Identification Of Reference Genes For RT-qPCR Analysis And Analysis Of Expression Pattern Of Entire Cytochrome P450s In Polypogon Fugax In Wheat Field

Asia minor bluegrass(Polypogon fugax Nees ex Steud.)is an annual herbaceous plant species of the Poaceae family and is widespread in most regions of China.It is a hygrophilous species that seriously infests wheat(Triticum aestivum L.)fields with a rice(Oryza sativa L.)rotation,causing a great reduction in grain yield.In the past 20 years,the acetyl-Co A carboxylase(ACCase)-inhibiting herbicide fenoxaprop-P-ethyl has been frequently used for control of grass weeds in wheat fields such as P.fugax.However,herbicides in this class generally act on a single target site,and their repeat uses have greatly facilitated the resistance evolution.Previously,our research team has collected a P.fugax population AHHY with suspected resistance to fenoxaprop-P-ethyl.Preliminary studies have revealed that Trp-1999-Ser mutation of ACCase and cytochrome P450monooxygenases(P450s)-mediated enhanced metabolism were simultaneously involved in the resistance phenotypes.However,which P450(s)were responsible for the metabolic resistance are still unknown.In this study,based on the gene resources established by a thirdgeneration full-length transcriptome,the stalely expressed reference genes were detailedly identified in P.fugax under different experimental conditions through functional annotation,BLAST alignment,and expression analysis for accurate real-time quantitative PCR(RTq PCR)assay.The temporal expression patterns of entire genes annotated as P450 s were then systematically compared between resistant and susceptible populations of P.fugax.The P450 s stably up-regulated in the resistant than in the susceptible biotype and/or could be induced up-regulated by herbicide treatment were also determined,which will lay a foundation for uncovering the mechanisms of P450-mediated metabolic-herbicideresistance in P.fugax.The main results are as follows:1.Identification of reference gene(s)in P.fugaxSeven reference genes stably expressed in other plant species were selected as candidate genes,including actin(ACT),eukaryotic initiation factor 4a(EIF4A),elongation factor 1α(EF1α),18 S r RNA(18S),25 S r RNA(25S),ribulose-1,5-bisphosphate carboxylase oxygenase(RUBP),and glyceraldehyde-3-phosphate dehydrogenase(GAPDH).Based on the gene resources from full-length transcriptome of P.fugax,the sequences of above seven genes were obtained through functional annotation and BLAST alignment.Thereafter,their amplification specificity and expression stability were analyzed in the susceptible(S,SDTS)and resistant(R,AHHY)populations under different herbicide stresses,at different growth stages,or in different organs to determine stably expressed reference genes for accurate RTq PCR analysis.The results showed that in P.fugax,the stably expressed reference genes were ACT,18 S,and RUBP under fenoxaprop-P-ethyl stress,GAPDH and EF1α under mesosulfuron-methyl stress,and ACT,EF1α,EIF4 A,and 25 S at different growth stages.However,no stably expressed gene(s)was identified for accurate data normalization in the different organs of P.fugax.2.Gene isolation and expression assay of entire P450 s in P.fugaxBased on the gene resources from full-length transcriptome of P.fugax,169 isoforms annotated as related to putative P450 s were isolated by functional annotation,and a total of48 potential P450 s were successfully identified according to their sequence similarity by BLAST alignment.Gene primers were designed based on the conserved regions of different alleles,and RT-q PCR analysis was performed by using ACT,18 S,and RUBP as internal controls under fenoxaprop-P-ethyl stress.To determine the P450 s related to the metabolic fenoxaprop-P-ethyl resistance in P.fugax,the expression patterns of 48 P450 s were first analyzed in the S and R biotypes at different times after fenoxaprop-P-ethyl treatment.The results showed that in the R population,22 P450 s were up-regulated more than threefold for at least one time point after fenoxaprop-P-ethyl treatment,of which six genes,respectively,annotated as CYP709B1,CYP71A1-4,CYP711A1,CYP78A9,P450-11,and P450-39 were up-regulated more than 10-fold.In the S population,40 P450 s were up-regulated more than threefold,of which 18 genes were up-regulated more than 10-fold.Subsequently,the transcript levels of the 48 P450 s were compared between the S and R populations at different times after herbicide treatment.The results showed that under fenoxaprop-P-ethyl stress,24 genes were up-regulated more than threefold in the R relative to the S line for at least one time point,of which CYP57,P450-13,CYP97B2,CYP94B3-1,CYP71D8,CYP71A1-5,P450-39,and CYP92A44-2 were constitutively up-regulated in the R vs S plants.Also,three genes CYPRO4,CYP313A4,and CYP51H11 had constantly higher transcript abundances in the R than in the S line during 6 to 24 h.Combining all the above results,a total of nine genes separately annotated as CYPRO4,CYP313A4,CYP51H11,CYP709B1,CYP71A1-4,CYP711A1,CYP78A9,P450-11,and P450-39 were constitutively and/or herbicide-induced up-regulated in the R vs S plants of P.fugax.The up-regulated expression of above P450 genes was consistent with the higher P450 activity in the resistant plants,and thus these genes can be served as potential fenoxaprop-P-ethyl metabolism-related genes in P.fugax.In conclusion,this study determined suitable reference genes in P.fugax under different experimental conditions,laying foundation for the accurate RT-q PCR analysis of the target gene in this species.The temporal expression patterns of entire P450 s were detailedly compared between the Resistant and Susceptible populations of P.fugax,and the genes constitutively and/or herbicide-induced up-regulated in the R vs S plants were also determined,which can be served as a foundation for uncovering the mechanism for P450-mediated herbicide resistance.These results can also provide some references for investigating the metabolic-herbicide-resistance mechanisms in other grass weeds.