http://www.jstage.jst.go.jp/browse/jpsa doi:10.2141/ jpsa.0120133 Copyright Ⓒ 2013, Japan Poultry Science Association. The Identification of a Novel Transcript Variant of Chicken Lmbr1 and the Sequence Variation Analysis Wen Chen1, Xiaohui Du2, Ling Ling Hou2, Shuping Zhang1, Xiangtao Kang1, Ruili Han1, Guirong Sun1 and Yanqun Huang1 1 College of Animal Science and Veterinary Medicine, Henan Agricultural University, Zhengzhou, 450002, P. R. China 2 College of Animal Science, Sichuan Agricultural University, Yaan, 625000, P. R. China Lmbr1 has been reported to be associated with chicken polydactyly and carcass traits. Based on the hypothesis that chicken Lmbr1 should have multiple alternative splices as human and mouse Lmbr1/C7orf2, we successfully identified one novel chicken Lmbr1 transcript variant (designated as Lmbr1-2) by bioinformatics analysis and RT- PCR confirmation. Chicken Lmbr1-2 transcript was predicted to encode a 30 amino acid protein. Lmbr1-2 transcript was ubiquitously expressed in a range of chicken tissues. ASNP (rs14135851: G ＞T, also named as 288G＞T reference to 603234777F1) was found to be located at the 5′boundary (first base) of alternatively spliced exon (IN2) of chicken Lmbr1-2. Eight variants/10 haplotypes were detected from 4 breeds in a 300-bp genomic fragment surrounding 288G＞T. The 288T＞G and 331G＞Achanges were predicted resulting to the formation/loss of SRSF5 and SRSF6 motifs. The genotype/allele distribution for a G/Avariant (331G ＞A, reference to 603234777F1) and haplotype types in polydactylous Silkie presented distinct difference with that in other four-toed breed chickens. Association analysis showed that chicken 331G＞Avariation had significant effect on meat quality traits including breast muscle water loss rate, leg muscle water loss rate and leg muscle fiber density, and carcass traits including evisceration weight, pancreas weight in Gushi chicken F2 resource population. These data demonstrated that chicken Lmbr1 was alternatively spliced to produce multiple splice forms as was the case in mammals, and it suggested that chicken Lmbr1-2 variation might have important effect on the carcass and meat quality traits in chicken. Key words: chicken, Lmbr1/C7orf2, splice variant, tissue expression, variation analysis J. Poult. Sci., 50: 104-113, 2013 underlying limb abnormalities in mouse, human and chicken Introduction and was termed as “zone of polarising activity regulatory The nonsyndromic preaxial polydactyly (PPD) in human sequence” (ZRS) (Lettice et al., 2002; Goode et al., 2005; and mouse had been mapped to the homologous region that Dunn et al., 2011). Mutations in intronic ZRS region of contains C7orf2/Lmbr1, which showed C7orf2/Lmbr1 was an Lmbr1 were found to be associated with ectopic Shh important candidate gene for limb development among spe- expression in the limb and/or polydactyly in humans, mice, cies (Dobbs et al., 2000; Ianakiev et al., 2001; Sato et al., cats and chickens (Gurnett et al., 2006; Masuya et al., 2007; 2007). Huang et al. (2006, 2007) found the significant as- Lettice et al., 2008; Dunn et al., 2011). sociation of Lmbr1 variation with chicken polydactylous Alternative splicing is a common phenomenon of organ- phenotype and growth/carcass traits including shank and ism, which can modulate gene function, affect organismal claw weight (SCW) and shank girth (SG) and so on. Dunn phenotype (Cartegni et al., 2002; Lu et al., 2012). One gene et al. (2011) mapped dominant PPD locus in Silkie (SL) often produces multiple mRNAs and protein isoforms (Wang chickens to a single nucleotide polymorphism (SNP) in the et al., 2008). The expressions of these transcript variants intron 5 of Lmbr1. Ahighly conserved region of intron 5 in may be tissue and development-stage specific (Terenzi and Lmbr1/C7orf2 was identified as a cis-acting regulator of the Ladd, 2010). Searching EST database is an effective way sonic hedgehog (Shh) gene, a proposed candidate gene for finding potential splice forms (Eyras et al., 2004). The release of chicken EST database provides a great conven- Received: August 29, 2012, Accepted: November 27, 2012 Released Online Advance Publication: December 25, 2012 ience for searching chicken splice variants of specific genes Correspondence: Dr. Y. Huang, College of Animal Science and Veterinary by informatics methods. The online chicken EST databases Medicine, Henan Agriculture University, No. 95 Wenhua Road, Zheng- include NCBI database (http://blast.ncbi.nlm.nih.gov/Blast. zhou, 450002, P. R. China. (E-mail: [email protected]) cgi), BBSRC ChickEST Database (http://www.chick.man Chen et al.: Chicken Lmbr1 Isoform and the Variation 105 chester.ac.uk/) and U.D ChickEST Database (http://www. intestine, stomach, ovary, skin, crureus, pectoralis major, chickest.udel.edu/). Alternative splicing can be modulated abdominal fat and subcutaneous fat from above two 8- week- by variation both in the cis genomic splicing signals and in old hybrid chickens were also used for the tissue expression the cellular pathways that regulate splicing (Stamm et al., pattern analysis. The tissue samples for RNAextraction 2005). Human disease-causing splicing mutations often were collected and deep-frozen in liquid nitrogen, and then affect critical splicing regulatory signals in cis (e.g., mutating stored at −80℃. All chickens were cage-raised in Henan consensus splice site sequences at exon‒intron boundaries or Agricultural University Poultry Farms. All procedures splicing enhancer/silencer elements within exons or introns) followed the established protocols approved by the institu- (Cartegni et al., 2002). Examining whether SNPs that may tional animal care and use committee. cause splicing differences can be linked to phenotypic traits Total RNAs were extracted by Trizol reagent (Takara, is an alternative to gene-specific functional experiments (Lu Dalian, China) according to the instructions of manufacturer et al., 2012). Established GS chicken F2 resource population andstoredat−80℃. The quantity and quality of the iso- in our lab includes growth, carcass, meat quality, and muscle lated RNAwas determined by GeneQuant pro ultraviolet fiber traits (Han et al., 2011, 2012), it is an important re- spectrophotometry (Amersham Pharmacia, Bucks, UK) and source for to learn the association of gene variation with the agarose gel electrophoresis respectively. 1 μg total RNA related traits. was subsequently reversely transcribed by PrimeScript®RT Lmbr1 consists of 17 exons in human, mouse and chicken reagent kit with gDNAEraser (Takara, Dalian, China) (Clark et al., 2001; Ianakiev et al., 2001; Maas and Fallon, according to manufacturer’s instructions. The synthesized 2004; Huang et al., 2007). Till now, at least five and four first strand cDNAwas stored at −20℃ until use. alternative splicing transcripts of Lmbr1 gene have been Breed Samples found in human and mouse, respectively (Clark et al., 2000; In order to study the variation distribution among breeds, Ianakiev et al., 2001; Huang et al., 2006). In chicken, three blood were collected from the chicken wing vein in four Lmbr1 transcript variants have been reported (Huang et al., breeds including WPR (n＝10), GS (n＝8), SL (n＝10) and 2006, 2011). Human Acheiropodia (ACHP) was found to be Lushi green eggshell chicken (LS, n＝10), respectively. caused by an abnormal splice variant deleted exon 4 of Individuals were selected as unrelated as possible from each C7orf2/Lmbr1 (and surrounding genomic region), which breed. Genomic DNAwas extracted from blood by phenol- introduced a reading frame shift leading to a premature stop chloroform extracting method. Each breed has clearly dif- codon in exon 6 (Ianakiev et al., 2001). Asimilar ab- ferent characteristics. WPR is a cosmopolitan fast-growing normally spliced variant deleted 140 bp exon 4 of Lmbr1 has breed with white plumage, which was collected from China been detected too (Huang et al., 2006). It has been inferred Agricultural University Breed Resource Conservation Cen- that mutations and chromosomal deletions/rearrangements in ter; while SL, GS, and LS are the Chinese local breeds, which the Lmbr1 genic region may be associated with ACHP were collected from Henan Agricultural University Poultry phenotype (Lettice et al., 2003; Maas and Fallon, 2004). Breed Resource Conservation Center. SL is an officinal Based on the hypothesis that chicken Lmbr1 should have breed with black skin, feather-crest and threadlike plumage; much more transcript variants as human and mouse, we GS is a Henan native breed, with yellow skin, yellow plum- further identified one novel transcript isoform of Lmbr1 from age and green shank. In addition, SL is a polydactylous chicken EST database by EST searching and comparative breed, while the others are four-toed breeds. genomics analysis. Its tissue distribution patterns, the varia- GS chicken F2 Resource Population tion distributions among breeds in the surrounding retained The GS chicken F2 resource population was constructed intron sequence, and the association between the genetic by reciprocal crossing with GS chicken (a slow growing variation and performance traits were further analyzed, Chinese native breed, 24 hens and 2 roosters ) and Anka which would build a foundation to further make clear the chicken (a fast growing broiler type, 12 hens and 4 roosters ) gene function of Lmbr1. as Han et al. (2011, 2012) described. It includes four direct cross-bred families (with Anka roosters mating with GS Materials and Methods hens) and two reciprocal families (with GS roosters mating RNA Samples with Anka hens). To construct the F2 population, 9 F1 Considering the potential spatiotemporal-specificity of females and 1 F1 male were selected from each of 7 families chicken Lmbr1 transcript variant, the samples from multiple (6 unrelated rooster families and 1 half sib). The F2 resource tissues/breeds/growth-stages were collected for use. The population was established by two hatching batches at 2- heart tissue of one one-day-old Arbor Acres commercial week intervals. It consists of 42 grandparents, 70 F1 parents, broiler and the mixed tissue samples (including heart, liver, and 860 F2 chickens.