were similar with that of CC genotype (P>0.05). For A279T, breast muscle weights (whole) of TT was far higher than that of AT genotype (P<0.05). For C281T, Liver weight of CC genotype was far higher than that of TT genotype (P<0.05), but no significant difference was observed between CC, TT and CT genotypes (P>0.05). For C281T, dressing percentage of CC genotype was significantly higher than those of CT and TT genotype (P<0.05), but no significant difference was observed between CT and TT genotypes (P>0.05). For C339T, liver weight of TT genotype was far higher than that of CC genotype (P<0.05), but CT genotype showed no significant difference with TT and CC genotypes (P>0.05). For C339T, dressing percentage of TT genotype was far higher than those of CC and CT genotypes (P<0.05), but no significant difference was observed between CC and CT genotypes (P>0.05).
 

Table 1: Association analysis of GnRH gene polymorphism with carcass traits of quail.


 
The association between GnRH receptor gene polymorphism and testicular parameters, and found that GnRH receptor gene was significantly correlated with testicular length (TL) (P<0.05) (Iyer et al., 2019). The kisspeptin (KiSS)/GnRH1 system was expressed in horse Leydig cells and modulated their endocrine activity (Petrucci et al., 2020). That VAL-opsin not only affected activity of the HPG axis but also acted on the pituitary gland to directly stimulate a new sexual maturation pathway that promoted the secretion of GTHs independent of GnRH in goldfish (Choi et al., 2020). There existed a remarkable association between the SNPs of GnRH and sperm quality traits of Chinese water buffalo (Wang et al., 2020). A highly significant association between G840327C of GnRH-I gene and age at first egg (AFE) in a Chinese chicken population (P<0.01) (Xu et al., 2011a). For mutation site T68G of GnRH gene detected in Rongjiang little sweet sheep, lambing number of GG genotype was far higher than those of the rest two genotypes (P<0.05) (Li et al., 2016). That 3 SNPs of GnRH gene influenced laying start age of Erlangshan chicken significantly and SNP5 played an important role in body weight at beginning of laying (Hu et al., 2015). All These studies showed that GnRH gene could regulate the production performance of poultry. In this study, five SNPs including A209C,  C215T,  A279T, C281T and C293G of GnRH-1 gene were significantly correlated with the breast muscle weight (whole), liver weight, dressing percentage, body weight, dressed carcass weight and whole net carcassweight of quail (P<0.05), which supported the studies mentioned above.
Totally 14 SNP mutation sites of GnRH-1 were detected in China yellow quail, Beijing white quail and Korean quail. 5 SNP loci such as A209C, C215, A279T, C281T and C293G were significantly correlated with breast muscle weight (whole), liver weight, dressing percentage, body weight, dressed carcass weight and whole net carcass weight of quail (P<0.05).
Sincere gratitude goes to the sponsor of National Natural Science Foundation (31201777) and Industry-University-Research Cooperation Project in Henan Province (152107000095.0).

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