Analysis Of Transcriptome Of Broomcorn Millet Based On Rna-seq And Condon Usage Bias In The Chloroplast Genome Of Broomcorn Millet (Panicum Miliaceum L.)

Broomcorn millet(Panicum miliaceum L.),also known as proso millet or hog millet,is one of the oldest domesticated crops,and is mainly distributed in semi-arid areas of Asia and Europe.As a short-day C4 crop,broomcorn millet has many excellent agronomic traits,such as high yield,relatively short growing season,lower demanding of water,and it is suitable to grow in various soil,especially in the barren sandy loam,thus it can be well adapted to the extreme environment.The RNA-seq mainly based on NGS(Next generation sequencing)technology can be high-quality and provide a comprehensive understanding of all transcriptional elements,including mRNAs,small RNAs and other non-coding RNAs.The results of the transcriptome could provide detailed description for expression patterns of the organs,tissues or cell gene in different physiological states,which is vital in understanding the biological development and the process of diseases attacking.The codon usage bias(CUB)is mainly due to difference of the usage frequency of the synonymous codon,which is an important evolutionary feature,and it is of great significance in exploring evolutionary mechanism between species,gene function and exogenous gene expression.In this study,the mixed RNA samples of Yumi 2 an Yumi 3 were subjected to RNA-seq sequencing,and the data were filtered and analyzed.RT-PCR was also performed on the candidate differential expressed genes.At the same time,some bioinformatic softwares were used to analyse codon usage bias of chloroplast genome of broomcorn millet,such as Mobyle.The results obtained are as follows:1.A total of 113,643 contigs were obtained from the mixed samples of four tissues using Illumina sequencing platform with the total length of 164,535,293 bp.A total of 65,470 unigenes longer than 1 kb were identified by sequence assembled;2.The blast similarity analysis results of the protein sequences of the assembled unigenes with the NR database,the Swiss-Prot database,the COG database,and the KEGG database revealed that a total of 60,352 unigene had been annotated.At the same time,it was found that the unigenes and other monocotyledons share higher homology,including sorghum,rice,Brachypodium distachyon,Triticum urartu,switchgrass.3.The results of GO,KOG and KEGG metabolic pathways analysis of unigenes showed that a total of 62,543 unigene sequences were annotated into 3 major GO classes.The 33,671 unigenes matched to the KOG database were classified into 25 different functional groups.15,514 unigenes are mainly assigned to 202 KEGG metabolic pathways.4.A total of 35,216 SSR loci were identified in the 27,055 sequences of 113,643 unigenes in broomcorn millet.Among the 2,536 sequence have more than one SSR.4 Repeated SSRs were the highest repeat type(49.99%),followed by 5,10,6 and over 20 repeated SSRs,and the proportion was reapectively about 14.45%,13.38%,8.79 % and 0.05%.In these different types of SSRs,the trinucleotides are the most abundant repeat units,accounting for about 66.72% of all SSRs,where CCG / CGG is the most abundant structural unit.Also,the length of most SSRs is 12bp(51.39%).5.A total of 292 differential expression genes were identified and classified into 88 GO and 12 KEGG metabolic pathways,and four differentially expressed genes were identified by qRT-PCR,which were unigene34608,unigene35973,unigene41558,and unigene33484;6.53 coding DNA sequences were chosen to be candidate genes.The results showed that the proportion of GC content at the third position was 30.67% in the cp genome of Broomcorn millet,which had the lowest GC content;Effective number of codons(ENC)ranged from 37.14 to 61,indicating that codon bias was weak;Relative synonymous codon usage(RSCU)results implied that nearly all of them ended with A or T,thus,those were preferable codons in the chloroplast genome of Broomcorn millet;Neutrality analysis,ENC-plot(ENC vs GC3S)analysis and correspondence analysis of relative synonymous codon usage(RSCU)together suggested that natural selection,mutation and other factors all contributed to the formation of the codon usage in the Broomcorn millet cp genome.and 9 optimal codons were identified within Broomcorn millet.