Molecular cloning,sequence characterization,polymorphism and association analysis of porcine ROPN1 gene

The full-length cDNA sequence of one porcine gene, ROPN1, was isolated using the rapid amplification of cDNA ends (RACE) method based on one pig EST sequence which was highly homologous to the coding sequence of human ROPN1 gene. The porcine ROPN1 gene encodes a protein of 212 amino acids which shares high homology with the rhophilin associated protein 1 (ROPN1) of eight species: gray short-tailed opossum (96%), horse (95%), cattle (94%), mouse (93%), rat (92%), chimpanzee (85%), human (85%) and rhesus monkey (85%). Phylogenetic analysis revealed that the porcine ROPN1 gene has a closer genetic relationship with the ROPN1 gene of gray short-tailed opossum. Polymorphism analysis showed that there was a T/C mutation at the position of 536 bp of mRNA and this leaded to the amino acid alteration from the Arg residue to the Cys residue. PCR-Hae III-RFLP was established to detect this T/C mutation and eight pig breeds display obvious genotype and allele frequency differences at this mutation locus. Association of this SNP with litter size traits was assessed in Large White (n = 100) and Landrace (n = 100) pig populations, and results demonstrated that this polymorphic locus was significantly associated with the litter size of first parity (P < 0.01) and all parities (P < 0.05) in Large White sows, and also significantly associated with the litter size of all parities in Landrace sows (P < 0.01). Therefore, ROPN1 gene could be a useful candidate gene in selection for increasing litter size in pigs. These data serve as a foundation for further insight into this novel porcine gene.     

Molecular characterization, polymorphism and association of porcine SPATA19 gene

Spermatogenesis associated 19 (SPATA19) is an important reproduction related gene. In this study, we cloned the full-length cDNA sequence of porcine SPATA19 gene through the rapid amplification of cDNA ends (RACE) method. The porcine SPATA19 gene encodes a protein of 154 amino acids which shares high homology with the SPATA19 of ten species: giant panda (87?%), dog (86?%), cattle (84?%), rabbit (78?%), sumatran orangutan (72?%), human (71?%), rhesus monkey (71?%), chimpanzee (70?%), mouse (71?%) and rat (69?%). The phylogenetic analysis revealed that the porcine SPATA19 gene has a closer genetic relationship with the SPATA19 gene of dog. This gene is structured in six exons and five introns as revealed by computer-assisted analysis. PCR-RFLP was established to detect the GU475012:c.515T>C substitution of porcine SPATA19 gene mRNA and association of this mutation with litter size traits was assessed in Large White (n?=?100) and Landrace (n?=?100) pig populations. Results demonstrated that this polymorphic locus was significantly associated with the litter size of all parities in Large White sows and Landrace sows. Therefore, SPATA19 gene could be an useful candidate gene in selection for increasing litter size in pigs. These data serve as a foundation for further insight into this novel porcine gene.     

Molecular Cloning of Porcine SUN5 Gene and Association between a SNP with Litter Size Trait

SUN domain-containing protein 5 (SUN5) is an important reproduction related gene. In this study, we cloned the full-length coding sequence of porcine SUN5 gene through RT-PCR. Sequence analysis of this gene revealed that the pig SUN5 gene encodes a protein of 383?amino acids that has high homology with the SUN5 protein of eight species: wild Bactrian camel (95%), alpaca (95%), Yangtze River dolphin (94%), sperm whale (94%), sheep (93%), black flying fox (93%), goat (92%), and horse (91%). This gene is structured into 13 exons and 12 introns as revealed by computer-assisted analysis. The prediction of transmembrane helices showed that pig SUN5 protein might be a transmembrane protein. PCR-Taq I-RFLP was established to detect the GU475008:c.138 G>A substitution of porcine SUN5 gene coding sequence and eight pig breeds displayed obvious genotype and allele frequency differences at this mutation locus. Association of this SNP with litter size traits was assessed in Large White (n?=?200) and Landrace (n?=?200) pig populations, and the results demonstrated that this polymorphic locus was significantly associated with the litter size of all parities in Large White and Landrace sows (P?相似文献   

Molecular characterization, polymorphism and association of porcine MYST2 gene

MYST histone acetyltransferase 2 (MYST2) is an important reproduction related gene. In this study, we cloned the full-length cDNA sequence of porcine MYST2 gene through the rapid amplification of cDNA ends method. The porcine MYST2 gene encodes a protein of 611 amino acids which shares high homology with the MYST2 of six species: cattle (99%), rabbit (99%), human (99%), rat (99%), mouse (99%) and chicken (98%).The open reading frame of this gene is structured in 15 exons and 14 introns as revealed by computer-assisted analysis. The phylogenetic analysis revealed that the porcine MYST2 gene has a closer genetic distance with the MYST2 gene of cattle. PCR-RFLP was established to detect the GU373686:c.2872G > A substitution of porcine MYST2 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 in Large White sows and Landrace sows. These data serve as a foundation for further insight into this porcine gene.     

Molecular Characterization,Polymorphism, and Association of Porcine GADD45G Gene

Growth arrest and DNA-damage-inducible gamma (GADD45G) is a reproduction related gene. In this study, the full-length cDNA sequence of porcine GADD45G gene was cloned through rapid amplification of cDNA ends (RACE) method. The porcine GADD45G gene encodes a protein of 159 amino acids that shares high homology with the GADD45G of nine species: chimpanzee (97%), sumatran orangutan (97%), white-tufted-ear marmoset (97%), northern white-cheeked gibbon (97%), cattle (97%), human (97%), rhesus monkey (97%), rat (96%), and mouse (95%). This novel porcine gene was assigned to GeneID: 100152997. Phylogenetic analysis revealed that the porcine GADD45G gene has a closer genetic relationship with the GADD45G gene of cattle. Computer-assisted analysis indicated that porcine GADD45G gene is structured in four exons and three introns. PCR-Rsa I-RFLP was established to detect an A/G mutation on the position of 294-bp of coding sequence and eight pig breeds display obvious genotype and allele frequency differences at this mutation locus. Association of this SNP with litter size traits was assessed in Large White (n = 100) and Landrace (n = 100) pig populations, and result demonstrated that this polymorphic locus was significantly associated with the litter size of all parities in Large White and Landrace sows (P < 0.01). Therefore, porcine GADD45G gene could be a useful candidate gene in selection for increasing the litter size. These data serve as a foundation for further insight into this novel porcine gene.     

A Colon Cancer Related Gene-MLH1, Significantly Affecting the Pig Litter Size

MutL homolog 1, colon cancer, nonpolyposis type 2 (MLH1) is a tumor related gene. In this study, the full-length cDNA sequence of porcine MLH1 gene was cloned through rapid amplification of cDNA ends (RACE) method. The porcine MLH1 gene encodes a protein of 757 amino acids which shares high homology with the MLH1 of five species: chiru (96%), sheep (95%), cattle (93%), southern-white-rhinoceros (92%) and goat (92%). This novel porcine gene was assigned to GeneID: 100337665. Phylogenetic analysis revealed that the porcineMLH1 gene has a closer genetic relationship with theMLH1 gene of southern- white-rhinoceros. PCR-HhaI-RFLP was established to detect GU373696:c.395 G > A substitution of porcine MLH1 gene and eight pig breeds display obvious genotype and allele frequency differences at this mutation locus. Association of this SNP with litter size traits was assessed in Large White (n = 200) and Landrace (n = 200) pig populations, and result demonstrated that this polymorphic locus was significantly associated with the litter size of all parities in Large White and Landrace sows (P < 0.01). Therefore, MLH1 is also a reproduction related gene.     

Effect of polymorphism in the leukemia inhibitory factor gene on litter size in Large White pigs

DNA polymorphism of the porcine leukemia inhibitory factory (LIF) was investigated and used to study the effects on litter size in Large White pigs. A total of 2,167 litter records from 420 sows genotyped at two SNP loci (LIF1 and LIF2) within LIF gene were analyzed to determine whether LIF influenced total number born (TNB) and number born alive (NBA). The results indicated that B allele at LIF1 locus and A allele at LIF2 locus seem to have advantageous effects on litter size. However, the combined analyzed results demonstrated that genotype AAAA, ABBB, and BBBB are better than genotype AAAB, AABB, and ABAB for TNB and NBA in either third to eighth parity or all parities. In all parities, the sows with AAAA genotype had an advantage of 1.76 piglets (P < 0.001) for TNB and 1.44 piglets (P < 0.01) for NBA per litter over the AAAB sows, respectively. The results in this study demonstrated that LIF gene was significantly associated with litter size in pigs. H. C. Lin and G. F. Liu contributed equally to this work.     

Effect of VNTR polymorphism of the Muc1 gene on litter size of pigs

An investigation was undertaken to study the association between the variable number of tandem repeats polymorphism of the Muc1 gene and the litter size in pigs. Four different alleles were found in three breeds. The sequence analysis shows that the repetitive region of pig Muc1 gene is an array of 108-bp repeats. A total of 2,430 litter records from 897 sows genotyped at Muc1 gene were used to analyze the total number born (TNB) and number born alive (NBA). The study of the effects on litter size suggests that TNB and NBA of genotype AA are the highest in Large White, and the TNB and NBA of the third to ninth parities are 1.61 and 2.29 piglets per litter higher (P < 0.05) than those of the genotype DD, respectively. In Landrace, TNB and NBA of the genotype AA are 1.68 (P < 0.01) and 1.58 (P < 0.05) piglets per litter higher than those of the BB genotype in the third to ninth parities, but for all parities the TNB of genotype AA were 0.76 piglets per litter (P < 0.05) higher than BB. In Duroc, the TNB and NBA of genotype AA are about 1.5 piglets per litter more than those of DD in the third to ninth parities, though not significantly. The research suggests that the smaller allele tends to have higher litter size. The results indicate that Muc1 gene is significantly associated with litter size in pigs.     

Identification of polymorphism and association analysis with reproductive traits in the porcine RNF4 gene

The ring finger protein 4 gene (RNF4), which might play a role in fetal germ cell development as well as in oocyte and granulosa cell maturation, was one of the potential candidate genes for reproductive traits. In the present work, we isolated the complete coding sequence of porcine RNF4 gene, identified a single nucleotide polymorphism (SNP: T/C) in intron5, and developed a PCR-SacII-RFLP genotyping assay. Association of this SNP with reproductive traits was assessed in three populations with diverse genetic backgrounds. One was Chinese Qingping sows. Another was consisted of crossbred sows derived from Landrace, Large White, Chinese Tongcheng and/or Chinese Meishan (Line DIV). The third is Large White × Meishan (LW × M) F₂ slaughtered population. Statistical analysis demonstrated that, in the first parity, the difference between RNF4 genotypes and reproductive traits of both Qingping and Line DIV sows was not significant. In the second and subsequent litters, CC animals in Qingping population had more piglets born (+1.74 piglets) and piglets born alive (+2.02 piglets) than sows with the TT genotype (P < 0.05). Line DIV sows inheriting the CC genotype had additional 0.69 piglets born compared to the TC animals (P < 0.05) in second and subsequent litters. No significant difference was observed between genotypes and reproductive tracts components in F₂ animals. In addition, we found RNF4 gene has a significant additive effect on both piglet born and piglet born alive in Qingping animals (P < 0.05). Results here suggested that the RNF4 SNP was significantly associated with litter size in two populations and could be useful in selection for increasing litter size in pigs. Further studies were needed to confirm these preliminary researches.     

Effect of polymorphism in the peroxisome proliferator-activated receptor gamma gene on litter size of pigs

The association of polymorphisms in peroxisome proliferator-activated receptor γ (PPARγ) gene with litter size was studied in Large White and Landrace pig. Three SNP loci (P1, P2 and P7) on PPARγ₂ gene were determined by PCR–SSCP and the results showed that there were A → G mutations at 220 and 324 bp in 5′-regulator region and at 147 bp in exon 6, respectively. Allele frequencies were analysed in two breeds. Information on 2341 litter records from 564 sows was used to analyse the trait total number born (TNB) and number born alive (NBA). In Large White, TNB and NBA of genotype BB for P2 locus were the lowest, and the TNB and NBA of third and following parities and all parities were 0.74 and 0.51 piglets per litter less (P < 0.001) than those of the highest genotype AB, respectively, but for P1 and P7 locus the beneficial genotype AA were more 0.4–0.8 piglets per litter (P < 0.05) than the inferior genotype AB. In landrace, TNB and NBA of the first parity of genotype BB for P1 locus were 2.0 piglets per litter higher than AA (P < 0.05), but for all parities the TNB and NBA of genotype BB were 0.66 and 0.97 piglets per litter (P < 0.05) higher than AA, respectively. At P2 locus, the TNB and NBA of the second parity of genotype AA were obviously higher than those of AB (P < 0.05). And at P7 locus, the TNB and NBA of each parity of genotype AA were both about 2 piglets per litter more than those of BB (P < 0.05). The results indicated that PPARγ gene was significantly associated with litter size in pigs.     

SLA-11 mutations are associated with litter size traits in Large White and Chinese DIV pigs

Litter size is an important economic traits in pigs. SLA-11 gene is a member of SLA (swine leukocyte antigen) complex. In our previous study, the SLA-11 gene was differentially expressed in PMSG-hCG stimulated preovulatory ovarian follicles of Chinese Taihu and Large White sows. Here, we identified two mutations (c.754-132 T?>?C and c.1421?+?38 T?>?C) in SLA-11 gene and analyzed the associations of two SNPs with litter size traits in Large White (n?=?263) and DIV (n?=?117) sows. The results showed that in Large White pigs, SLA-11 c.754-132?CC sows produced 0.74 and 0.87 more pigs per litter for TNB and NBA of all parities than did TT sows (p?<?.05); In DIV pigs, SLA-11 c.754-132?CC sows produced 1.17 more pigs per litter for TNB of all parities than did TC sows (p?<?.05). In Large White pigs, SLA-11 c.1421?+?38?CC sows produced 0.9 more pigs per litter for TNB of all parities than did TT sows (p?<?.05), while in DIV pigs SLA-11 c.1421?+?38?CC sows produced 0.84 and 0.7 less pigs per litter for TNB and NBA of all parities than did TT sows (p?<?.05). Our research indicated that SLA-11 mutations were potential molecular markers for improving the litter size traits in pigs.     

Polymorphism of the pig 17beta-hydroxysteroid dehydrogenase type1 (HSD17B1) gene and its association with reproductive traits

17beta-Hydroxysteroid dehydrogenase type 1 (HSD17B1) is a key enzyme of 17beta-estradiol biosynthesis, which might play an important role in follicular development of the ovary. In this study, we isolated the complete coding sequence of porcine HSD17B1 gene and its unique intron sequences of porcine HSD17B1 gene, identified a single nucleotide polymorphism (SNP: A/C) in intron 4, and developed a PCR-MvaI-RFLP genotyping assay. Association of the SNP and litter size was assessed in two populations (purebred Large White and a experimental synthetic Line (DIV) sows). Statistical analysis demonstrated that, in the first parity, AC animals in experimental synthetic Line (DIV) sows had 0.52 more piglets born compared to the CC animals (P<0.05). In the all parities, pigs with the AA genotype had an additional 1.11 and 0.96 piglets born alive compared to the CC animals (P<0.05) in both experimental synthetic Line (DIV) and purebred Large White, respectively. Experimental synthetic Line (DIV) sows inheriting the AC genotype had additional 0.84 piglets born alive compared to the CC animals (P<0.01) in all parities. In addition, significant additive effect of -0.55+/-0.24 piglets/litter and -0.48+/-0.22 piglets/litter on piglet born alive was detected in both experimental synthetic Line (DIV) sows and purebred Large White lines (P<0.05), respectively. Therefore, HSD17B1 gene was significantly associated with litter size in two populations and could be a useful molecular marker in selection for increasing litter size in pigs.     

FSHβ subunit gene is associated with major gene controlling litter size in commercial pig breeds

An insertion fragment in porcine FSHβ subunit gene was cloned by PCR. Sequencing data show that the insertion is a retroposon of 292 bp siting in intronⅠ at the site between +809 and +810 base. Based on these results, a PCR programme was created to genotype animal individuals in different pig breeds at FSHβ locus and polymorphism of FSHβ gene was analyzed. With the combination of genotype and litter size of sows, it was demonstrated that FSHβ locus is closely associated with major gene controlling litter size in commercial pig breeds, such as Yorkshire, Landrace, Durco. Averagely the AA sows give more 1.5 piglets than BB sows do per litter.     

FSHB subunit gene is associated with major gene controlling litter size in commercial pig breeds

An insertion fragment in porcine FSHβ subunit gene was cloned by PCR. Sequencing data show that the insertion is a retroposon of 292 bp siting in intron I at the site between + 809 and +810 base. Based on these results, a PCR programme was created to genotype animal individuals in different pig breeds at FSW locus and polymorphism of FSHP gene was analyzed. With the combination of genotype and litter size of sows, it was demonstrated that FSHβ locus is closely associated with major gene controlling litter size in commercial pig breeds, such as Yorkshire, Landrace, Durco. Averagely the AA sows give more 1.5 piglets than BB sows do per litter.     

The influence of ovulation rate on subsequent litter size in sows

Data were collected from 318 pregnant sows (Large White, Landrace or crossbred), which had been weaned from their first litter, to determine the relationship between ovulation rate (OR) and subsequent litter size (total born alive - TBA). Laparoscopy was performed on sows 7 to 14 days after mating to determine OR, and subsequent litter size (TBA) was recorded. Mean (+/-SE) ovulation rate, litter size (TBA) and foetal survival (FS) was 14.56+/-0.19, 9.00 +/- 0.16 and 63.5+/-1.1%, respectively. Regressions of TBA against OR and FS against OR were best fitted by a linear function which indicated that litter size of first-litter sows was limited by ovulation rate up to at least 23 ova. The respective linear regressions were:     

A SNP in the miR-27a gene is associated with litter size in pigs

MicroRNAs (miRNAs) are 20–25 nt, endogenous non-coding RNA molecules that act by binding to the complementary sequence of target messenger RNAs. Many evidences showed that miRNAs were involved in the process of germ proliferation and differentiation. In the present study, miR-27a gene was selected as a candidate gene for litter size due to its biological function, its location near a mummified pigs QTL, and its differentially expressed profile in Large White and Chinese Erhualian PMSG-hCG stimulated preovulatory ovaries. By comparative sequencing of miR-27a gene in Large White and Chinese Meishan pigs, one SNP (T/C) which created an additional HpaII site was detected. Then associations of this SNP with litter size traits were assessed in Large White (n = 142) and DIV (n = 140) pig populations. The statistical analysis demonstrated that AA differed from AB (P < 0.01) and BB (P < 0.05) for total number of piglets born in the first parities, and also differed from AB (P < 0.01) for the number of piglets born alive in all parities (P < 0.05) in DIV pigs. No significant difference was observed between different genotypes in Large White pigs.     

猪整合素β1基因CRS-PCR多态性与产仔数的关联性分析

文章采用CRS-RFLP技术对长白猪、大白猪和杜洛克猪3个品种的整合素β1基因第5外显子T32207C位点及第7外显子A35230G位点进行单核苷酸多态性分析,并将基因多态性与猪的产仔数进行关联分析。结果表明:32207多态位点的基因型效应对3个品种的总产仔数(TNB)和产活仔数(NBA)影响均不显著;35230多态位点的基因型效应对大白猪和长白猪头胎、二胎及所有胎次的TNB和NBA的影响达到显著(P<0.05)或者极显著水平(P<0.01),基因型GG、AG与AA对产仔数的影响存在差异,其效应为GG,AG>AA。可见整合素β1基因35230位点的G等位基因对大白猪和长白猪的产仔数性状有显著影响。