Differentiation Of Sitobion Avenae Biotypes And The Underlying Genetic And Molecular Bases

The English grain aphid,Sitobion avenae?Fabricius?,is a significant cereal crop pest worldwide,and it can survive on many poaceous plants?cereal crops or grasses?.Studies have shown that there exists some degree of differentiation among different geographic populations and aphid clones on different host plants,indicating that the aphid has the potential to differentiate into different biotypes.However,direct genetic evidence for S.avenae's differentiation between different host-associated clones has been rare.The current research on the divergence of S.avenae biotypes and their geographical distribution is still extremely limited,and genetic and molecular mechanisms of biotype differentiation of this aphid are not yet clear.To address the issues,we genotyped S.avenae clones on barley and wheat from nine provinces of China by using microsatellite markers,and performed biotype identification on different S.avenae genotypes by using variable wheat/barley varieties.The genetic basis and molecular mechanisms of biotype differentiation of S.avenae were explored using a combination of techniques in ecology,behavior,molecular biology and transcriptomics.The main results are as follows:1. Microsatellite analyses showed that there existed differences in genetic diversity among different geographic populations of S.avenae.High genetic diversity of S.avenae populations was found in Jiangsu,Qinghai and Hubei,based on gene diversity?Hs?,number of effective alleles?Ne?and Shannon's information index?I?.The result of molecular variance analysis?AMOVA?showed genetic divergence among different geographic populations of S.avenae was statistically significant.The analysis of pairwise F_ST,principal coordinate analyses?PCo A?,unrooted NJ tree and STURCTURE also showed that there were large differences in genetic structure between the populations of S.avenae in the western?e.g.,Qinghai,Gansu and Xinjiang?and central-eastern regions?i.e.,Zhejiang,Jiangsu,Henan,Anhui,and Hubei?of China.Based on the Bayes Ass analysis,there was no significant gene flow between aphid populations of two regions.Significant differences in genetic diversity were detected between different host-associated clones of S.avenae.The inbreeding coefficient(F_IS)of barley clones was significantly higher than that of wheat clones.Pairwise F_ST analyses showed that there was significant genetic differentiation between different host clones of Jiangsu,Anhui,Gansu,Zhejiang and Xinjiang(F_ST=0.070,0.098,0.109,0.090 and 0.286,respectively).The plots for PCo A,unrooted NJ tree and analyses in STURCTURE showed a clear separation between barley and wheat clones in the above-mentioned provinces.2. Identification of S.avenae biotypes:using the ratio of aphid counts,10 resistant barley/wheat varieties?i.e.,Zhong 4 wumang,Zhengmai 366,186-TM12-34,Yanyin2129-56,Dulihuang,Zhudamai No.4,Zaoshu No.3,Zangqing 25,Ganpi No.6,and Xiyin No.2?were identified from 25 barley and 33 wheat varieties.Based on the damage performance of S.avenae genotypes on the five barley/wheat resistant varieties?i.e.,Zhong 4 wumang,186-TM12-34,Dulihuang,Zaoshu No.3 and Xiyin No.2?,six biotypes of S.avenae were identified,and a biotype identification system based on these five barley/wheat varieties was established.All the above mentioned five varieties in this system were resistant to biotype 1.Biotypes 2 and4 were able to overcome the resistance of Zaoshu No.3 barley and 186-TM12-34 wheat,respectively.Biotype 3 was virulent to all the three barley varieties,but not to the two wheat varieties,whereas biotype 5 was virulent to two?i.e.,Zaoshu No.3 and Xiyin No.2?of the three barley varieties.Biotype 6 was characterized by its virulence to the wheat variety 186-TM12-34.Principal component analyses?PCA?also showed clearly the differences in performance among different biotypes.Biotype 1 is the dominant biotype?90.5%?.In addition,considerable geographic variation was found among these biotypes.For instance,biotype 2 occurred only in western provinces?i.e.,Shaannxi,Qinghai,Gansu,and Xinjiang?of China.Biotypes 5 and 6were found only in Xinjiang and Zhejiang,respectively.3. Based on comparisons of life history traits of this aphid on different host plants,the biotype differentiation of S.avenae was verified.Biotype 1 on Mingxian 169,Aikang 58 and Xinong 979 showed shorter developmental time of nymphs,larger adult weight and higher 10-d fecundity than on Zhong 4 wumang,186-TM12-34,Dulihuang,Zaoshu No.3 and Xiyin No.2.The fecundity of biotype 3 on all three barley varieties?i.e.,Dulihuang,Zaoshu No.3 and Xiyin No.2?was higher than that on wheat varieties.In addition,the identification of six S.avenae biotypes was confirmed in the principal component analysis of aphid life-history traits.Choice bioassays showed that,at 6h,the selection rates of all six biotypes for the three barley varieties were higher,indicating the antixenosis of these barley varieties are low for all biotypes of S.avenae.With the exception of biotype 6,the choice of all other biotypes for Zhong 4 wumang was low,indicating that biotype 6 could overcome both antibiotic and antixenotic effects of Zhong 4 wumang,and this variety had strong antixenotic effects on all biotypes except biotype6.In addition,the selection rates of biotype 3 for Aikang 58 were also low,as well as those of biotype 3,5 and 6 for 186-TM12-34,showing that Aikang 58 and 186-TM12-34 had some degree of antixenotic effects on corresponding biotypes.4. Feeding behavior of S.avenae was observed using EPG?electrical penetration graph?technique.The results showed that,compared with wheat variety Aikang 58,biotype 1 on barley variety Xiyin No.2 showed lower duration of E2 wave?phloem sap ingestion?,and higher number and duration of waves NP?non-probing?and C?pathway phase?,while the performance of biotype 3 on the two host plants was opposite.This indicated that barley and wheat had higher resistance to biotypes 1 and 3,respectively.The abovementioned differences in E2duration may be related to different detoxification abilities of the two biotypes for specific secondary metabolites in barley and wheat.There were no significant differences in frequency and duration of wave F?stylet derailment?between the two biotypes on barley and wheat,indicating that there could be limited difference in the physical resistance of the wheat variety Aikang 58 and the barley variety Xiyin No.2.Collectively,our results indicated that feeding behaviors and the plasticity of life history traits for S.avenae might be closely related to biotype differentiation of this aphid.5. Microsatellite analyses showed that,based on parameters like number of effectivealleles?Ne?,Shannon's information index?I?,and expected heterozygosities?He?,biotype 1had highest genetic diversity among all biotypes,whereas biotype 6 had lowest genetic diversity.Pairwise F_ST values indicated significant genetic differentiation between biotypes of this aphid.The PCo A analysis and neighbor-joining tree also showed the difference in genetic structure between different biotypes.However,close genetic and evolutionary relationships were found between some biotypes?e.g.,biotypes 1 and 2,and biotypes 3 and 6?.In addition,the genetic differentiation between six S.avenae biotypes is consistent with their damage performance on different host plants,indicating it is possible to distinguish different biotypes with molecular markers such as microsatellites.The 16S r DNA diagnostic PCR was used to detect secondary symbionts in the six S.avenae biotypes.The compositions and contents of secondary symbiotic bacteria varied in different biotypes.Regiella insecticola was not detected in biotypes 3 and 6,but it was detected in all clones of biotype 4.Hamiltonella defense had a higher infection rate in biotypes 3 and 6.Correspondence analyses between biotypes and the infection of secondary symbionts showed that the infection of secondary symbionts might play a role in the biotype differentiation of S.avenae.6. Transcriptome analysis showed that 3392 and 2246 DEGs?differentially expressed genes?were detected between biotypes 1 and 3 on wheat and barley,respectively.Among them,1528 genes were differentially expressed between the two biotypes both on the two host plants.GO and KEGG enrichment analysis showed a large number of genes related to digestion and defense existed in these 1528 common DEGs.Top defense-related common DEGs with the most significant expression changes included three PODs?peroxidases?,two UGTs?UDP-glycosyltransferase?,two CPs?cuticle proteins?,one glutathione S-transferase?GST?,one superoxide dismutase,and one esterase,suggesting their potentially critical roles in the divergence of S.avenae biotypes.When feeding on wheat,there were 36 defense-related genes specifically expressed between biotypes 1 and 3,such as PODs,P450s,UGTs,and CPs.On barley,only 13 defense-related genes were specifically expressed between the two biotypes,including four P450s,four UGTs,two CPs,one ABC transporter,and one POD.Wheat and barley specifically induced the expression of different number and function of defense-related genes,which may reflect the differences in the composition and content of secondary metabolites in wheat and barley.7. In response to host plant shift,39 plastic genes were shared by biotypes 1 and 3,whereas 127 and 865 plastic genes occurred only in biotypes 1 and 3,respectively.Among all these plastic genes,defense-related ones underwent intensive expression restructuring,such as UGTs,GSTs,alkaline phosphatase,and cysteine proteases.In addition,the expression of common plastic genes?e.g.,cysteine proteases and alkaline phosphatase gene?were significantly correlated with the fecundity of S.avenae.Specific plastic genes of biotypes 1 and3 clustered into five and nine transcriptional modules,respectively.The fecundity of biotype 1was significantly related to the expression of cuticle protein gene?CP5?in the module P1 and P450 gene?CYP6DA2?in the module P3,while the fecundity of biotype 3 was correlated with the expression of UGT gene?UGT2B2?in the module T8.These genes and transcription modules may have significant functional implications for colonization of alternative plants for biotypes 1 and 3.GO and KEGG enrichment analysis revealed that many specific plastic genes were related to detoxification and defense.In addition,differential expression of transcripts between both biotypes was not only reflected in the dramatic difference in the number of plastic genes,but also in the expression pattern and degree of these genes.Transcriptional plasticity of biotype 3 was lower than that of biotype 1 in response to host transfer.The expression plasticity of some defense-related genes?e.g.,CYP6DA2 and UGT2B2?was positively and negatively correlated to aphid fecundity for biotype 1 and 3,respectively,indicating that phenotypic plasticity could be adaptive for biotype 1,but maladaptive for biotype 3.This can explain the different level of phenotypic plasticity between the two biotypes.Our results have significant implications for phenotypic plasticity's roles in adaptive evolution of insect populations.Our results showed that phenotypic plasticity could have significant implications for the development and evolution of different biotypes in S.avenae.