The Research Of The Missense Mutation RRM1^I241V Contributes To Goat Tameness

Animal domestication has changed the way and means by which humans obtain production materials,and to some extent has driven the rapid development of human agricultural society.Goats are one of the earliest domesticated animals and are now widely distributed around the world with population growth.The physiological and biochemical characteristics of goats have undergone significant changes during the domestication process,such as coat color,body size,reproduction,and temperament.Due to the rapid development of sequencing technologies,a vast amount of sequencing data has been released,greatly advancing the study of goat domestication at the population genetic level.However,the genetic analysis of specific domestication traits related to the domestication selection mode,such as the domestication phenotype,remains unclear.Therefore,this study mainly conducted extensive and in-depth research and discussion on the genetic mechanisms related to domestication traits during the goat domestication process.In this study,through population differentiation analysis and QTL analysis of larger-scale resequencing data of goats worldwide,including various wild goats,a large number of modern domestic goats with different geographical distributions,and ancient goats from different historical periods,we identified and screened the domestication loci STIM1-RRM1^dom and the important variant site RRM1^I241V related to goat domestication.We also constructed RRM1^I241V mutant mice and conducted multi-omics analysis and experiments to further validate the role of RRM1^I241V in animal domestication.The results of this study are as follows:1.Through selection sweep analysis of domestic goats and bezoars on a global scale,199 domestication selection windows were identified,mainly related to animal metabolism,immunity,physiological hormone release,and synaptic function.The strongest selection signal during goat domestication was further verified and determined to be located on chromosome 15 of domestic goats(Chr15:32.24Mb to 32.37Mb,windowed F_ST=0.87,πln-ratio=3.94,XP-EHH=8.12).This locus is associated with ADHD+DBDs in children and contains two genes,STIM1 and RRM1,related to neural development.The correlation between the STIM1-RRM1 locus and goat domestication behavior was then tested and analyzed through FD behavioral experiments.Compared with STIM1-RRM1^dom/dom BC individuals,STIM1-RRM1^dom/wild BC individuals exhibited a shorter FD distance（P=4.24×10-6,one-way ANOVA）.Furthermore,QTL analysis of whole-genome resequencing data from 16 experimental goats further confirmed that the STIM1-RRM1 locus is located within the strongest association signal with goat domestication behavior（Chr15:31.65-34.92 Mb,LOD=5.41,-log10（P）=8.97）.2.Within the STIM1-RRM1 locus,there are 15 high-frequency SNPs（>0.96）in modern domestic goat populations.By analyzing the evolutionary history of these SNPs,we inferred that the domestication haplotype may have existed in the early stages of goat domestication,but with a low frequency,and then it was selected and gradually increased in frequency during the domestication process.Moreover,the only missense mutation RRM1^I241V among these 15 SNPs was first detected 6,500 years ago,and its appearance was accompanied by a rapid increase in the frequency of the entire domestication haplotype,which quickly spread throughout the modern goat population.Therefore,we speculated that this missense mutation may have accelerated the fixation of the domestication haplotype,and it was considered a candidate causal variant for further analysis.Then,through ASE analysis of hybrid goat brain tissue,we found that STIM1 and RRM1 did not show significant differential expression,suggesting that STIM1 and RRM1 may play a role through changes in protein structure and function rather than changes in expression levels.Comparative genomics analysis showed that this missense mutation is highly conserved among species and only occurs in goats.In addition,RRM1^I241V is located on the dimer interface and near the S-site of the RRM1.The hydrophobicity prediction results showed that the missense mutation altered the hydrophobicity of nearby amino acid sequences.Furthermore,the functional analysis of the missense mutation on RRM1 protein damage showed that RRM1^I241V has a moderate effect on RRM1.Therefore,this site was selected as a candidate causative variant for further validation and analysis.3.In this study,we used CRISPR/Cas9 gene editing technology to generate RRM1^I241Vmissense mutant mice.The RRM1^I241V mutant mice were viable and fertile,and did not show significant differences in body size or weight compared to wild-type mice.We then conducted three different mouse domestication experiments to test the effect of RRM1^I241Vmutation on domestication behavior.The results showed that the RRM1^I241V mutation significantly increased the tolerance of mice to human handling,and they exhibited more active approach towards humans.This study used CRISPR/Cas9 gene editing technology to create RRM1^I241V missense mutation mice,which were able to survive and reproduce normally without significant differences in body size or weight.Subsequently,the study investigated changes in the mice’s taming behavior through three mouse taming experiments.The results showed that RRM1^I241V missense mutation significantly increased the mice’s tolerance to human manipulation and displayed more active approach behaviors towards humans.The study also conducted transcriptome analysis of the mouse brain and identified377 differentially expressed genes,some of which have been reported in other animal domestication studies,such as GRM3.Additionally,KEGG enrichment analysis showed that the most enriched signal pathway was the glutamatergic synaptic signaling pathway,as well as several other neural synaptic signaling pathways that are closely related to animal domestication.Furthermore,the study used several tests,such as the OFT and EPM,to evaluate the mice’s anxiety-like behavior and found that RRM1^I241V missense mutation reduced the mice’s anxiety-like behavior to a certain extent.The study also tested the effects of RRM1^I241V missense mutation on the mice’s social behavior through a multiple-chamber social test and found that it did not affect the mice’s social level with the same species but increased their tolerance to other animals.The water maze experiment showed that RRM1^I241V missense mutation did not affect the mice’s learning and memory level or the generation of hippocampal neurons.In summary,this study provided a relatively comprehensive analysis of animal domestication characteristics and identified and validated key genetic variations that contribute to animal domestication traits.And this study provides a molecular basis and theoretical foundation for the generation of more domesticated animals in the future.