Genome Wide Identification Of Wheat AP2 Subfamily Gene And Molecular Basis Of Wheat Architecture Regulared By Gene Q

Q gene is an important domestication gene originated from a spontaneous mutation in the process of wheat hexaploidization.It makes wheat from speltoid spike to quadrangle spike,conferring wheat the characteristics of easy threshing and plant height dwarfing.It is convenient for farmers to harvest and grind in the production and responsible for the rapid and widespread cultivation of wheat worldwide.Q and its homologous genes encode a class of AP2 transcription factors and belong to the euAP2 group in the AP2 subfamily.It possesses a binding site（21-bp in length）for miR172 which could regulate its expression.For the domestication from q to Q,it was previously considered that Q had a G>A mutation at the nucleotide of 2123bp compared with q.This mutation made the coding amino acid at position 329 vary from Valine to Isoleucine,enabling the Q protein to form a more stable homodimer.In recent research,it is acknowleged that the key mutated site for its domestication is not at 2123bp,while in the tenth exon which is a C>T nonsense mutation.The mutation is located in the regulatory region of miR172,which results in reduced miRNA targeting,and therefore lead to the increased expression level of Q.Since the hexaploidy nature of common wheat,Q which is located at the end of the long arm of the wheat 5A chromosome has homologous genes on the 5B and 5D chromosomes,respectively.For convenience of description,we assigned 5AQ ψ5Bq（it is a pseudogene）and 5Dq to each of them.In previous research,5AQ has been extensively studied,and there was few report on 5Dq.NAUH164,obtained from the mutagenesis by EMStreatment of wheat variety Sumai 3,has a dwarf plant height and compact spike morphology.It was officially named Rht23 by the International Wheat Gene Nomenclature Committee.Gene cloning showed that Rht23 is an allelic variation of 5Dq,denoted as 5Dq’.The mutated locus of 5Dq’ occurred at 3147bp within miR172 binding site.This mutation in the miR172 binding site reduces the efficiency of miR172’s cleavage and results in the upregulation of its expression level,causing a series of changes on the agronomic traits,such as dwarfing of wheat plants,compact spikes,and grain threshing.The mutation from 5Dq to 5Dq’ simulates the domestication process of Q.In order to analyze the function of Q genes from different wheat subgenomes and their interaction effects in regulating wheat plant type and spike type,in the present research,we will performed work from three aspects:（1）genome wide identification of AP2 subfamily in wheat;（2）We will derived various Q/q recombination for different subgenomes by hybridization experiment with Chinese Spring structural aberrant at Q/q loci for different Q/q haplotypes.Based on this genetic resources,we phenotype related agronomic traits and survey the developmental processes through micro-observation.The interactive and coordinated effects among the Q/q allele will also be investigated.We will also evaluate the genetic effect of 5Dq’ for digging out its probably new function and potential application in wheat breeding;（3）To identify Q regulated genes at transcriptional levels through RNA-seq methods.The Q regulated network and biological processes will be deduced.The main results were as follows.1.Identification of AP2 subfamily genes in wheatIn this study,a total of 68 AP2 subfamily genes were identified in the wheat genome.Through chromosome compositon analysis,AP2 genes are distributed on all of 21 chromosomes.According to the gene structure analysis,the genes of the wheat AP2 subfamily can be divided into three groups,of which 10 genes have miR172 binding sites and belong to the euAP2 group.The euANT group and the basalANT group both include 29 genes.Both Q and 5Dq are included in the euAP2 group.Through phylogenetic analysis,these genes can be divided into three branches,and the euAP2 group genes are clustered into a single branch,which is consistent in evolution.According to the expression patterns of euAP2 group genes in different wheat tissues at different stages,we deduced these genes might be involved in wheat growth and development at a specific period.2.The genetic effect between 5Dq’ and 5AQ and their interaction in determinating plant architectureIn order to study the genetic effect between 5Dq’ and 5AQ and their interation in determinating plant architecture,NAUH164 was crossed with Chinese spring（CS）and its deletion lines 5AL-10,5BL-9 and 5DL-5 to obtain hybrid F1.Relative expression of 5AQ and 5Dq/5Dq’ were analyzed for the spikes of F1 and the parents at the heading stage.To our expection,expression of 5AQ is significantly higher than that of 5Aq,and expression of 5Dq’ is significantly higher than that of 5Dq.Q/q with more copies result in higher expression level,confirming the dosage effect of Q.Various phenotypes were observed and measured at the heading/flowering stage for the parents and F1 of different cross combinations,including plant height,stem internode length,spike type,spike length,and spike width.The plant height of F1 and the internode length of the stem are between the parents.The spike type exhibits different degrees of compactness,and there is no obvious difference in spikelet number.In addition,the individuals with different spike types were investigated in the F2 population.Individuals having both 5AQ and 5Dq’ exhibited compact spike type,and when each of them was present alone the plants had square spike type.Those plants without any of the two genes had speltoid spike type.These indicated the 5AQ and 5Dq’ had additive effect.Correlation analysis between agronomic traits and Q/q/q’gene expression showed Q/q/q’ expression was negatively correlated with spike length and plant height,and positively correlated with spike width and stem thickness.In conclusion,the abundance of Q/q transcripts affect wheat growth and development and hence determine the plant architecture,including plant height,spike density,threshability and so on.3.The genetic effect of 5Dq’ in wheat growth and developmentThe growth of leaves,stems and spikes of Sumai 3 and NAUH164 were compared at various development stages of wheat.No obvious difference in plant morphology was observed at the seedling stage,including leaf,tillers,etc.Since the joining or elongation stage,they showed difference in stem elongation and spike development,and the difference became more obvious henceforth.As a result,compared with wildtype Sumai 3,NAUH164 showed dwarfness due to the reduced length of each internode,especially for peduncle（internode below spike）which was 61%reduction in length.The genetic effects of 5Dq’ in different background was further investigated.In the 5AL-10×NAUH164 F2:3 population,plants which were null for 5AQ and homozygous for 5Dq’ were identified by marker analysis.These lines showed square spike,indicating similar genetic effect of 5AQ and 5Dq’ and even the 5Dq’ can enhance the effect 5AQ.The 5Dq’ was transformed into the rice variety Nipponbare.The rice growth was compared for the transgenic plants and the receptor or negative control.No significant difference was observed at the seedling stage.At the heading stage,the transgenic plant showed reduced plant height and shortened erected panicle.It implied 5Dq’ can also reduce the plant height and shorten the panicle under rice background.4、5Dq’/Rht23 regulats wheat growth and development through the age pathwayTranscription factors of the euAP2 group are strictly regulated by miRNA（miR172-AP2）,and occur during a specific period of reproductive development.AP2 negatively regulates the flowering process.Normally,when the plant develops to a certain"age" stage,this inhibition can be released by the activated expression of miR172.The point mutations of 5AQ and 5Dq’ in the miRNA regulatory region lead to a significant reduction in the efficiency of miR172 recognition and splicing.5Dq’/Rht23 is involved in the regulation of growth and development through the age pathway,which makes the plants dwarfing.This study also found that 5Dq’ suppresses the elongation of stem and spike and promotes leaf growth after jointing.It is speculated that the increase in 5Dq’ gene expression inhibits reproductive development and promotes vegetative development.5.Transcriptome analysis reveals molecular pathways of Q regulating plant growth and developmentThe ten developmental stages for the spike from the young spike differentiation stage to heading stage were observed and compared.Obvious differences were observed between NAUH164 and Sumai 3 in five stages from floret differentiation stage to heading stage.Therefore,gene expression patterns of wheat spike at these five stages were compared between them.Heat-map clustering analysis showed that trend of genome-wide gene expression for NAUH164 and Sumai 3 was generally identical.However,NAUH164 displayed a delayed expression for a portion of genes,including 40 MADS-box genes in the developmental processes.GO and KEGG analysis indicated most of these genes are involved in nucleotide metabolism,amino acid metabolism and genetic information processing.Moreover,these genes showed opposite gene expression pattern to that of 5AQ or 5Dq’ and are deduced to be suppressed by Q/q/q’.We deduced Q/q/q’ regulated plant growth and development through the inhibition of MADS-box genes which subsequently affected the expression of a large number of responsive genes associated with reproductive growth.We have also identified a gene A which are down-regulated simultaneously at five time points in NAUH164 compared with wild type Sumai 3.This gene is involved in the regulated ubiquitination of specific protein substrates and targets them for degradation bythe proteosome.DELLA protein is a key negative regulator of gibberellin（GA）signaling and targeted by SCF complexes.We deduced Q gene could regulate GA pathway through the inhibition of Gene A which results in the accumulation of DELLA protein and the blocking of GA signaling.