Identification And Comparative Analysis Of MiRNAs During Seed Germination Of Wheat Varieties With Different Pre-harvest Sprouting Resistance

Wheat(Triticum aestivum L.)is an important source of human healthy diet,rich in a variety of nutrients,including carbohydrates,proteins,and minerals.In recent years,due to the frequent occurrence of global extreme weather,pre-harvest sprouting has caused enormous economic losses to wheat crop production,which seriously affects the yield and quality of wheat.Micro RNAs(miRNAs)are a very important transcription level regulator in organisms and play an indispensable role in plant growth and development.Studies have shown that miRNAs play a key role in the process of seed germination,and seed germination is one of the important factors affecting pre-harvest sprouting.At present,research on miRNA-mediated plant seed germination is mainly focused on crops such as rice,corn,and rapeseed,while the molecular mechanism of miRNA-mediated wheat seed germination is less studied.Therefore,exploring miRNAs and genes related to wheat pre-harvest sprouting resistance and clarifying the molecular mechanism of wheat seed germination mediated by miRNAs is of great significance for genetic improvement of new wheat varieties with pre-harvest sprouting resistance.In this study,resistant pre-harvest sprouting germplasm Huamai 1168 and sensitive pre-harvest sprouting germplasm HZ1005 were used as experimental materials.18 miRNA libraries were constructed by high-throughput sequencing of their seeds after 12 h,18h,and 36 h of germination.The miRNAs and target genes that are significantly differentially expressed during seed germination in two varieties have been comprehensively mined and identified.Further,through functional enrichment and differential expression analysis,the functions of candidate miRNAs related to wheat germination and the regulatory mechanisms involved were preliminarily explored.The main results in this research were as follows:1.Phenotypic identification of pre-harvest germination: The whole pre-harvest germination experiment in 2021 and 2022 showed that the pre-harvest germination rate of Huamai 1168(1.8 % and 7.2 %)was significantly lower than that of HZ1005(48.2 % and56.7 %).The seed germination rates revealed the 4-fold and 2-fold changes between HZ1005 and Huamai 1168 from 24 h to 48 h.At 72 h,the germination rate of both wheat varieties reached over 97 %.The results showed that the seeds of the two varieties had the same germination potential,but the germination time of the Huamai 1168 was significantly delayed.2.Analysis of miRNAs sequencing results: By comparing 18 sRNA libraries,a total of 1104 miRNAs were identified(856 known miRNAs and 248 new miRNAs).A total of1036 miRNAs were identified in HZ1005(including 832 known miRNAs and 204 new miRNAs),and 751 miRNAs were identified in Huamai 1168(including 601 known miRNAs and 151 new miRNAs).The number of miRNAs identified during the germination of HZ1005 seeds was 285 more than that of Huamai 1168.Randomly select some known and new miRNAs and use real-time fluorescence quantitative PCR to verify the expression level.The quantitative results show that the sequencing results are reliable.3.Expression profile analysis of differentially expressed miRNA:By comparing the differentially expressed miRNAs in the two varieties at three time points(SG12vs RG12,SG18 vs RG18 and SG36 vs RG36),it was found that the expression of 30 miRNAs in the three periods was always up-regulated and the expression of 49 miRNAs was consistently down-regulated in the three periods under the condition of p <0.05.The number of down-regulated miRNAs was significantly higher than that of up-regulated miRNAs.94 miRNAs were differentially expressed in three periods(SG12vs RG12,SG18 vs RG18,and SG36 vs RG36).By comparing the miRNAs expressed in the two materials(SG12vs SG18 vs SG36 and RG12 vs RG18vs RG36),85 miRNAs were specifically expressed in Huamai 1168,262 miRNAs were specifically expressed in HZ1005,and 364 miRNAs are co-expressed in both germplasm.Based on the clustering of the expression levels of these miRNAs during the germination of Huamai 1168 and HZ1005,47 miRNAs that were highly expressed in Huamai1168 and 43 miRNAs that were highly expressed in HZ1005 were selected.4.Enrichment analysis and functional validation of miRNA target genes: Through GO and KEGG enrichment analysis of differentially expressed and highly expressed miRNAs regulated target genes in Huamai 1168 and HZ1005,it was found that pathways related to glucose metabolism were co enriched.At the same time,6 miRNAs related to sugar metabolism were screened and their expression levels during seed germination were quantified using q RT-PCR.Among them,it was found that the target genes regulated by ata-mi R166c-3p＿R-2,Traes CS3D02G359300.1(starch phosphorylase)and Traes CS1B02G172400.1(malate dehydrogenase MDH),may be key genes regulating the germination rate of wheat seeds.In addition,5 miRNAs related to plant hormone signaling and their regulated target genes also have an impact on the rate of wheat seed germination.In summary,miRNA mediated carbohydrate metabolism and hormone signal transduction are important factors affecting wheat seed germination.This study provides a new idea for the study of wheat seed germination mechanism and the mining and utilization of wheat pre harvest sprouting resistance genes.