Identification Of Candidate Genes Associated With Genetic Resistance To Gastrointestinal Nematodes(GINs) Infection In Goat

Gastrointestinal nematodes(GINs)are one of the most economically important parasites of small ruminants and a major animal health concern in many regions of the world.The resistance or susceptibility against this parasitic infection are related to the genetic factors and varies between and within breeds,even for individual to individual.Understanding the mechanisms of genetic resistance to GINs infection is important for effective and sustainable control measures.However,the molecular mechanisms of the host response to GINs infection in goat are still little known.In this study,the genetic resistance associated candidate genes,pathways,specific antibody responses and their possible mechanisms to GINs infection in goat were investigated.(1)Resistant and susceptible goat individuals were evaluated in Yichang White Goats(YWGs)on the basis of high and low fecal egg count(FEC).The selected goats were monitored for parasitological and hematological parameters and the extreme resistance and susceptible individuals to GINs infection was used for artificial challenge trial and transcriptome analysis study.The local mIgA and systemic s Ig E against specific antigens were also evaluated to know the protective mechanisms and modulating activities against GINs infection.In addition,a total of 384 goats from different location were also used to identify single nucleotide polymorphisms in the ATP2A3 gene and investigated their polymorphisms association with GINs infection traits.The field experiment showed lower worm burden,delayed and reduced egg production in the relatively resistant group than the susceptible group.The hematological parameters during the browsing period in susceptible goats had significantly lower(p< 0.01)hemoglobin(Hgb)and lower(p< 0.05)packed cell volume(PCV)percentage compared to resistant goats.It was also found that the susceptible goats had lower hemoglobin than the normal hemoglobin value(90–150g/L).After the nematode challenge trial,the mean number of total Haemonchus contortus worms were recovered from resistant goats was significantly less(p< 0.05)than those of susceptible goats.Among the three selected traits for this study,negative correlation showed between fecal egg count(FEC)and Hgb(r =-0.94)and FEC and PCV(r =-0.90),where as positive correlation was shown between Hgb and PCV(r = 0.94).(2)Two genetically distinct goat groups,one relatively resistant and the other susceptible to GINs infection were identified in Yichang white goat and then four extreme individuals in each group were chosen to compare blood m RNA expression profiles using RNA sequences.The analysis of RNA sequencing showed that 2369 genes,1407 of which were up-regulated and 962 down-regulated,were significantly(p< 0.001)differentially expressed between these two groups.Functional annotation of the 298 genes more highly expressed in the resistant group yielded a total of 46 significant(p< 0.05)functional annotation clusters including 31 genes(9 in innate immunity,13 in adaptive immunity,and 9 in innate immune responses)related to immune biosynthetic process as well as some immune system related pathways i.e.transforming growth factor(TGF)-?,mitogenactivated protein kinase(MAPK),and cell adhesion molecules(CAMs).These findings provide insights that are immediately relevant for the improvement of host resistance to GINs infection and which will make it possible to know the mechanisms underlying the resistance of goats to GINs infection.(3)Twelve up and down-regulated genes were selected to validate the expression profile by the qRT-PCR with the obtained RNA sequencing expression.Among the selected genes,six up-regulated genes(ITGA4,C3,BCL2,ATP7 A,ERAP1,and ST6GAP1)were higher expressed in susceptible goats.Another six down-regulated genes(LST1,IFI44 L,IL1R2,F12,CCL27,and IFI6)were lesser expressed in susceptible goats,compared with resistant goats.The genes ITGA4,C3,BCL2,ST6GAL1,LST1,IFI44 L,IL1R2,and IFI6 were significantly differentially expressed at p< 0.01 levels and ATP7 A,ERAP1,F12,and CCL27 were significantly differentially expressed at p< 0.05 levels,as expected from the RNA sequenced expression data.The qRT-PCR log2 expression(foldchange)data and m RNA sequence log2 expression(fold-change)data were fitted in the line plot and expression pattern,i.e.the correlation coefficient was 0.912 and the linear regression model was Y = 0.839X-2.252,were showed strong correlation with the sequencing expression results.(4)The local mucosal Immunoglobulin-A(mIgA)and systemic serum Immunoglobulin-E(s Ig E)are the most important antibody response to protect against GINs infection.The local mIgA and systemic s Ig E were investigated to know their correlation and possible resistant mechanisms to GINs infection with infection-related genes.We monitored local mIgA and systemic s Ig E against specific antigens and found a negative correlation with both of them and fecundity as well as worm length at adult stage of GINs infection suggesting protective antigens in this stage modulating parasites infection.(5)We investigated relative expression profiles of 5 infection-related genes using qRT-PCR in goat demonstrating higher expression levels of C3,LTBP2,ABCB1,and SMAD3 genes in susceptible goats,but IL1R2 gene was highly expressed in resistant goats.The relative expression profiles in the m RNA of C3 and IL1R2 genes in abomasal tissues(tissues of abomasal fundus and pylorus),lymph nodes(abomasal and mesenteric),and tissues of small intestine were showed significantly differences,whereas genes LTBP2,ABCB1,and SMAD3 showed no differences in lymph nodes and in tissues of small intestine compared between susceptible and resistant goats.Collectively,antigenspecific local mIgA and systemic s Ig E correlation results as well as tissues specific genes responses,especially C3 and IL1R2 responses suggesting important effectors in the mechanism of genetic resistance to GINs infection in goat.These data indicate that resistant goats can better maintain GINs responses at the local site of infection and also provide us to make an effective hypothesis on mechanisms of resistance against GINs infection in goat.(6)We also identified polymorphisms in the ATP2A3 gene and investigated their association with GINs infection traits measured in three Chinese indigenous goat populations.The seven novel variants were identified in the goat ATP2A3 gene,in where five mutations located in 3? UTR regions and two synonymous mutations located in exon region.Genotypic and allelic frequencies of seven variants were consistent with HardyWeinberg equilibrium and the population genetics parameters,i.e.Polymorphism information contents(PIC),Heterozygosity(He),and Effective allele numbers(Ne)values were 0.26,0.32 and 1.54 in ESB(Enshi black);0.28,0.35 and 1.60 in NJY(Nanjiang yellow);and 0.28,0.34 and 1.55 in YCW(Yichang white)goat populations,respectively.(7)The association analysis revealed that out of seven polymorphisms,four polymorphisms had high significant effects on GINs infection traits in goat.Populations specific association analyses also were showed similar significant association effects with GIN infection traits.From haplotype and LD structures of seven SNPs,two pair variants,C24361893 T and C24379974 T from exon region as well as C24358400 A and G24358441 C from 3? UTR regions showed significant LD with each other with strong coefficient of linkage disequilibrium(D?)and spanning 18.081 kb(~18 kb)and 0.041 kb(~0 kb)blocks,respectively.From four polymorphic sites of ATP2A3 gene and their haplotype combinations,eight previously undiscovered haplotypes were constructed from these four mutations,where the AG and CC haplotypes showed the highest frequency 65.0% and 53.3%,followed by other haplotypes.The relative expression of m RNA in ATP2A3 gene by the qRT-PCR analysis was higher in susceptible goats compared to that of resistant goats and the values were significantly differed at p< 0.05 levels as expected similar relative fold change value from the RNA sequenced data.These results indicated that the ATP2A3 gene is an important candidate gene in GIN infection traits in the goat.For more clarify,further studies are needed on a large number of goat populations considering different genetic backgrounds.