Whole-genome de novo sequencing reveals genomic variants associated with differences of sex development in SRY negative pigs

Abstract

Background: Differences of sex development (DSD) are congenital conditions in which chromosomal, gonadal, or phenotypic sex is atypical. In more than 50% of human DSD cases, a molecular diagnosis is not available. In intensively farmed pig populations, the incidence of XX DSD pigs is relatively high, leading to economic losses for pig breeders. Interestingly, in the majority of 38, XX DSD pigs, gonads still develop into testis-like structures or ovotestes despite the absence of the testis-determining gene (SRY). However, the current understanding of the molecular background of XX DSD pigs remains limited.

Methods: Anatomical and histological characteristics of XX DSD pigs were analysed using necropsy and HE staining. We employed whole-genome sequencing (WGS) with 10× Genomics technology and used de novo assembly methodology to study normal female and XX DSD pigs. Finally, the identified variants were validated in 32 XX DSD pigs, and the expression levels of the candidate variants in the gonads of XX DSD pigs were further examined.

Results: XX DSD pigs are characterised by the intersex reproductive organs and the absence of germ cells in the seminiferous tubules of the gonads. We identified 4,950 single-nucleotide polymorphisms (SNPs) from non-synonymous mutations in XX DSD pigs. Cohort validation results highlighted two specific SNPs, "c.218T > C" in the "Interferon-induced transmembrane protein 1 gene (IFITM1)" and "c.1043C > G" in the "Newborn ovary homeobox gene (NOBOX)", which were found exclusively in XX DSD pigs. Moreover, we verified 14 candidate structural variants (SVs) from 1,474 SVs, identifying a 70 bp deletion fragment in intron 5 of the WW domain-containing oxidoreductase gene (WWOX) in 62.5% of XX DSD pigs. The expression levels of these three candidate genes in the gonads of XX DSD pigs were significantly different from those of normal female pigs.

Conclusion: The nucleotide changes of IFITM1 (c.218T > C), NOBOX (c.1043 C > G), and a 70 bp deletion fragment of the WWOX were the most dominant variants among XX DSD pigs. This study provides a theoretical basis for better understanding the molecular background of XX DSD pigs. DSD are conditions affecting development of the gonads or genitalia. These disorders can happen in many different types of animals, including pigs, goats, dogs, and people. In people, DSD happens in about 0.02-0.13% of births, and in pigs, the rate is between 0.08% and 0.75%. Pigs have a common type of DSD where the animal has female chromosomes (38, XX) but no SRY gene, which is usually found on the Y chromosome in males. XX DSD pigs may look like both males and females on the outside and have testis-like or ovotestis (a mix of ovary and testis) gonads inside. XX DSD pigs often lead to not being able to have piglets, slower growth, lower chance of survival, and poorer meat quality. Here, we used a method called whole-genome de novo sequencing to look for variants in the DNA of XX DSD pigs. We then checked these differences in a larger group of pigs. Our results reveal the nucleotide changes in IFITM1 (c.218T > C), NOBOX (c.1043 C > G), and a 70 bp deletion fragment in intron 5 of the WWOX, all linked to XX DSD pigs. The expression levels of these three genes were also different in the gonads of XX DSD pigs compared to normal female pigs. These variants are expected to serve as valuable molecular markers for XX DSD pigs. Because pigs are a lot like humans in their genes, physiology, and body structure, this research could help us learn more about what causes DSD in people.

Keywords: SRY negative pig; De novo sequencing; Single-nucleotide polymorphism; Structural variation; XX DSD.

Fig. 1

Characteristics of 1-month-old XX DSD pigs. (A) External genitalia characteristics of XX DSD pig. (B) Internal genitalia characteristics of XX DSD pig. Red arrow stands for ovary; yellow arrows stand for uterine horns; blue arrows stand for the vulva; purple arrows stand for testes; green arrows stand for the penis. (C) Histological analysis of the gonads in XX DSD pigs. HE staining was used to analyse the histological characteristics of the ovaries of normal female pigs, the testes of normal male pigs and the testis-like gonads of XX DSD pigs. Black stars indicate primary follicles; black triangles indicate the lumen of the seminiferous tubules. (D) Duplex PCR for SRY gene detection using GAPDH as an internal reference gene; M stands for the marker, RM stands for reference male, RF stands for reference female, NF stands for normal female pig, and D1 and D2 stand for XX DSD pigs

Fig. 2

Distribution characteristics of SNPs on chromosomes in three Yorkshire pigs (NF, D1, and D2). (A) The number of SNP distribution on chromosomes; (B) Distribution of SNPs on chromosomes. (C) Number of SNPs with different mutation types in the exon region

Fig. 3

SNPs screen according to recessive inheritance pattern. (A) Screening for homozygous nonsynonymous mutations only present in XX-DSD pigs; NF_all stands for all non-synonymous SNPs in normal female pigs; D1_hom and D2_hom stand for homozygous SNPs with non-synonymous mutations in D1 and D2, respectively (same as below). (B) Screening for homozygous nonsynonymous mutations only present in normal females. NF_hom stands for homozygous SNPs with non-synonymous mutations in normal female pigs; D1_all and D2_all stand for all non-synonymous SNPs in D1 and D2, respectively (the same as below). (C) Screening for nonsynonymous mutations that are homozygous in XX-DSD pigs and heterozygous in normal female. NF_het stands for heterozygous SNPs with non-synonymous mutations in normal female pigs (the same as below). (D) Screening for heterozygous nonsynonymous mutations only present in normal female pigs

Fig. 4

Comparison of SNPs and relative mRNA expression of candidate genes. (A) The SNP (c.218T > C) in the IFITM1. (B) The SNP (c.1043 C > G) in the NOBOX. (C) The relative expression of IFITM1 and NOBOX mRNA was detected by qRT-PCR (n = 3 for each group; ** P < 0.01, ns P > 0.05)

Fig. 5

Characteristics analysis of SVs in three Yorkshire pigs (NF, D1, and D2). (A) Distributions of SVs on chromosomes. (B) The proportion of different SVs

Fig. 6

Validate candidate SVs in the XX DSD pig cohort. (A) The six SVs displayed altered copy numbers in the XX DSD pig cohort. (B) A total of 22 individuals with only one SV were detected in the XX DSD pig cohort. SOX9 stands for CNV in the region upstream of the SOX9 gene

Fig. 7

PCR and Sanger sequencing validated the 70 bp deletion of the WWOX gene. (A) Gel electrophoresis of PCR products derived from normal female pig exclusively displayed wild-type bands (referred to as band A, 298 bp). Conversely, in XX DSD pigs, discernible deletion bands surfaced, encompassing homozygous (referred to as band B, 228 bp) as well as heterozygous (228 bp and 298 bp) deletions. (B) The sanger sequencing results showed a 70 bp deletion between the wild type and homozygous type. The sequencing results of the heterozygous bands were manually corrected to show two strands with a 70 bp difference. Black boxes indicate the 70 bp deletion. WT indicates wild-type, Het indicates heterozygous, and Hom indicates homozygous

Fig. 8

Comparison of WWOX immunofluorescence staining in gonads of XX DSD, normal female and normal male pigs. Bar = 50 μm