Molecular Cloning of Porcine DIS3L2 Gene and Association between a SNP with Litter Size Trait Molecular Cloning of Porcine DIS3L2

this work was to clone the full-length porcine DIS3L2 mRNA sequence, search for polymorphism within this gene and perform association analyses between a gene-tagged SNP and litter size traits in the Large White and Landrace pig breeds. ABSTRACT The aim of this work was to clone the full-length coding sequence of porcine DIS3L2 (DIS3 mitotic control-like protein) gene, search for polymorphism and perform association analyses between a gene-tagged SNP and litter size traits in the Large White and Landrace pig breeds. The full-length cDNA sequence of porcine DIS3L2 gene was cloned through the rapid ampliﬁcation of cDNA ends (RACE) method. Sequence analysis revealed that the pig DIS3L2 gene encodes a protein of 883 amino acids which has high homology with the DIS3L2 protein of seven species: cattle (92%), giant panda (92%), horse (91%), dog (91%), human (89%), rat (85%) and mouse (85%). This novel porcine gene was assigned to GeneID: 100153005. This gene is structured in 20 exons and 19 introns as revealed by computer-assisted analysis. Phylogenetic analysis indicated that the pig DIS3L2 gene has a closer genetic relationship with the DIS3L2 gene of cattle. PCR– RFLP was established to detect the GU373702:c.1999C>A substitution of porcine DIS3L2 gene mRNA and association of this mutation with litter size traits was assessed in Large White (n = 200) and Landrace (n = 200) pig populations. Results demonstrated that this polymorphic locus was significantly associated with the litter size of all parities (P<0.01) in Large White and Landrace sows. Therefore, DIS3L2 gene is also a reproduction related gene.

were collected and snap frozen in liquid nitrogen then stored at -80 °C. The total RNA was extracted using the Total RNA Extraction Kit (Gibco, USA). These RNA samples were used to perform RACE PCR. Ear samples were collected from 995 unrelated animals belonging to eight swine populations presented in Table 1 Table 1 were recorded for 700 litters. The litter size traits data and genomic DNA of these pigs would be used to perform association analyses.

Sequence Analysis
The gene analysis was conducted by GenScan software (http:// genes.mit.edu/GENSCAN.html). The protein comparison was performed using the Tool of BLAST at the National Center for Biotechnology Information (NCBI) server (http://www.ncbi.nlm. nih.gov/BLAST) and the ClustalW software (http://align.genome. jp/). The theoretical isoelectric point (pI) and molecular weight (Mw) of proteins was computed using the Compute pI/Mw Tool (http://www.expasy.org/tools/pi_tool.html).

PCR-RFLP
The DNA from the above pigs Table 1  water. PCR was run as follows: 94°C for 4 min, followed by 35cycles of 94°C for 50 s, 57°C for 50 s, 72°C for 1 min, then 72°C extension for 10min, finally 4°C to terminate reaction. The 31 μL PCR-RFLP reaction volume was: 10 μL PCR product, 18 μL sterile water, 1 μL Taq I (10 U), 2 μL10 × buffer. The mixture was incubated in an air incubator at 65°C for 4 h, and then the genotypes were analyzed on the agarose gel (2.5%) containing ethidium bromide.

Statistical Analysis
The relationships between DIS3L2 genotypes and litter size traits of Large White (n=200) and Landrace (n=200) sows were evaluated with the general linear model (GLM) procedure of SAS version 8.0. Both additive and dominance effects were also estimated using REG procedure, where the additive effect was estimated as -1, 0 and 1 for AA, CA and CC genotype, respectively; and the dominance effect represented as 1, -1 and 1 for AA, CA and CC genotype, respectively [9]. The model: Yijkl =μ+ Pi +Sj + Fk+ Gl + eijkl, where Yijkl is the observation of the trait, μ is the least square means, Pi is the effect of ith parity (i =1, 2, 3, 4,5,6,7 (parity ≥ 7)), Sj is the effect of jth season, Fk is the effect of kth farm (k=1, 2), Gl is the effect of lth genotype (l = 1-3) and eijkl is the random residual [10].

RACE Results for Pig DIS3L2 Gene
For pig DIS3L2 gene, through 5'-RACE, one PCR product of 1656bp was obtained. The 3'-RACE product was 2214 bp. These products were then cloned to T-vector and sequenced. These products were then cloned to T-vector and sequenced. Taken together, a

Sequence Analysis
The cDNA nucleotide sequence analysis using the BLAST software revealed that this gene was not homologous to any of the known pig genes and it was then deposited into the GenBank database (Accession number: GU373702 jp/), as shown in (Figure 2).    In addition, for all parities, in the purebred Landrace sows, AA and CA animals had 1.069 and 1.002 more piglets born than the CC animals, respectively (P<0.01), and 1.895 and 1.676 more piglets born alive than the CC animals, respectively (P<0.01).

Conclusions
In conclusion, we first isolated the pig DIS3L2 gene and performed necessary sequence analysis, polymorphic analysis and association analysis. Our results suggest that DIS3L2 is not only a tumor related gene but also a fertility related gene. This established the primary foundation for further insight into this novel pig gene.