| Aluminum(AI)toxicity is one of the important factors that restricts the yield of crops in acidic soils.Studies have shown that planting Al-resistant/tolerant varieties is one of the most economical and effective approaches to increase crop yield in acid soils.Tibetan wild barley,has abundant genetic diversity and is an important germplasm resource for studying the mechanism of tolerance.At present,there has been reported that the acid/Al resistance of barley is revealed from the perspective of physiology and candidate genes.However,there are few identifications of microRNAs(miRNAs)related to acid/AI stress in barley.This study intended to use high-throughout sequencing technology to investigate the genotype differences of miRNA expression in barley roots of Tibiten wild and cultivated genotypes in response to acid/Al stress.The expression pattern of genes was expected to provide the basis for the analysis of barley miRNA-mediated acid/Al stress regulation networks.The main results were summarized as follows:The hydroponic experiment was carrired out using two Tibiten wild barley genotypes XZ16(acid/Al-tolerant)and XZ61(acid/Al-sensitive)and one acid/Al-tolerant cv.Dayton,treated with 50 μM Al(pH4.3,24 h)and control conditions(CK1,basic nutrient solution(BNS)at pH6;CK2,BNS at pH 4.3).A total of 216,404,167 clean reads were obtained from 9 root libraries(XZ16-CK1,XZ16-CK2,XZ61-CK1,XZ16-A1,XZ61-CK2,XZ61-Al,Dayton-CK1,Dayton-CK2,Dayton-Al)using high-throughput sequencing.Statistical analysis was performed on the length distribution of small RNAs from wild barley under different treatments.The results showed that the sequence length of small RNA was mainly 21nt and 24nt,among which 24nt sequences occupyed the majority.Compared with the miRNA database,there were 70 perfectly matched conserved miRNAs,of which 27 miRNAs belonged to 13 miRNA families,and the remaining 43 miRNAs were still unknown.Besides,1113 novel miRNAs were found.Among them,45 conserved miRNAs and 77 new miRNAs were significantly differently expressed under acid/Al stress and control conditions.These miRNAs were involved in plant growth,antioxidant defense,and stress response,implying that miRNAs and their target genes might play an important role in acid/Al resistance.Analyzing and comparing the genotypic differences of root miRNA changes before and after acid/AI stress,we identified 83 miRNAs involved in the A1 response process,including 28 conserved miRNAs(from 10 families)and 55 novel miRNAs;39 miRNAs participated in the acid response,including 17 conserved miRNAs(from 8 families)and 22 novel miRNAs.Under A1 stress,11 miRNAs(hvu-miR 166b,hvu-miR168-3p,Novel-m0252-5p,Novel-m0333-5p,Novel-m0406-5p,Novel-m0636-3p,Novel-m0796-3p,Novel-m1074-3p,Novel-m 1236-5p,Novel-m1328-3p)were down-regulated in XZ16 and up-regulated/unchanged in XZ61.5 miRNAs(hvu-miR6186,Novel-m0164-5p,Novel-m0380-3p,Novel-m0513-3p,Novel-m 1426-3p)remained unchanged in XZ16 and were up-regulated in XZ61.Under acid stress,13 miRNAs(hvu-miR159b,hvu-miR168-5p,Novel-m0432-3p,Novel-m0456-3p,Novel-m0547-3p,Novel-m0748-3p,Novel-m0800-3p,Novel-m0833-3p,Novel-m1032-3p,Novel-m1060-3p,Novel-m1132-3p,Novel-ml257-3p,Novel-ml454-3p)were down-regulated in XZ16,up-regulated/unchanged in XZ61.4 miRNAs(hvu-miRl 56a,Novel-m0403-5p,Novel-m0636-3p,Novel-m0960-3p)were unchanged in XZ16 and up-regulated in XZ61.These miRNAs were considered to be acid/Al stress-related miRNAs.70 conserved miRNAs and 1113 novel miRNAs were predicted by using psRobot and TargetFinder.14535 target genes were found through 1107 miRNAs,and finally 10832 target genes(1620 from conserved miRNAs,10155 from novel miRNAs)were obtained after filtering.The functions of these target genes were annotated with GO and KEGG,and it was found that genes had different functions and they were related to transcriptional regulation,protein transportion,and energy metabolism.Through the functional prediction of target genes,the results showed that most target genes of barley miRNAs were plant-specific transcription factors,such as MYB,AP2,HD-zip,SP family.It was also found that the target genes of several conserved miRNAs in barley had homology among different plant species,such as hvr-miR166 target genes were HD-Zip,hvr-miR159 target genes were MYB,hvr-miR397 target genes were laccase,hvr-miR168 target genes were EREBP,and the other target genes regulated by miRNAs included the MFS transporter family(hvr-miR397),transporter SLC2 A13(hvr-miR397),hydrolase(hvr-miR6179),and ATP-dependent RNA helicase(hvr-miRNA156).In addition,there were some unknown-function genes that were predicted as target genes for miRNAs.There might be closed related to the differences of acid/Al stress among the three barley genotypes,and we believed that these miRNAs also played an important role in the life activities of barley.Through the analysis of root transcriptions of XZ16,XZ61 and Dayton in response to Al stress,54 genes were identified.The expression of XZ16 was up-regulated and XZ61 was down-regulated/unchanged,or XZ16 was unchanged and XZ61 was down-regulated(Al vs CK2).The results showed that these genes were mainly involved in energy metabolism,hydrolase activity,stress stress,cell growth and other biological processes.Comparing the two acid/AI-tolerance genotypes XZ16 and Dayton,we found that the expression of these genes was different between the two genotypes in response to acid/Al stress.For example,MLOC 53797.5 was significantly up-regulated in XZ16 and down-regulated in Dayton.Based on the inconsistency of gene expression patterns between XZ16 and Dayton,we speculated that there were different mechanisms of acid/Al tolerance between XZ16 and Dayton. |
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