Polymorphism Of MHC-DRB Gene And Toll-like Receptor 4 Gene In Shinisaurus Crocodilurus Bred In Captivity And In The Wild In LuoKeng Nature Reserve

The crocodile lizard(Shinisaurus crocodilurus)is a first-class protected animal in China.The population of S.crocodilurus in China is rapidly decreasing due to habitat fragmentation,disease outbreak and human killing.However,with the increasing awareness of conservation in China,the population of S.crocodilurus has also picked up.For some endangered wild animals,artificial breeding has become an important way to protect more species.Our previous studies showed that the swimming ability of the artificial breeding S.crocodilurus was significantly different from that of the wild,and the phenomenon of bacterial infection of S.crocodilurus in the artificial breeding environment was significantly increased.To study the difference of immune ability between artificial breeding and wild S.crocodilurus can further understand the influence of artificial breeding on S.crocodilurus,and also provide reliable opinions and suggestions for the artificial breeding and wild release of S.crocodilurus in China in the future.Studies on reptile immune genes mainly focus on Toll like receptor and major histocompatibility complex.At present,a large number of experimental studies have proved that TLR4 gene has important immune function in reptiles.MHC gene family has a rich research history,and also has a certain research in reptiles.Previous studies have shown that the polymorphisms of TLR4 and MHC genes in different geographical populations of S.crocodilurus are low,but there is no comparison between artificial breeding and wild S.crocodilurus.In order to fill the gap and fully understand the influence of artificial breeding on the immune ability of S.crocodilurus,we chose the artificial breeding and wild S.crocodilurus in Luokeng nature reserve of Guangdong Province to study.We selected the DRB gene of MHC class II family to study,and obtained 285 bp MHC-DRB exon3 gene sequence and 270 bp MHC-DRB Exon2 gene sequence.We found a single nucleotide polymorphism(SNP)in the exon3 gene sequence of 20 artificially bred S.crocodilurus and a single nucleotide polymorphism in the exon3 gene sequence of 27 wild S.crocodilurus.We found no single nucleotide variation in Exon2 gene sequence in both artificial breeding and wild S.crocodilurus.It was found that both wild and artificially bred S.crocodilurus showed low single nucleotide polymorphisms,and there was no significant difference between them.By comparing the exon3 gene sequences and calculating the genetic diversity index,we found that there were three polymorphic gene loci and three haplotypes.The haplotype diversity index was 0.353,the nucleotide diversity index PI was 0.00192,and the average nucleotide difference K was 0.537,Tajima's D was-0.975.The results showed that there were 5 polymorphic loci and 4 haplotypes.The haplotype diversity index was 0.430,the nucleotide diversity index PI was 0.00312,the average nucleotide difference K was 0.940,and the Tajima's D value was-0.772.Although the MHC-DRB gene polymorphisms of both artificial and wild S.crocodilurus were low,the MHC-DRB gene polymorphisms of wild S.crocodilurus were slightly higher than those of artificial S.crocodilurus.From the point of view of average nucleotide difference,the average nucleotide difference of wild S.crocodilurus is also higher than that of artificially bred S.crocodilurus.The results showed that the MHC-DRB exon3 gene polymorphism of wild S.crocodilurus was slightly better than that of artificially bred S.crocodilurus.The results of TLR4 gene analysis showed that there were 9 SNPs shared by artificial breeding and wild S.crocodilurus,and 2 snp8 and snp11 were unique to artificial breeding S.crocodilurus.Among the common SNPs,the number of SNPs in wild S.crocodilurus was slightly more than that in artificially bred S.crocodilurus.Only snp4 had slightly more SNPs in artificially bred S.crocodilurus than that in wild.By comparing the gene sequence variation sites and genetic diversity index of artificial breeding and wild S.crocodilurus,we found that there were 11 gene sequence variation sites in 28 artificial breeding S.crocodilurus gene sequences,with nucleotide diversity index PI = 0.00273 and Tajima's D value of 1.97104.There were 10 variation sites in 29 wild S.crocodilurus gene sequences,with pi = 0.00234 and Tajima's d =1.72912.In general,the genetic diversity of artificially bred S.crocodilurus was slightly higher than that of wild,but the difference was not significant.However,there is a unique SNP phenomenon in the artificial breeding of S.crocodilurus,which indicates that the artificial breeding of S.crocodilurus is facing a new pathogen invasion.Studies on artificial breeding and wild S.crocodilurus show that the genetic diversity of S.crocodilurus population is low on MHC-DRB gene and toll like receptor 4 gene.Although wild S.crocodilurus may be slightly better than artificial,its overall disease resistance is not optimistic.At the same time,artificial breeding also brings new pathogenic pressure to S.crocodilurus.Low disease resistance is not conducive to the survival of the S.crocodilurus,which will significantly affect the survival rate of the rejuvenated population.In the future,the artificial breeding work needs to focus on improving the immune ability of the S.crocodilurus to ensure the recovery of the S.crocodilurus population.