Single-nucleotide polymorphisms in the promoter of the growth hormone-releasing hormone receptor gene are associated with growth and reproduction traits in chickens

Growth hormone‐releasing hormone receptor (GHRHR) plays a critical role in growth hormone (GH) synthesis, release and regulation in animals. The objective of this study was to investigate variations of the chicken GHRHR gene and their associations with growth and reproduction traits in 768 Beijing You chickens. Results revealed three single nucleotide polymorphisms (SNPs) in the promoter region of the gene (g.‐1654A>G, g.‐1411A>G and g.‐142T>C). Association analysis revealed that the novel SNP g.‐1654A>G had significant effects on chicken body weight at 7, 9, 11, 13, 17 weeks of age and the age of first egg as well as egg number at 32, 36 and 40 weeks. Significant association was also observed between g.‐1411A>G and g.‐142T>C with EN24. Moreover, the age of first egg was distinctly related with g.‐142T>C (P < 0.05). Although significant statistical difference was not detected in GHRHR mRNA levels among genotypes of the SNPs (P > 0.05), strong expression variations of the gene were found between the ages 17 and 20 weeks in the population (P < 0.05). These results suggest that the three SNPs in the GHRHR promoter could be used as potential genetic markers to improve the growth and reproductive traits in chickens.     

A novel 62‐bp indel mutation in the promoter region of transforming growth factor‐beta 2 (TGFB2) gene is associated with body weight in chickens

Transforming growth factor‐beta 2 (encoded by TGFB2) is a growth factor that regulates a plethora of cellular functions. In this study, we sequenced the promoter and full‐length exon region of the chicken TGFB2 and found two mutations (g.‐640C>T and g.‐851_‐790del) within the promoter. The two polymorphisms were genotyped in 1030 pedigreed hens recorded for body weight at 7 (BW7), 9 (BW9), 11 (BW11), 13 (BW13), 17 (BW17) weeks old, egg weight at 36 weeks of age (EW36) and egg numbers from the age at first egg (AFE) to 40 weeks of age (EN40). Despite the fact that no mutations were found to have statistically significant genetic effects on egg production, the association results of growth traits showed that both g.‐640C>T and g.‐851_‐790del had significant effects on body weights and that both genotype g.‐640TT and g.‐851_‐790wt/wt were positive for body weight performance. Therefore, the polymorphisms of TGFB2, especially the g.‐851_‐790del mutation associated with body weight at almost all periods, could be potential useful genetic markers to improve the growth of Beijing You chickens.     

Genome‐wide association study reveals novel variants for growth and egg traits in Dongxiang blue‐shelled and White Leghorn chickens

This study was designed to investigate the genetic basis of growth and egg traits in Dongxiang blue‐shelled chickens and White Leghorn chickens. In this study, we employed a reduced representation sequencing approach called genotyping by genome reducing and sequencing to detect genome‐wide SNPs in 252 Dongxiang blue‐shelled chickens and 252 White Leghorn chickens. The Dongxiang blue‐shelled chicken breed has many specific traits and is characterized by blue‐shelled eggs, black plumage, black skin, black bone and black organs. The White Leghorn chicken is an egg‐type breed with high productivity. As multibreed genome‐wide association studies (GWASs) can improve precision due to less linkage disequilibrium across breeds, a multibreed GWAS was performed with 156 575 SNPs to identify the associated variants underlying growth and egg traits within the two chicken breeds. The analysis revealed 32 SNPs exhibiting a significant genome‐wide association with growth and egg traits. Some of the significant SNPs are located in genes that are known to impact growth and egg traits, but nearly half of the significant SNPs are located in genes with unclear functions in chickens. To our knowledge, this is the first multibreed genome‐wide report for the genetics of growth and egg traits in the Dongxiang blue‐shelled and White Leghorn chickens.     

Polymorphisms of Flanking Region of the ASB15 Gene and Their Associations with Performance Traits in Chicken

Research on the identity of genes and their relationship with traits of economic importance in chickens could assist in the selection of poultry. In this study, an F₂ resource population of Gushi chickens crossed with Anka broilers was used to detect single-nucleotide polymorphisms (SNPs) in the flanking region of the ASB15 gene by DNA sequencing and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). One SNP of ?1271 C>T in 5′ flanking region of the chicken ASB15 gene and two SNPs of the 10618 A>G and 10716 G>A in 3′ flanking region were identified. Furthermore, the 10618 A>G and 10716 G>A in 3′ flanking region were in complete linkage. Association analysis results showed that ?1271 C>T was not associated with performance traits, while the 10618 A>G and 10716 G>A were significantly associated with BW2, 4, 6, 8, 10, 12, SL12, CD8, CW4, 8, 12, BSL4, 8, 12, and SEW, EW, WW, BMW, LW, CW, SFT. Our results suggest that the ASB15 gene profoundly affects chicken performance traits.     

A comprehensive,sensitive and economical approach for the detection of mutations in the <Emphasis Type="Italic">RB1</Emphasis> gene in retinoblastoma

Retinoblastoma (Rb) is the most common primary intraocular malignancy in children. It is brought about by the mutational inactivation of both alleles of RB1 gene in the developing retina. To identify the RB1 mutations, we analysed 74 retinoblastoma patients by screening the exons and the promoter region of RB1. The strategy used was to detect large deletions/duplications by fluorescent quantitative multiplex PCR; small deletions/insertions by fluorescent genotyping of RB1 alleles, and point mutations by PCR-RFLP and sequencing. Genomic DNA from the peripheral blood leucocytes of 74 Rb patients (53 with bilateral Rb, 21 with unilateral Rb; 4 familial cases) was screened for mutations. Recurrent mutations were identified in five patients with bilateral Rb, large deletions in 11 patients (nine with bilateral Rb and two with unilateral Rb), small deletions/insertions were found in 12 patients all with bilateral Rb, and point mutations in 26 patients (14 nonsense, six splice site, five substitution and one silent change). Three mutations were associated with variable expressivity of the disease in different family members. Using this method, the detection rates achieved in patients with bilateral Rb were 44/53 (83%) and with unilateral Rb, 5/21 (23.8%). This approach may be feasible for clinical genetic testing and counselling of patients.     

The timing of molecular and morphological changes underlying reproductive transitions in wild tomatoes (Solanum sect. Lycopersicon)

Molecular mechanisms underlying the transition from genetic self‐incompatibility to self‐compatibility are well documented, but the evolution of other reproductive trait changes that accompany shifts in reproductive strategy (mating system) remains comparatively under‐investigated. A notable exception is the transition from exserted styles to styles with recessed positions relative to the anthers in wild tomatoes (Solanum Section Lycopersicon). This phenotypic change has been previously attributed to a specific mutation in the promoter of a gene that influences style length (style2.1); however, whether this specific regulatory mutation arose concurrently with the transition from long to short styles, and whether it is causally responsible for this phenotypic transition, has been poorly investigated across this group. To address this gap, we assessed 74 accessions (populations) from 13 species for quantitative genetic variation in floral and reproductive traits as well as the presence/absence of deletions at two different locations (StyleD1 and StyleD2) within the regulatory region upstream of style2.1. We confirmed that the putatively causal deletion variant (a 450‐bp deletion at StyleD1) arose within self‐compatible lineages. However, the variation and history of both StyleD1 and StyleD2 was more complex than previously inferred. In particular, although StyleD1 was statistically associated with differences in style length and stigma exsertion across all species, we found no evidence for this association within two species polymorphic for the StyleD1 mutation. We conclude that the previous association detected between phenotypic and molecular differences is most likely due to a phylogenetic association rather than a causal mechanistic relationship. Phenotypic variation in style length must therefore be due to other unexamined linked variants in the style2.1 regulatory region.     

Single nucleotide polymorphisms in chicken <Emphasis Type="Italic">lmbr1</Emphasis> gene were associated with chicken growth and carcass traits

Lmbr1 is the key candidate gene controlling vertebrate limb development, but its effects on animal growth and carcass traits have never been reported. In this experiment, lmbr1 was taken as the candidate gene affecting chicken growth and carcass traits. T/C and G/A mutations located in exon 16 and one A/C mutation located in intron 5 of chicken lmbr1 were detected from Silky, White Plymouth Rock broilers and their F₂ crossing chickens by PCR-SSCP and sequencing methods. The analysis of variance (ANOVA) results suggests that T/C polymorphism of exon 16 had significant association with eviscerated yield rate (EYR), gizzard rate (GR), shank and claw rate (SCR) and shank girth (SG); A/C polymorphism of intron 5 was significantly associated with SCR, liver rate and head-neck weight (HNW), while both sites had no significant association with other growth and carcass traits. These results demonstrate that lmbr1 gene could be a genetic locus or linked to a major gene significantly affecting these growth and carcass traits in chicken.     

Phylogenetic analysis of DNA length mutations in a repetitive region of the Hawaiiandrosophila yolk protein geneYp2

Nucleotide sequence analysis has demonstrated that interspecific size variation in the YP2 yolk protein among HawaiianDrosophila is due to in-frame insertions and deletions in two repetitive segments of the coding region of the Yp2 gene. Sequence comparisons of the complex repetitive region close to the 5′ end of this gene across 34 endemic Hawaiian taxa revealed five length morphs, spanning a length difference of 21 nucleotides (nt). A phylogenetic character reconstruction of the length mutations on an independently derived molecular phylogeny showed clade-specific length variants arising from six ancient events: two identical insertions of 6 nt, and four deletions, one of 6 nt, one of 12 nt, and two identical but independent deletions of 15 nt. These mutations can be attributed to replication slippage with nontandem trinucleotide repeats playing a major role in the slipped-strand mispairing. Geographic analysis suggests that the 15 nt deletion which distinguishes theplanitibia subgroup from thecyrtoloma subgroup occurred on Oahu about 3 million years ago. The homoplasies observed caution against relying too heavily on nucleotide insertions/deletions for phylogenetic inference. In contrast to the extensive repeat polymorphisms within otherDrosophila and the human species, the more complex 5′Yp2 repetitive region analyzed here appears to lack polymorphism among HawaiianDrosophila, perhaps due to founder effects, low population sizes, and hitchhiking effects of selection on the immediately adjacent 5′ region. Correspondence to: M.P. Kambysellis     

Single nucleotide polymorphisms in chicken lmbr1 gene were associated with chicken growth and carcass traits

Lmbr1 is the key candidate gene controlling vertebrate limb development, but its effects on animal growth and carcass traits have never been reported. In this experiment, lmbr1 was taken as the candidate gene affecting chicken growth and carcass traits. T/C and G/A mutations located in exon 16 and one A/C mutation located in intron 5 of chicken lmbr1 were detected from Silky, White Plymouth Rock broilers and their F₂ crossing chickens by PCR-SSCP and sequencing methods. The analysis of variance (ANOVA) results suggests that T/C polymorphism of exon 16 had significant association with eviscerated yield rate (EYR), gizzard rate (GR), shank and claw rate (SCR) and shank girth (SG); A/C polymorphism of intron 5 was significantly associated with SCR, liver rate and head-neck weight (HNW), while both sites had no significant association with other growth and carcass traits. These results demonstrate that lmbr1 gene could be a genetic locus or linked to a major gene significantly affecting these growth and carcass traits in chicken. Supported by the State Major Basic Research Development Program (Grant No. G20000161) and Beijing Natural Science Foundation (Grant No. 5011002)     

Novel SNPs in the <Emphasis Type="Italic">PRDM16</Emphasis> gene and their associations with performance traits in chickens

The PR domain containing 16 (PRDM16) is a member of the Prdm family, and is known to regulate cell differentiation. In the present study, DNA pool sequencing methods were employed to screen genetic variations in the chicken PRDM16 gene. The results revealed four novel single nucleotide polymorphisms (SNPs): NC_006108.2: g.92188G>A, XM_417551: c.1161C>T (Ala/Ala, 387aa), c.1233C>T (Ser/Ser, 411aa) and c.1433G>A (Ser/Asn, 478aa). The BglI polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP) was used to detect c.1161C>T, while HhaI Forced PCR-RFLP methods were used to detect 1233C>T and c.1433G>A in 964 chickens. The chickens comprised 38 grandparents, 66 F₁ parents and 860 F₂ birds derived from an F₂ resource population of Gushi chickens crossed with Anka broilers. The associations of the polymorphisms in the chicken PRDM16 gene with performance traits were analyzed in the 860 F₂ chickens. The results indicated that the three SNPs were significantly associated with growth, fatness and meat quality traits in the chickens. In particular, the polymorphisms of the missense SNP (c.1433G>A) had positive effects on chicken body weight and body size at different stages. It affected also fatness traits significantly. Comparison of the different genotypes of c.1433G>A showed that the GG genotype favored chicken growth and fatness traits.     

A missense mutation in TYRP1 causes the chocolate plumage color in chicken and alters melanosome structure

The chocolate plumage color in chickens is due to a sex‐linked recessive mutation, choc, which dilutes eumelanin pigmentation. Because TYRP1 is sex‐linked in chickens, and TYRP1 mutations determine brown coat color in mammals, TYRP1 appeared as the obvious candidate gene for the choc mutation. By combining gene mapping with gene capture, a complete association was identified between the chocolate phenotype and a missense mutation leading to a His214Asn change in the ZnA zinc‐binding domain of the protein. A diagnostic test confirmed complete association by screening 428 non‐chocolate chickens of various origins. This is the first TYRP1 mutation described in the chicken. Electron microscopy analysis showed that melanosomes were more numerous in feather follicles of chocolate chickens but exhibited an abnormal structure characterized by a granular content and an irregular shape. A similar altered morphology was observed on melanosomes of another TYRP1 mutant in birds, the roux mutation of the quail.     

Non‐target sites with single nucleotide insertions or deletions are frequently found in 16S rRNA sequences and can lead to false positives in fluorescence in situ hybridization (FISH)

Fluorescence in situ hybridization (FISH) has impacted profoundly on our knowledge of the in situ ecophysiology and biodiversity of bacteria in natural communities. However, it has many technical challenges including the possibility of false positives from the binding of probes to non‐target rRNA sequences. We show here that probe target sites containing single‐base insertions or deletions can lead to false FISH positives, the result of hybridization with a bulge around the missing base. Experimental and in silico data suggest this situation occurs at a surprisingly high frequency. The existence of such sites is not currently considered during most FISH probe design processes. We describe software to identify potential non‐target sites resulting from single‐base insertions or deletions in rRNA sequences. This software also provides an estimate of the FISH probe hybridization efficiency to these sites.     

Polymorphism of the bovine ADRB3 gene

The β₃-adrenergic receptor (ADRB3) is predominantly expressed in white and brown adipose tissue and mediates the lipolytic and thermogenic effects of high catecholamine concentrations. Variation in the ADRB3 gene (ADRB3) has been associated with obesity and the earlier onset of non-insulin-dependent diabetes mellitus in some ethnic groups, as well as some production traits of sheep, but to date variation of bovine ADRB3 has not been reported. In this study, variation in the promoter region of bovine ADRB3 was investigated in 737 cattle by polymerase chain reaction-single strand conformational polymorphism (PCR-SSCP) analysis. Six PCR-SSCP patterns representing six allelic variations and containing four single nucleotide polymorphisms (SNPs) and three nucleotide deletions/insertions were observed. Allele A was the most common allele (93.83%), whereas alleles C, D, E and F were rare (0.07, 1.09, 0.41, and 0.34%, respectively). The variation identified here might have an impact on both the function and level of expression of bovine ADRB3.     

SNP and INDEL detection in a QTL region on chicken chromosome 2 associated with muscle deposition

Genetic improvement is important for the poultry industry, contributing to increased efficiency of meat production and quality. Because breast muscle is the most valuable part of the chicken carcass, knowledge of polymorphisms influencing this trait can help breeding programs. Therefore, the complete genome of 18 chickens from two different experimental lines (broiler and layer) from EMBRAPA was sequenced, and SNPs and INDELs were detected in a QTL region for breast muscle deposition on chicken chromosome 2 between microsatellite markers MCW0185 and MCW0264 (105 849–112 649 kb). Initially, 94 674 unique SNPs and 10 448 unique INDELs were identified in the target region. After quality filtration, 77% of the SNPs (85 765) and 60% of the INDELs (7828) were retained. The studied region contains 66 genes, and functional annotation of the filtered variants identified 517 SNPs and three INDELs in exonic regions. Of these, 357 SNPs were classified as synonymous, 153 as non‐synonymous, three as stopgain, four INDELs as frameshift and three INDELs as non‐frameshift. These exonic mutations were identified in 37 of the 66 genes from the target region, three of which are related to muscle development (DTNA, RB1CC1 and MOS). Fifteen non‐tolerated SNPs were detected in several genes (MEP1B, PRKDC, NSMAF, TRAPPC8, SDR16C5, CHD7, ST18 and RB1CC1). These loss‐of‐function and exonic variants present in genes related to muscle development can be considered candidate variants for further studies in chickens. Further association studies should be performed with these candidate mutations as should validation in commercial populations to allow a better explanation of QTL effects.