Study On SSR And SNP Of Blue Fox Based On Next Generation Sequencing And Its Genetic Identification

The blue fox(Vulpes lagopus)is one of the rare furry animals widely reared in many countries in the world.Accurate genealogical records are particularly important for the determination of the descent of blue foxes and the application of BLUP method in animal model,which is the key to the successful breeding of excellent blue foxes.With the popularization of artificial insemination technology,blue foxes generally adopt the mating method of two or three times to improve the success rate of female foxes.Therefore,it is difficult to determine the paternal parent of young foxes,which leads to a certain error rate in pedigree records.Inbreeding is often caused by the disorder,lack of pedigree management and breeding registration or unscientific genetic management,and the introduced provenances have deteriorated seriously after several generations of reproduction and production,so the breeders have to continue to introduce new provenances from abroad,which makes them suffer huge economic losses.DNA molecular markers have widely used in individual identification and paternity identification,among which SSR markers and SNP markers were the most common.At present,the whole genome sequence information of blue fox is unknown,and the traditional methods for mining SSR and SNP markers in blue fox are inefficient.With the rapid development of second-generation sequencing technology,the ways of mining molecular markers have become diversified.In order to find more effective SSR and SNP markers for parental identification of blue foxes,this study took blue foxes from three breeding farms in the Greater Hinggan Mountains as the research subjects,adopted simplified genome sequencing and transcriptome sequencing strategies,and excavated blue foxes SSR and SNP at genomic DNA level and transcriptional level respectively.The main results are as follows:(1)The simplified genome sequencing of genomic DNA of 5 male and 5 female blue foxes resulted in a total of 35.83 GB of high quality sequence data,with an average of 3.56 GB per sample.The average comparison rate between the tested individuals and the reference genome was 70.28%.Using MISA to detect SSR,the total number of detected sequences was 13734842741 bp,the total number of detected sequences was 56529385,and the total number of consensus SSR was 6430271,among which,the SSR with single base repetition was 2661184,and the SSR with two base repetition units was 2357013.There were 268067 SSRs with three base repeating units,420418 SSRs with four base repeating units,135377 SSRs with five base repeating units,and 36183 SSRs with six base repeating units.BCFtools was used to detect SNPs,and the conventional filtering standards were used to filter SNP sites.The 10 samples were tested for SNP polymorphism,and a total of 13559700 SNP sites were detected,including 7065365 non-deletion sites and 6494335 deletion sites,with a proportion of 47.89%.The number of homozygous and heterozygous SNPs detected was 5266232 and 1799133,and the heterozygous rate was 21.67%.SNP of conversion type was 2234596,SNP of inversion type was 1361332,and the average conversion/inversion ratio was1.67.BCFtools was used to detect InDel,and 104246 InDel were detected,with an average InDel miss rate of 23.78% and an average InDel heterozygosity rate of 12.67%.(2)The RNA transcriptome of bone marrow of 9 male blue foxes was sequenced,and a total of 71.68 Gb Clean Data was obtained,and 99293 Unigene were assembled.A total of 28134 SSR were obtained by using MISA to analyze the SSR of Unigene sequences above 1kb,including 16942 SSR with single base repetition,4371 SSR with double base repetition,2573 SSR with three base repetition,642 SSR with four base repetition,202 SSR with five base repetition and 120 SSR with six base repetition.By comparing the Reads and Unigene sequences of each sample with STAR,1284989 SNP markers were obtained,including 886516 homozygous SNP markers and 398473 heterozygous SNP markers.(3)Some of the SSR and SNP markers in the simplified genome and transcriptome were screened,and 11 polymorphism SSR markers and 5 polymorphism SNP markers were obtained.Five SSR markers and three SNP markers were selected to identify the male parent of a male foxes with unclear parental information in Huangbin breeding farm,and the male parent of this male foxes was identified from six candidate males.In conclusion,simplified genome sequencing and transcriptome sequencing strategies can effectively mine SSR and SNP markers in blue foxes.In the process of mining the transcriptome SSR and SNP of blue fox,it is more appropriate to use the genome of domestic dog with clear annotated information as a reference.The SSR and SNP markers excavated in this study can effectively identify the parental relationships of blue foxes.