S0021859622000028jra 762..768

Insulin-like growth factor 1 receptor (IGF1R) is a cell surface receptor, belonging to the tyrosine kinase receptor superfamily. IGF1R plays a role not only in normal cell development but also in malignant transformation, which has become a candidate therapeutic target for the treatment of human cancer. This study aimed to explore insertions and deletions (indels) in IGF1R gene and investigate their association with growth traits in four Chinese cattle breeds (Xianan cattle, Jinnan cattle, Qinchuan cattle and Nanyang cattle). The current paper identified a 28-bp indel by polymerase chain reaction within IGF1R gene. The analysis showed that there was a significant correlation between the locus and the hucklebone width of Nanyang cattle in four periods, in which it was highly correlated at 6, 12 and 18 months. At the age of 6 months, it was also significantly correlated with body height, body weight and body length. Association analysis showed that the locus in Jinnan cattle was extremely significantly correlated with body slanting length and body weight, and significantly correlated with chest circumference. There was no significant correlation between this locus and growth traits of Xianan cattle and Qinchuan cattle. The detected indel in the IGF1R gene was significantly associated with growth traits in Jinnan and Nanyang cattle, and could be used as a molecular marker for growth trait selection. Introduction Insulin-like growth factor 1 receptor (IGF1R) is a cell surface receptor, belonging to the tyrosine kinase receptor superfamily. It has been shown that the normal secretion of hormones such as mammalian insulin, IGF1 and IGF2 is essential to safeguard growth and development, whereas their counterpart IGF1R and other cell membrane surface receptors play key regulatory roles. Insulin-like growth factor (IGF) is essential for normal foetal and postnatal growth and development. Silencing of the mouse IGF1 gene results in embryonic development deficiency, manifested by dwarfism at birth, postnatal growth impairment and infertility (Baker et al., 1993; Liu et al., 1993). Functional inactivation of the IGF2 gene also impairs embryonic growth but has relatively little effect on postnatal growth (DeChiara et al., 1990). IGF1R binds IGF1 with high affinity and initiates physiological responses in vivo (LeRoith et al., 1995). In endocrinology, the signal transmitted by IGF1R can understand the effect on somatic cell growth (Isaksson et al., 1987). Moreover, IGF1 was found to potently disarm cells such as neuronal cells, hematopoietic cells and fibroblasts from programmed cell death (Gluckman et al., 1992; Rodriguez-Tarduchy et al., 1992; Harrington et al., 1994). Silencing the expression of IGF1R under serum-rich conditions significantly impaired cell cycle progression of fibroblasts, preventing their entry into S phase under the presence of mitogenic growth factors. The establishment of IGF1R knockout mice into fibroblast lines is resistant to the transformational carcinogenic of a variety of viruses and cancer cells (Sell et al., 1994). These results reveal that the IGF1R axis has a role not only in normal cell development but also in malignant transformation (Valentinis, 1997). At present, IGF1R has become a candidate therapeutic target for the treatment of human cancer. With the emergence of sequencing technology, the third generation of genetic markers were found out, which usually have two alleles including single nucleotide polymorphism (SNP) and insertion/deletion (indel), also known as biallelic genetic markers (Sheng et al., 2018). Indel refers to the insertion or deletion of a certain number of DNA sequences in the genome. Indel genetic marker is numerous and has the characteristics of STR and SNP genetic markers, the distribution density of which is second only to SNP. In addition, indel is more distributed in autosomes than sex chromosomes and characterized by uneven distribution (Sjödin et al., 2010). Indel markers have been widely used in molecular breeding and medicine due to their wide distribution and simple typing. Indels are widespread in the genomes of various organisms, with large numbers of indels initially discovered in humans v. agricultural crops. It has begun to be applied in molecular breeding for livestock production in recent years. Studies have shown that indel mutations play important roles for animal economic traits. A 5-bp insertion mutation in goat MSTN gene is proven to be significantly associated with the body height, height at hip cross and chest width index in SBWC (P < 0.05), hinted that this insertion could be assigned to an effective molecular marker for growth trait in goat rearing (Bi et al., 2020). A 31-bp indel in the 5′ UTR region of GNB1L is significantly associated with chicken body weight and carcass traits (Ren et al., 2020).Two indels in the ACTL8 gene are identified to act as DNA markers for beef cattle breeding to select for increased body size (Cai et al., 2019). Marker-assisted selection (MAS) has occupied an important position in molecular breeding in recent years. Therefore, in this study, Qinchuan cattle, Jinnan cattle, Xianan cattle and Nanyang cattle were used as the research objects to screen and analyse whether IGF1R gene insertion-deletion polymorphism affects the growth traits of cattle, in order to provide a new theoretical basis for the genetic improvement of cattle. Materials and methods Numbers and description of experimental animals The current research selected the following four Chinese cattle breeds: Xianan cattle (XN) (Biyang, Zhumadian, Henan), Jinnan cattle (JN) (Yuncheng, Shanxi), Qinchuan cattle (QC) (Qinchuan beef improvement centre, Yangling, Shaanxi) and Nanyang cattle (NY) (Nanyang, Henan). Blood samples and ear tissue were collected from four breeds of Chinese cattle, a total of 537 female adult cattle (XN = 183, JN = 173, QC = 102, NY = 79). In order to demonstrate the relationship between polymorphisms and performance traits, more than 2000 growth traits were measured and calculated, including the body size data of XN (body height (BH), hip height (HH), body slanting length (BSL), heart girth (HG), cannon circumference (CC) and body weight (BW)), JN (body height (BH), hip height (HH), body slanting length (BSL), heart girth (HG) and body weight (BW)), QC (body height (BH), hip height (HH), heart girth (HG), rump length (RL), hip width (HW) and body weight (BW)) and NY (body height (BH), body weight (BW), body slanting length (BSL), heart girth (HG) and hucklebone width (HBW)). Screening of IGF1R gene The expression level of IGF1R gene in muscle was found through bovine muscle transcriptome sequencing in the early stage of the laboratory, and bioinformatics analysis was conducted on it by animal quantitative trait loci (QTL) Database and NCBI. The previous study found the mRNA of IGF1R was widely expressed in heart, liver, kidney, muscle, fat, stomach, spleen and lung (from female cattle) and testis (from male cattle) in adult cattle, with the highest level of expression in testis and the middle-level expression in muscle (Ma et al., 2019). Primer design The IGF1R gene region (AC_000178.1) was located according to the bovine reference genome, and the indel site (AC-000178.1: g.8086620-8086830) on the second intron was found. The specific primers were designed based on PrimerPremier5.0 software. The primer sequence is as follows. Forward primer: 5′-CGAAGTG GGACTTGAGGC-3′; Reverse primer: 5′-AGACAACTGGGAG AAGGGAC-3′. DNA extraction and detection DNA was isolated from whole blood or tissue by phenol chloroform method. The concentration and quality of DNA were detected by NanoDrop2000 spectrophotometer and agarose gel electrophoresis. PCR amplification In this study, polymerase chain reaction (PCR) was used to detect indel genotyping. The reaction system and procedure are shown in Table 1 and Table 2. Agarose gel electrophoresis detection and Sanger sequencing of the target fragment The PCR product was detected by 2% agarose gel. The indel typing was determined according to the image. The insertion and deletion products were sequenced to further verify the authenticity of the product. Statistical analysis The genotype and allele frequencies of IGF1R gene were calculated by Microsoft Excel 2013. Distribution differences for them were analysed according to the Hardy–Weinberg equilibrium using χ test. The population genetic parameters were calculated by Nei’s method, containing genetic homozygosity (Ho), heterozygosity (He), effective number of alleles (Ne) and polymorphism information content (PIC) (Nei and Roychoudhury, 1974).