Dissecting The Natural Variations Of Maize SRNA And Their Functions In Drought.response By High-throughput Sequencing

As one of the main abiotic stresses,drought negatively influences plant growth and yield.There are a lot of studies that have revealed the changes of genes at transcriptional,post-transcriptional and post-translational levels under drought stress.Small RNAs(sRNAs)are very important regulators of gene expression,thus play critical roles in plant development and stress responses.Previous studies have shown the function of some mi RNAs in stress responses,but the roles of the sRNA majority in stress responses remain largely unknown.Huge variations exist among various maize natural accessions,and are associated with gene expression and agronomical traits.But whether these variations are associated with maize sRNA expression is unknown yet.To dissect the associations between natural variations and sRNA expression,and further drought tolerance,we performed sRNA sequencing(sRNA-seq)based a population panel consist of 338 maize natural accessions and RNA-seq based on 197 lines from this panel with or without drought stress.We detected a lot of sRNAs with changed expression upon drought treatment(DR sRNAs),and found that 21-22 nt sRNAs tend to be up-regulated,while 24 nt sRNAs tend to be down-regulated.We performed statistical and regression analyses,and found that the putative target genes of DR sRNAs were significantly enriched in GO terms of stress response and metabolic related pathways.We performed GWAS to these DR sRNAs and identified 4265 and 4509 eQTLs in the population under well-watered and drought conditions respectively.Most of the eQTLs could be only detected in control or drought condition,suggesting that the expression variation of sRNAs is affected by both drought stress and sequence variation.We further analyzed an eQTL hotspot on chromosome 8(located in the region of ?140 Mb),which was only detected under drought stress.Further analysis showed that there is an inverted repeat(IR,?21.4kb)inserted upstream of the eQTL,and we named this insertion IR6642.IR6642 was located in gene Zm PP2C16,a member of Zm PP2 CA gene family.IR6642 was co-transcripted with Zm PP2C16 and finally processed into sRNAs.Insertion of IR6642 did not change the expression of Zm PP2C16,but altered thealternative splicing of Zm PP2C16.Statistic and linkage analyses revealed that IR6642 was significantly associated with drought sensitivity.Further,we found that the expression of many target genes could be regulated by IR6642-derived sRNAs through post-transcriptional gene silencing(PTGS)mechanism.As a proof of concept,IR6642-derived sRNAs digested Zm00001d016784,encoding a MYB protein,and overexpressing this gene enhanced plant drought tolerance.Besides drought tolerance,IR6642 was also correlated with maize kernel length.Moreover,the expression levels of many genes involved in kernel size regulation were significant correlated with IR6642.However,interestingly,no gene was predicted to be the target of IR6642-derived sRNAs,suggesting that IR6642 control kernel length via regulating these genes indirectly.Genotyping of IR6642 among teosintes,landraces and inbred lines suggested that IR6642 plays an important role in maize domestication and is selected during maize improvement due to various levels of precipitation.To further dissecting the relationship between IR and sRNA expression,we investigated genome-wide IR structures and identified 8261 IRs in maize genome.A large number of sRNAs can be mapped to these IRs,and interestingly,21-22 nt sRNAs were enriched.We further found that the expression of IR-derived sRNAs of different species were significant correlated with the lengths of IRs.By using sRNA sequencing data of mutants of sRNA processing genes,we found that IR-derived 21-22 nt sRNAs were processed in different pathways compared with IR-derived 24 nt sRNAs.We found that many eQTLs had IR structure in its flanking region,suggesting important roles of IRs in sRNA production.Further,we performed association analysis and found that the variations of IRs(in the flacking regions of eQTLs associated with DR sRNAs)were significant associated with many important agronomic traits.The comparison of IRs between modern maize and teosintes suggested that IRs in maize genome are rapidly gained and lost during domestication and improvement.In summary,we performed large-scale high-throughput sRNA-seq and RNA-seq for large maize populations in this study.We detected a large amount of drought-responsive sRNAs and identified many eQTLs associated with these sRNAs.We deeply studied thefunction,mechanism and evolution of an eQTL hotspot IR6642.Finally,we investigated the variations of genome-wide IR structures,and studied their associations with sRNA expression and maize agronomic traits.The huge sequence and expression variations detected in this study have the potential to be used as molecular markers in maize breeding.