Application Of Combining Conventional PCR And CNVplex Technology In Detecting CNVs Of Chicken

A large number of mutations have appeared in the genome of chicken along the long-time evolution, which provide a guideline on studying gene function and biological mechanisms of chicken. Some of the traits, such as chicken meat quality, egg quality, plumage color, comb and beak color are always the studying hotspots of the researchers. CNVs refer to a genomic segment of at least 50 bp that differs in copy number based on the comparison with the reference genomes. Compared with SNP, CNVs contain more genetic information which make it much more possible to affect the individual phenotype through the alteration on its expression level of genes.The third generation of Hubei Xinhua chicken were used as experimental materials in our study. Firstly we used CNVplex technology to detect the copy number of the 17 candidate CNVs which we found through previous studies. And then the conventional PCR was used to correct the copy number, with the purpose of finding a method to detect chicken CNVs which combines conventional PCR and CNVplex technology. Finally, we analyzed the date to understand the relationship between CNV with chicken egg traits. The present research is important for gaining an insight into the molecular basis of breeding and lying the groundwork for subsequent research. The main research was as follows:(1)Firstly we screened 17 high reliability CNVs through previous studies, and then detected the copy number of those 17 candidate CNVs. Secondly we selected CNVs with variant copy number. The results revealed that probes of 02 A, 05 A, 14 A, 14 B showed remarkable variations in copy number representatively.(2)Detecting the SNP locus of those four probes and assessing the impact of the SNP on CNVplex technology. The results showed that three SNPs(G>A、C>T、T>A) existed in 02 A probe sequence, two SNPs(A>T、C>T)existed in 05 A probe sequence, and these SNP locus with the same sequence were linkage inheritance in the experimental chickens. There were no SNPs in 14 A and 14 B probe sequences.(3)In order to correct the decimal copy number to an integer copy number, primers were designed to include 02A、05A、14A or 14 B probe sequences. The results showed that the samples whose copy number were 0(including copy number closed to 0) which detected by 02 A and 05 A probe could get the objective strap; Meanwhile, the sample whose copy number were 0(including copy number closed to 0) which detected by 14 A and 14 B probes could not get the objective strap.(4)We designed a pair of primers every 1kb around the 14 B probe sequence which aimed to find the boundary of 14 BCNV. Finalized we identified that about 6kb in region of chr13: 286368-292611 was missing in Xinhua chickens, and a 1.5kb fragment was found in downstream of 14 BCNV, the fragment was first found in Xinhua chickens.(5)GLM of soft SAS9.2 was used to analyze the association between copy number with egg traits. The results showed that two kinds of copy number variation of 14 ACNV, uneven copy number group(1、3、5) and even copy number group(0、2、4)were association with egg shell thickness and yolk color(P<0.05). Six kinds of copy number variation including 0、1、2、3、4、5 of 14 BCNV were highly significant correlation with age at first egg(P<0.01). Age at first egg 127.1 day of Samples whose copy number were 5 were significantly earlier than age at first egg 131.9 day whose copy number were 0.