| The geese have strong broodiness and poor egg performance.The egg-laying performance is a very important economic trait,because of its influences in the development and economic benefit of geese production.Laying performance is of low heritability,which is impacted by many factors.As the heritability of reproduction is low,it is hard to improve reproductive traits using traditional selection methods.Marker-assisted selection（MAS）is an effective way to improve such traits with low heritability.MAS can accelerate the genetic progress of target trait in goose breeding.In order to identify the molecular genetic markers of egg-laying in geese,we developed a pooled RAD sequencing strategy to detect geese laying-related SNPs that exhibited distinct allele distributions in high egg production and low egg production cohorts.These SNPs were further genotyped in a goose population to verify the association with egg numbers.Novel genes harboring laying-related SNPs were cloned for the further study in geese.Finally,two promising laying-related SNPs were applied in goose breeding.The main results obtained were as follows:1.Identification of laying-related SNP markers in geese using RAD sequencingA total of 492 female Yangzhou geese from the breeding farm of Jiangsu Lihua Animal Husbandry CO.,LTD were employed in this study.Three higher egg-laying groups（HIPV,HEBV and HF）and three lower egg-laying groups（LIPV,LEBV and LF）were designed by individual selection,individual estimated breeding values selection and within-family selection.In HIPV and LIPV groups,RAD sequencing generated 3.8 Gb of data containing more than 42.29 million single-end reads,The number of RAD tags per group is 893,579 and 992,670 for HIPV and LIPV group respectively.After the filtering steps,a total of 96,848 SNPs were detected.In HEBV and LEBV groups,RAD sequencing generated 4.0 Gb of data containing more than 43.55 million single-end reads.The number of RAD tags per group is 884,827 and 942,117 for LEBV and HEBV group,respectively.After the filtering steps,a total of 139,013 SNPs were detected.In HF and LF groups,RAD sequencing generated 4.4 Gb of data containing more than 47.95 million single-end reads.The number of RAD tags per group is 898,219 and 953,964 for LEBV and HEBV group,respectively.After the filtering steps,a total of 95,889 SNPs were detected.The chi square test of independence was used to test the difference of allelic frequencies of RAD-tags between HIPV and LIPV,HEBV and LEBV,HF and LF DNA pools,respectively.After Bonferroni correction,25 SNP were significant between HIPV and LIPV(P<5.2×10-6).467 SNP were significant between HEBV and LEBV(P<3.69×10-7),817 SNP were significant between HF and LF(P<5.2×10-6).Based on the SNP F estimates,the genetic difference was evaluated between high egg-laying and lower egg-laying groups,and to compare the effect of three grouping methods for egg-laying in goose.The results showed that the FST of individual selection（0.073）is lower than that of individual estimated breeding values selection（0.093）and within-family selection（0.101）.Compared with the other two grouping methods,the smaller genetic difference was observed between HIPV and LIPV groups.The higher selected accuracy was obtained by individual selection.So we checked the SNPs from individual estimated breeding values selection and within-family selection in the next step.AS-PCR was applied to individual genotyping.The results of further individual-based genotyping showed that 10 out of 55 SNP had significant(P<2.4×10-4.63×10-10)different allelic frequencies in the HEBV（n=10）and LEBV（n=10）cohorts.The results of further individual-based genotyping showed that 5 out of 37 SNPs had significantly(P<1.8×10-8.04×10-10)different allelic frequencies in the HF（n=14）and LF（n=10）cohorts.These 11 of 15 SNPs were further genotyped in a goose population of 492 geese to verify the association with egg numbers.The result showed that 8 of 11 SNPs were associated with egg numbers.Additionally,liner regression analysis revealed that SNP Record-111407,106975 and 112359 were involved in a multiple gene network affecting laying performance.We used IPCR to extend the unknown flanking regions of the candidate RAD tags.The obtained sequences were subjected to BLAST to retrieve the orthologous genes in either ducks or chickens.Five novel geese genes which harbored the candidate laying-related SNPs（Record-106975,134172,112359, 106582 and 111407）were cloned,including membrane associated guanylate kinase（MAGI-1）,KIAA1462,Rho GTPase activating protein 21（ARHGAP21）,acyl-CoA synthetase family member 2（ACSF2）,and astrotactin 2（ASTN2）,respectively.The information of laying-related SNP and gene will give insight into goose breeding.2.Molecular cloning and expression analysis of the ACSF2 gene in geese.The coding region of ACSF2 gene contains 1770 base pairs,and encodes 589 amino acids in geese.The nucleotide sequence of ACSF2 was high homology with those reported in other avian species（duck 88.20%,chicken 76.55%）.Four alternative splice variants were identified in the ovary of geese and were named as ACSF2-1,ACSF2-2,ACSF2-3 and ACSF2-4,ACSF2-1 is the predominant isform.The coding region of ACSF2-2,ACSF2-3 and ACSF2-4 are 692bp,1599 bp and 1917 bp,and they encode 563,532 and 638 amino acids,respectively.Compared with ACSF2-1,the type of ACSF2-2 is alternative 5’ splice site and lose the nucleotide sequences of 46bp on exon 14.ACSF2-3 loss whole exon7,8 and 9,and at the same time,127bp（E14a）was inserted between exon 13 and 14.For ACSF2-4,127bp（E14a）was also inserted between exon 13 and 14.We confirmed that the goose ACSF2 was a mitochondrial matrix protein through subcellular localization.ACSF2-1,ACSF2-3 and ACSF2-4 were ubiquitously expressed in the kidney,ovary,small investine,liver,abdominal fat,muscular stomach,breast musle,heart,hypothalamus,pituitary gland and granulosa cell,but ACSF2-2 was not be expressed in hypothalamus,pituitary gland and granulosa cell.The mRNA of three alternative splice variants（ACSF2-1,ACSF2-3 and ACSF2-4）was detected in granulosa cell of various size ovarian follicles（F1-F5,syf,lwf,swf）.ACSF2-1 was higher expressed in granulosa cell of various size ovarian follicles compared with the other two alternative splice variants（ACSF2-3 and ACSF2-4）.Quantitative real-time PCR was performed to determine the mRNA level of the ACSF2 gene in ovary tissues of high egg production（HEP）and low egg production（LEP）groups.The mRNA expression of ACSF2 in the ovaries of HEP group is lower than that of LEP group（P<0.01）.So did the mRNA expression of the other three alternative splice variants（ACSF2-2,ACSF2-3 and ACSF2-4）（P<0.05）.The expression constructs of three alternative splice variants（ACSF2-1,ACSF2-3 and ACSF2-4）were transiently transfected into the goose granulosa cells.After treatment for 24 h,the mRNA level of Caspase-3 was significantly higher than that of the two control groups（P<0.01）.We tested the efficiency of three sets of ACSF2 siRNAs and finally found that siRNA771 and siRNA1381 could significantly reduce the amount of ACSF2 mRNAs in granulosa cells.The Caspase-3 mRNA level were significantly reduced when the ACSF2 mRNA was knocked down by siRNA771（P<0.05）and siRNA1381（P<0.01）.The mRNA level of ACSF2 in the ovaries of goose was proved correlating with egg-laying performance.So ACSF2 may play a role in apoptosis of granulosa cell by regulating Caspase-3 expression.3.Molecular cloning and expression analysis of the MAGI1 gene in geese.The coding region of MAGI1 gene is 4152bp,and encodes 1383 amino acid protein in geese.The nucleotide sequence was high homology with those reported in other avian species.The sequences identity value of MAGI2 between geese and ducks reached 90.98%.Five alternative splicing were identified in the ovary of geese and were named as MAGI1-1,MAGI1-2,MAGI1-3,MAGI1-4 and MAGI1-5,while MAGI1-1 was the predominant isoform.The coding sequences of MAGI1-2,MAGI1-3,MAGI1-4 and MAGI1-5 are 4116bp,3945bp,4149bp and 2805bp,which encode 1371,1314,1382 and 934 amino acids,respectively.The ovary,abdominal fat and muscular stomach displayed higher expression levels of MAGI1 when compared with kidney,hypothalamus and pituitary.Follicle F2 displayed the highest mRNA levels of MAGI1 when compared with F1,F3,F4 and F5.The same levels of mRNA of MAGI1 were observed in syf,lwf,swf.Quantitative real-time PCR was performed to determine the mRNA levels of the MAGI1 gene in ovary tissues of high egg production（HEP）and low egg production（LEP）groups,and it turned out that it was higher in the HEP group and was lower in the LEP group（P =0.05）.The expression construct of MAGI1 were transiently transfected into the goose granulosa cells.After treatment for 24 h,the mRNA levels of Caspase-3 were significantly higher than that of two control groups（P<0.01）.We tested the efficiency of three sets of siRNAs for MAGI1 and finally found that siRNA507 and siRNA847 could significantly reduce the amount of MAGI1 mRNA,and siRNA847 worked better.The mRNA level of Caspase-3 was significantly increased when the MAGI1 mRNA was knocked down by with siRNA847.The expression of MAGI2 was proved correlating with egg numbers,and it influenced Caspase-3 expression in granulosa cells.These results showed that MAGI1 may play a role in the normal growth of granulosa cell that may regulate the development of follicle.4.Molecular cloning and expression analysis of the PAPPA gene in geese.The translation region of PAP PA gene is 4884bp with 24 exons,and encodes 1627 amino acid protein in geese.The nucleotide sequence was high homology with those reported in other avian species.The sequences identity value of PAPPA in geese and duck reached 98%.The protein subcellular localization prediction showed PAPPA is a secreted protein,and locates in the endoplasmic reticulum and extracellular.PAPPA are ubiquitously expressed in all tested tissues excepted liver,the mRNA level of PAPPA in the ovary is the highest,followed by heart and breast muscle.The pre-hierarchical follicles（syf,lwf,swf）displayed higher levels of expression of PAPPA when compared with hierarchical follicles（F1-5）.The mRNA levels of PAPPA were reduced according to size of hierarchical follicles from F5 to F1.The pattern expression of PAPPA in the follicular granulosa cell further testified the regulation function of PAPPA in the development of follicle.Quantitative real-time PCR was performed to determine the mRNA levels of the PAPPA gene in ovary tissues between high egg production（HEP）and low egg production（LEP）groups.The mRNA expression of PAPPA in the ovaries of HEP group is higher than that of LEP group（P>0.05）.Up-regulation of PAPPA expression in granulosa cell significantly decreased the mRNA level of Caspase-3（P<0.01）.Collectively,PAPPA was correlated with the growth of granule cells,and may play a important role in the development of follicle.5.Molecular marker assisted selection in goose breedingA total of 400 female Yangzhou geese from seventh generation（2014）,and another 596 Yangzhou geese（221 male geese and 375 female geese）from ninth generation（2015）in the breeding farm of Jiangsu Lihua Animal Husbandry CO.,LTD were employed in this study.3 SNP（Record-106582,106975 and 111407）were further genotyped in a goose population of seventh generation to verify the association with egg numbers.The AA and CA genotypes of Record-106582 had significantly higher egg productionthan those with CC genotype（P<0.01）.No significant difference in egg production was observed between the AA and CA genotypes geese（P>0.05）.No significant association between the genotypes of Record-106975 and egg production was found（P>0.05）,but the egg number of GG genotype is 4.4 higher than that of AA（P>0.09）.No significant association between the genotypes of Record-111407 and egg production was found（P>0.05）.Record-106582 and 106975 were identified as molecular marker for egg-laying performance of goose.The estimated laying performance of male geese of seventh generation was calculated according to daughter’s egg numbers.The estimated laying performance was calculated for male geese of eighth generation according to egg number of female geese in seventh generation.The egg number of female geese in first 20 weeks were present on eighth generation.The mate of 39 male geese and 78 female geese of eighth generation with high egg number were selected,respectively,and 596 offspring（211 male geese and 375 female geese）for seed stock was produced in the ninth generation.Two populations of homozygous genotype with high laying performance were grouped by the genotypes of Record-106582 and 106975,including 141（39 male geese and 102 femal geese）and 280（76 male geese and 204 female geese）geese. |
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