Genome-wide Dissection Of Genetic Mechanisms Underlying Intraspecific Variation Of Small Rnas In Maize

Small RNAs(s RNAs)with a length of 18-30 nucleotides(nt)are widely found in animals,plants,and fungi.They are an important class of RNA molecules and play a vital role in many biological processes in organisms.As early as in the 1980 s,research on s RNA was conducted and found that s RNAs are produced by the endonuclease DICER-LIKE(DCL).In 1999,s RNAs related to gene silencing were first discovered in plants.Subsequent studies have shown that s RNAs can regulate gene expression at transcription,post-transcription or translation level by complementary pairing with target gene sequences,thereby participating in plant growth and development,metabolic processes,and signal pathway regulation in response to various stress reactions important biological processes.There are wide variations in s RNAs in plants,and variations between different materials within the same species.Furthermore,it is reported that the intraspecific variation of s RNAs may be closely related to phenotypic variation within species.However,the types of s RNAs within the species and the underlying molecular mechanisms of the intraspecific variation are poorly understood,which to a large extent,limits our understanding of plant phenotypic variation and the practical application of molecular breeding.In order to systematically profile the types of intraspecies variation of s RNAs and dissect the molecular mechanism of their variations,we collected a deep s RNA-sequencing data from the leaf tissues of two widely-used maize elite inbred lines B73 and Mo17 and collected a maize population with extensive genetic variation(338 excellent inbred lines).The s RNAs data set contains 1.553 billion effective s RNAs sequences.After screening the sequence quality,we obtained 308.7 million high-quality s RNAs sequences of 18?26nt.In the B73 and Mo17 leaf tissue depth s RNAs data sets we obtained,154.6 million s RNAs sequences were uniquely mapped to B73 reference genome,154.1 million s RNAs sequences were uniquely mapped to the Mo17 maize genome,and the B73 mapped reads cover 6.98% and Mo17 cover 7.06% of their respective genomes.According to the distribution of s RNAs in the genome,in B73,38.47% of the s RNA is distributed in the intergenic region,followed by 30.37% is the gene region;and in the genome of Mo17,42.78% of the s RNAs are distributed in the intergenic region,the gene region accounts for 28.43%.This result shows an extensive intraspecific variation in s RNAs between both genomes in maize.Based on the criterion that the expression abundance(RPM)is greater than or equal to 0.6 and detected simultaneously in at least 60 different inbred lines,we have obtained a total of 339,797 s RNAs species,of which 171,813 s RNAs were detected in the B73 genome There were 167,984 s RNAs detected in,and there were 156,422 s RNAs detected in both genomes.Further analysis revealed that the expression of s RNAs between B73 and Mo17 changed to some extent.s RNA species 15391 and 11562 specifically obtained in both respective genomes.Additionally,about 3537 differentially expressed s RNAs were identified between two inbreds.In order to analyze the molecular mechanism of the extensive intraspecific variations of s RNAs between B73 and Mo17,we conducted a genome-wide expression association analysis on 1999 differentially expressed s RNAs between B73 and Mo17 in an association mapping population with extensive genetic variation.Up to 72,671 genomic SNP sites that were significantly associated with intraspecies s RNAs variation were identified.Further deredundancy analysis of related sites yielded 5744 trans-e QTLs that affected s RNA variation.These e QTLs have a certain preference in the distribution of 10 maize chromosomes.Among them,most trans-e QTLs(1633)were identified on chr8,followed by chr2(1077)and chr1(731),and chr10(183)has the least trans-e QTLs.In order to uncover the key regulatory factors that control the intraspecific variation of s RNAs within the maize genome,we found by Mont Carl simulation analysis that the trans-e QTL in these 5744 e QTLs were significantly enriched in the 26.37 Mb genome region,of which 25 enriched peaks were obtained as trans-e QTL hotspots spanning 2.2 Mb genomic region.Among these trans-e QTL hotspots,three major trans-e QTL hotspots are distributed on chr1,chr2 and chr8.In the trans-e QTL hotspot region of chr8,13 genes have a significant correlation with the expression of 428 of the 469 intraspecies variant s RNAs they regulate.It is worth noting that most of the 469 s RNAs regulated by the trans-e QTL hotspot on chr8 are 24 nt s RNAs,and there is significant s RNAs length enrichment.These 24 nt s RNAs are mainly related to the expression of three genes in the trans-e QTL hotspot region of chr8.These three genes are Zm00001d008982,Zm00001d008983,Zm00001d008984,which may participate in the biosynthesis and regulation of 24 nt s RNAs.Similarly,the trans-e QTL hotspot on chr2 regulates the expression variation of 180 s RNAs,of which 75 s RNAs are 22 nt s RNAs.These 22nt-length s RNAs are mainly related to the expression of a gene of unknown function in the hotspot region of transe QTL on chr2.Through similar correlation analysis and correlation analysis of expression abundance,we have identified ten functional gene loci that can regulate the expression variation of s RNAs,which provide us the understanding of the molecular mechanism of s RNA variation within crop species.In conclusion,this s RNA study provides us with a rich distribution of the whole genome and a landscape of s RNA variation between two genomes within a species.At the same time,genome-wide s RNAs expression correlation analysis identified 25 trans-e QTL hotspots that regulate s RNAs variation.These results deepen our understanding of s RNA variation within species and provide a new perspective for a more comprehensive understanding of phenotypic variation within species.