- Identification of miRNA-eQTLs in maize mature leaf by GWAS. - Background: MiRNAs play essential roles in plant development and response to biotic and abiotic stresses through interaction with their target genes. - Little is known about the content within the plant genome contributing to the variations in plants. - This study aims to identify miRNA expression-related quantitative trait loci (miR-QTLs) in the maize genome.. - Investigation of the effects of these miRNAs on the expression levels and target genes provided evidence that miRNAs control the expression of their targets by suppression and enhancement.. - Conclusions: These identified significant miR-eQTLs contribute to the diversity of miRNA expression in the maize penal at the developmental stages of mature leaves in maize, and the positive and negative regulation between miRNA and its target genes has also been uncovered.. - In plants, pri-miRNA is transcribed by RNA polymerase II from the miRNA gene with a 5′ cap and 3′ polyadenylation modification and subsequently removed by a microprocessor while cutting the end of the stem-loop structure to produce pre- miRNA. - In the cytoplasm, only a single strand of the mature miRNA is loaded into the RNA-induced ribonucleoprotein silencing complex (RISC), which includes Argonaute (AGO1), to guide the miRNA by finding its target sites to cleave the corre- sponding mRNA or to inhibit translation [2]. - In plants, miRNAs regulate target genes by being nearly comple- mentary to the target sequences. - The identification of miRNAs and their target genes has been well. - The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. - Full list of author information is available at the end of the article Chen et al. - BMC Genomics . - Most of the targets are transcription factors, indicating the import- ance of plant miRNAs in gene regulation [7, 8]. - In addition, the global repression of miRNA expression has been reported in maize hybrid lines, which may be crit- ical for increasing yield [16, 17]. - low phosphorus-related miRNAs [20], and low nitrate-related miRNAs [21, 22] in maize seedling roots. - How- ever, it remains unclear how genomic loci affect miRNA expression in the maize genome.. - In addition, in humans, miRNA expression-related quantitative trait loci (miR-eQTLs) in different organs or stimulations have been reported, in- cluding human fibroblasts [33], glioblastoma [34], adi- pose tissue [35], infected dendritic cells [36], and 5239 human whole blood samples [37].. - Although the locations and functions of miRNAs in plants have been revealed by numerous studies, to the best of our knowledge, none of them have focused on miR- eQTLs in plants. - The object- ive of this study is to reveal this relationship by combining the maize HapMap and miRNA expression profile of a maize panel (200 maize lines) to identify miR-eQTLs. - This is the first report on the identification of four miR-eQTLs in maize and the finding that miRNAs regulate their target genes in both negative and positive ways.. - Identification of the highly expressed miRNAs in the maize panel. - A total of 31 miRNAs belonging to 17 miRNA families were identified and defined as highly expressed miRNAs based on the sum of total read counts greater than 2000 among 200 maize lines of the panel (Fig. - To understand the diversity of miRNA expression pat- terns in the maize panel, 31 highly expressed miRNAs were analyzed by multidimensional scaling (MDS) ana- lysis, and no cluster was detected (Fig. - Furthermore, the Boxcox transformation was applied for highly expressed miRNAs individually, which transformed the distribution of miRNA expression to a normal distribu- tion for GWAS analysis.. - The coexpression of highly expressed miRNAs in the maize panel. - The expression level of the 31 highly expressed miRNAs was input as phenotype data in GWAS analysis based on general linear model (GLM) plus 5 PCA and 2 latent PCA to control the population structure, and the Bon- ferroni correction level was used as criteria to identify miR-eQTLs. - The effect of miR-eQTLs on their target genes. - It is evident that miRNAs control plant development by regulating their target genes. - To understand whether miR-eQTLs identified in this study could affect the ex- pression of the associated miRNAs and their target genes, the expression levels of miRNA and their target genes in the maize panel were obtained from the same mature leaf RNA samples for both miRNA library and. - In this way, we could ensure the accuracy of the expression level of miRNA relative to their target genes. - To understand the regulation of miRNAs on their target genes, the expression levels of miRNAs and their target genes were collected, separated and compared based on the genotypes of significantly as- sociated SNPs in the identified miR-eQTLs individually (Table 4). - This indicated that miRNA might exert its control over target genes by suppression. - 6c) and its target genes GRMZM2G478709, GRMZM2G124566 (GRF-transcrip- tion factor 9, grftf9), and GRMZM2G033612 (GRF-tran- scription factor 5, grf5) (Fig. - The difference of miRNA expression level of miR156k-5p distinguished by most significant SNP type. - Highly expressed miRNAs and coexpressed miRNAs This study aimed to identify miR-eQTLs in a specific de- velopmental stage using the mature leaf tissue (leaf at the flowering time) of the maize panel as RNA sources for both mRNA and small RNA libraries. - In this study, miRNAs of the mature leaf tissue analyzed showed that the expression level of miR159a-3p was the highest, while miR171d-3p was the lowest, which was quite different from the reported miRNA types in a pre- vious study [18]. - This might be caused by the difference in environmental factors in the maize field including water, sunlight, winds, and soil conditions between ma- terials reported in Zhang et al. - The difference of miRNA expression level of miR159a-3p distinguished by most significant SNP type. - It was suggested that coexpression might occur among those miRNAs in the proximity of the genomic regions [39]. - To understand if 31 highly expressed miRNAs were response to the environment or the physically clustered, the coexpression of highly expressed miRNA were de- tected. - With regard to the coexpression of miRNAs, the function of the miRNA. - target gene is one of the possible reasons why miRNAs would be coexpressed within the same miRNA family or between members of different miRNA families. - The difference of miRNA expression level of miR390a-5p distinguished by most significant SNP type. - On the. - We have tested if the six genetic groups affect the miRNA expres- sion of the final four targeted miRNAs, and found that no differences was shown among six different maize subgroups.. - The difference of miRNA expression level of miR396e-5p distinguished by most significant SNP type. - Table 4 Expression trend between miRNA and its target genes Associated. - miRNA expression. - Maize growth in the field may owe its endurance to the above special en- vironmental factors. - A total of eight candidate genes were found within the genomic region of miR-eQTL that contribute to the ex- pression variance of miR156k-5p (Table 3). - One of the candidate genes, hagtf27 (GRMZM2G049730), showed homology to histone acetyltransferases involved in several metabolic pathways in plants [50], such as lipid metabol- ism and jasmonic acid biogenesis in cotton [51]. - It has been reported that DNA damage could regulate miRNA expression through a p53- dependent pathway or modulation of the steps of miRNA processing and maturation. - candidate genes were identified within the miR-eQTL genomic region and contribute to the expression of miR156k-5p in this study. - For another two miR-eQTLs, MiR390a-5p and miR396e-5p, no annotation of the candi- dates GRMZM2G108694 and GRMZM2G027282 in maize genome databases could be found. - The expression of these genes has no significant correl- ation to the miRNA expression after analysis by Pearson’s correlation coefficient. - This suggested that there might be other factors affecting miRNA expression in additon to the genes within the eQTL region.. - The possible regulatory mechanisms of the four miR- eQTLs and their target genes. - The relationship between miRNAs and their target genes is of great interest. - This study identified that miR-eQTLs contribute to miRNAs, which showed both negative (miR156k-5p, miR390a-5p) and positive (miR159a-3p, miR396e-5p) ways of regulating their target genes indi- vidually (Table 4, Fig. - In this study, the target binding sites of miR156k-5p and miR390a-5p were all at the 3’UTR of miRNA target genes.. - On the other hand, miR159a-3p and miR396e-5p were at the CDS of miRNA target genes. - For the negative regula- tion of miRNAs on their target genes, such as miR156k- 5p and miR390a-5p in this study, it has been reported that human miRNAs repress their target genes through RNA degradation and translational repression pathways. - For positive regulation, in humans, miRNA was reported to accumulate in the cell nucleus [59]. - However, this study identified positive regulation of miR159a-3p and miR396e-5p through binding the CDS of target genes that did not include any type of upregulated miRNA described above [60]. - Its biogeneration methods, functions, and target genes have also been investigated carefully. - expressed miRNAs using NGS on small RNA libraries derived from mature leaf samples of the maize panel (200 maize lines) to understand the network of miRNAs.. - A strong positive correlation was found within and be- tween miRNA families, and environmental factors might be the cause of the coexpression. - This study is the first report on the identifi- cation of maize miR-eQTLs and demonstrates that both negative and positive regulatory relationships exist be- tween miR-eQTLs and their target genes. - The positive regulation of miR159a-3p and miR396e-5p on the tar- gets suggests that some plant miRNAs might regulate their target genes positively by a mechanism different from that of humans.. - The maize 282 panel as mentioned in the previous study [61] was applied. - The second leaf from tassel was collected and the leaf section was made based on 1 cm square in the center of the leaf, three plants were com- bined per genotype for further analysis. - For developing small RNA libraries through the NEB small RNA library prep kit, a 2 ng fraction of the total RNA of each sample from 282 maize lines was prepared and shipped to the company “Global Biologics”. - Library analysis and estimation of miRNA expression A total of 200 out of the original 282 libraries derived from mature leaf samples were selected based on the quality of sequencing, and reads with lengths between 20 and 32 bp were kept after trimming the adapter.. - Reads from libraries were classified by length and matched with the relative length of the miRNA reference. - Highly expressed miRNAs were selected and defined as the sum of matched read numbers among all maize lines in the panel higher than 2000 and kept for further ana- lysis. - with the matched read number as a phenotype of the specific miRNA. - Regulation network among most significant SNP, miRNA and miRNA target genes. - Regulation network of miR156k- 5p and its target genes based on the most significant SNP. - Regulation network of miR159a-3p and its target genes based on the most signifi- cant SNP. - Regulation network of miR390a-5p and its target genes based on the most significant SNP. - Regulation network of miR396e-5p and its target genes based on the most significant SNP.. - miR-eQTL: miRNA expression-related quantitative traits loci. - SYC wrote the draft of the original manuscript and the experiments. - NKL helped to grow and took care of the maize plants. - This work was also par- tially financed to Hsin-Mei Ku by the funding from the Advanced Plant Bio- technology Center from The Featured Areas Research Center Program within the framework of the Higher Education Sprout Project by the Ministry of Education (MOE) in Taiwan.. - Genome-wide approaches in the study of microRNA biology. - BMC Genomics. - Identification and functional analysis of miRNAs in developing kernels of a viviparous mutant in maize. - A genome-wide characterization of microRNA genes in maize. - Genome-wide identification of microRNAs in response to low nitrate availability in maize leaves and roots. - Genome-wide expression quantitative trait loci (eQTL) analysis in maize. - A genomic portrait of the genetic architecture and regulatory impact of microRNA expression in response to infection. - Identification of miRNAs and their target genes in developing maize ears by combined small RNA and degradome sequencing. - BMC genomics . - Differential expression of miRNAs in response to salt stress in maize roots. - Analysis of UV-B regulated miRNAs and their targets in maize leaves. - Regulation of small RNA accumulation in the maize shoot apex. - Spanudakis and Jackson: The role of microRNAs in the control of flowering time. - Structure and functions of the GNAT superfamily of acetyltransferases. - Construction of the third generation Zea mays haplotype map
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