| Mules are known to be sterile,but in some cases a female mule or hinny may occasionally produce foals when mated with a male horse or donkey.Since 1928,there have been about 30 well-documented(karyotype analysis and photographs)of mule offspring worldwide(including four in our collection).The “affinity”hypothesis states that individual fertile female mule or donkey can produce eggs containing only her maternal genome,and thus be fertile.However,some studies show that the variation of phenotype(body size or hair color)and karyotype(chromosome number or chromosome structure)of some mule offspring were obviously inconsistent with the hypothesis of affinity.Due to the limitations of the technology at that time,their study also did not find any signs of recombination between horse and donkey chromosomes,but their articles mentioned the speculation of possible recombination of horse and donkey chromosomes.So which chromosome combinations or structural variations form fertile gametes? Due to the high similarity of some chromosomes of horse and donkey and the possible variation of karyotype in mule offspring,it is difficult for conventional cytogenetic methods to accurately identify the variation of chromosome structure.In this study,10 X Genomics sequencing technology and bioinformatics analysis methods were used to explore the genomic evolution of equine animals and the characteristics of karyotype variation of mule offspring,in order to have a deeper understanding of equine and donkey hybrid sterility and occasional fertility.The results are as follows:(1)Reconstruction of equine phylogenetic treeWe reconstructed the circular mitochondrial genome of the 16611 bp Equus hemionus hemionus.Based on this de nove circular mitochondrial genome,we reconstructed the equine phylogenetic tree using the maximum likelihood method combined with the circular mitochondrial genome data of other equine species.The research shows that the domestic donkey is the descendant of the African wild donkey,which is sister group to the Asian wild donkey.The donkey is relatively close to the zebra,followed by the horse.These results lay a foundation for understanding the evolution of equine animals and their reproductive isolation among species.(2)Recombination of donkey genome assembly at chromosome levelWe determined the cytogenetic standard number and sequence direction of donkey chromosome length sequence by collinearity analysis of horse and donkey genomes.On this basis,the structural rearrangement between the horse and donkey genomes was analyzed at the sequence level,and the evolution of the horse and donkey genomes in odd-hooded animals,especially the detailed evolution within chromosomes,was illustrated with the examples of the horse 5 and donkey 1chromosome.This study laid a solid foundation for the analysis of the chromosomal genome structure of mule offspring.(3)Genomic structure of mule offspringCombined with previous chromosome staining results,after deducting paternal genetic material,it is clear that there are two types of karyotypes in mule offspring.One is consistent with the affinity hypothesis,in which mare mules produce eggs that contain only a set of horse chromosomes,and then mate with donkeys to produce mule karyotype offspring.The other is not in accordance with this hypothesis,that is,part of the chromosomes of she-mules undergo recombination between orthologous chromosomes of horses and donkeys during oogenesis,resulting in a karyotype with a balanced total genetic material but heterogeneous mosaicism of some chromosomes.Large fragments of different provenances from the horse and the donkey appear alternately on the chromosome.Due to the difference in the karyotypes of mule ova,they formed a partially heterologous diploid karyotype with different provenance ratios but a balanced total genetic material after mating with horses or donkeys,which resulted in variation in the number of chromosomes in mule offspring(2n=60/62/63).In this study,we verified the correctness of the affinity hypothesis in some cases;it showed the recombination between orthologous chromosomes from different species of large mammals,explained the causes of karyotype variation of mule offspring,and confirmed predecessor’s speculation that there is another type of chromosomal mosaic karyotype in the mule offspring in some cases.(4)The paternity relationship of fertile mules and their offspring was confirmed at the sequence levelFor this(partial)heterodiploid of mule offspring,the general method of paternity testing is not applicable,so we decided to use genetic distance to infer the parentchild relationship.For the offspring of the mule karyotype,we calculated the mismatch rate of single-nucleotide variants(SNVs)between the offspring and the control samples on 31 horse autosomes and 30 donkey autosomes;for mule offspring with chimeric chromosomal karyotype,only the SNV mismatch rate between the maternal haploid region and the corresponding region of the control sample was counted.In the 31 horse autosomal regions in the samples of mule karyotype offspring,the pairwise comparison of SNVs found that the average mismatch rate between individuals was 0.3562,while the individuals with genetic relationship in the pedigree record were less than 0.02.However,in the 30 donkey autosomal regions of the same individual,the average mismatch rate between individuals was 0.3762,and the above-mentioned large deviations were not found among individuals with genetic relationships in the pedigree record.Similarly,there are similar results in the maternal haploid region of mule offspring with a chromosomal mosaic karyotype.The above results are all consistent with the results of mitochondrial genome analysis and pedigree records.Here,we rigorously demonstrated the parent-child relationship between the she-mules and their offspring from the nuclear genome and mitochondrial genome,dispelling people’s doubts about this rare phenomenon as much as possible. |
