Identification of SNP markers associated with antler growth rate in sika deer (Cervus nippon)

Abstract

Sika deer (Cervus nippon) constitutes one of the most valuable animal genetic resources in east Asia. The aim of this study was to identify and validate single nucleotide polymorphisms (SNPs) from antler growth-related genes of sika deer. The whole genome sequencing data of sika deer were used to identify SNP markers. Among them, 31 SNPs from antler growth-related genes exhibited significant polymorphism using genotyping by mass spectrometry. The observed and expected heterozygosities were ranged from 0.147 to 0.997 and 0.201 to 0.500, respectively. Significant deviation from the Hardy-Weinberg equilibrium was observed in 6 loci. These findings provide effective molecular detection markers for the study of variation in antler growth rate of sika deer.

Background

Sika deer (Cervus nippon) were once widely distributed in eastern Asian countries, and this species is one of the most commercially valuable species for production of high value velvet antler for medicinal use (Hu et al. 2019). There are large herds of farmed animals kept mainly for production of velvet antlers where the antler growth rate is the key factor that determines the profitability of deer farming. Therefore, the identification of molecular markers associated with antler growth-related genes offers potential for application in the selection of sika deer (especially females who do not grow antlers) for artificial breeding.

Single nucleotide polymorphisms (SNPs) have been widely used for investigating genetic diversity, species identification and for exploring potential applications in breeding for deer (Ba et al. 2017), cattle (Zhang et al. 2019) and goats (Ee et al. 2019). Using the genome sequencing data of the sika deer individuals with fast and slow growing velvet antler status (Genbank accession number：PRJNA541418), we have obtained a large number of SNPs.

We utilized the mass spectrometry SNP detection technology combined with multiplex PCR, single base extension and Matrix-Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry (MALDI-TOF-MS) to identify SNP variation in individual deer. This technology offers advantages as up to 40-fold multiplex PCR reactions and genotype detection can be run at one time, and can be used simultaneously on thousands of samples at numerous SNP sites, with an accuracy of up to 98% (Colombo et al. 2019; Jiménez-Sousa et al. 2019).

The genomic and transcriptomic sequencing data of fast growing velvet antlers compared with slow growing velvet antlers were analyzed and antler growth-related genes were identified. The gene sequence containing SNP sites was extracted from the sequencing data of our sika deer genome, and a total of 31 SNPs were selected. In order to validate the polymorphisms at these SNP loci, PCR reactions and single base extension primers were designed using the software MASARRAY assay design suite v3.1 (Table 1). Genomic DNA was extracted from peripheral venous blood of farmed sika deer (from northern China) using whole blood genome DNA isolation kit (BioTeke Corporation, Beijing, China). The PCR amplification, shrimp alkaline phosphatase (SAP) digestion, and extension reaction were carried out sequentially; the reaction product (9 μl in total) was diluted 3 times, desalinated with resin, and the desalted sample was placed on the sample target and allowed to crystallize naturally. Mass spectrometry detection was carried out and data collected. The original results were sorted out and detection rate of each SNP loci was calculated. Population genetic indexes including expected heterozygosity (He), observed heterozygosity (Ho), Hardy-Weinberg equilibrium (HWE) and Minor Allele Frequency (MAF) were calculated using Haploview 4.1 software.

Samples from a total of 384 farmed sika deer were analyzed. The detection rate for the 31 SNP loci was ranged from 83% to 100%. There was a high degree of polymorphism among the 31 loci and the MAF was ranged from 0.113 to 0.499. The He and the Ho were ranged from 0.201 to 0.500 and from 0.147 to 0.997, respectively. Significant deviation from Hardy-Weinberg equilibrium was observed in 6 loci (Table 2).

Our findings provide the basis for the investigation of the potential for the application of molecular markers in the selection of sika deer for breeding.

Declarations

Acknowledgements

This work was supported by the Scientific Research Starting Foundation, Changchun Sci-Tech University, China. We wish to thank Dr Peter Fennessy of AbacusBio Limited in New Zealand for his kind help in reviewing the manuscript.

References

1. Ba H, Jia B, Wang G, Yang Y, Kedem G, Li C (2017) Genome-Wide SNP Discovery and Analysis of Genetic Diversity in Farmed Sika Deer (Cervus nippon) in Northeast China Using Double-Digest Restriction Site-Associated DNA Sequencing G3 (Bethesda) 7:3169–3176 doi:10.1534/g3.117.300082
2. Colombo C et al. (2019) Impact of Mutation Density and Heterogeneity on Papillary Thyroid Cancer Clinical Features and Remission Probability Thyroid : official journal of the American Thyroid Association 29:237–251 doi:10.1089/thy.2018.0339
3. Ee G-X, Zhou DK, Yang BG, Duan XH, Na RS, Han YG, Zeng Y (2019) Association analysis of sixty-seven single nucleotide polymorphisms with litter size in Dazu Black goats Animal Genetics 51 doi:10.1111/age.12879
4. Hu P, Wang T, Liu H, Xu J, Wang L, Zhao P, Xing X (2019) Full-length transcriptome and microRNA sequencing reveal the specific gene-regulation network of velvet antler in sika deer with extremely different velvet antler weight Molecular genetics and genomics : MGG 294:431–443 doi:10.1007/s00438-018-1520-8
5. Jiménez-Sousa MA et al. (2019) VDR rs2228570 Polymorphism Is Related to Non-Progression to AIDS in Antiretroviral Therapy Naïve HIV-Infected Patients Journal of Clinical Medicine 8:311
6. Zhang F et al. (2019) Genome-Wide SNPs and InDels Characteristics of Three Chinese Cattle Breeds Animals 9 doi:10.3390/ani9090596

Tables

Table 1 Primer sequence and product length used in SNPs verification

Table 2 Summary of the 31 SNP markers in the farmed sika deer