Construction Of Spermatic MicroRNA Libraries For The Study Of Fertility In Different Ploidy Cyprinids

Allotetraploid lineage,an excellent experimental material for biological evolution and genetic breeding of fish,was propagated to 25 filial generations.Males and females tetraploid individuals are fertile.And also,we got sterile triploid fish(TCC)by crossing the male tetraploid(AT)with female red crucian carp(RCC).Triploids do not contaminate natural fish germplasm resources.In addition,they have a potentially rapid growth rate and strong disease resistance,so its application prospects must be very bright.MicroRNAs(miRNAs)were small non-coding regulatory RNAs(About 22 nucleotides)that can negatively control the expression of target genes by combining to the 3'-untranslated regions of mRNAs,and resulting in miRNA-mediated mRNA decay or translational repression in eukaryotes.Research increasingly suggests that miRNAs play a key role in the organ formation,embryonic development,tumorigenesis and cell proliferation,differentiation,apoptosis.In fishes,Hypothalamus-Pituitary-Gonad axis(HPG)also has a significant role to play in gonad development.Kisspeptin,a key factor in the neuroendocrinological regulation of animal reproduction,playsan important role in the regulatory of HPG.In this research,we established miRNA libraries of the spermary tissue,conducted bioinformatics analysis which is also verified by the relevant experiments,and carried out a primary study on kiss genes in red crucian carp,triploid fish and allotetraploid fish.These will help us to have a further understanding in molecular mechanism of the sterile triploids,as well as systematically study the reproduction and development processes of the vertebrates.1.Small RNAs,used for RNA-seq by using the Illumina HiSeq 2500 platform,were prepared from 9 samples from red crucian carp(RC1,RC2,RC3),triploid fish(TC1,TC2,TC3)and allotetraploid fish(AT1,AT2,AT3).Ultimately,we got 9,072,963 clean reads 5,170,431 uniq reads from RC1;8,189,738 clean reads 4,391,085 uniq reads from RC2;8,579,897 clean reads 4,608,008 uniq reads from RC3;9,448,066 clean reads 5,522,231 uniq reads from TC1;9,364,165 clean reads 5,940,300 uniq reads from TC2;9,082,507 clean reads 5,649,978 uniq reads from TC3;10,232,739 clean reads 5,147,281 uniq reads from AT1;9,431,584 clean reads 5,315,216 uniq reads from AT2;and 9,910,677 clean reads 4,994,043 uniq reads from AT3 after quality evaluation and length selection.2.According to the length selection of miRNAs(about 21?22nt),miRNA is 9.47%of sRNA in RC1,7.69%in RC2,12.86%in RC3,16.31%in TC1,7.43%in TC2,8.70%in TC3,20.60%in AT1,15.18%in AT2,26.01%in AT3.143 known miRNAs and 122 novel miRNAs were identified using a combination of high-throughput sequencing and bioinformatics approach.And we also counted every base preference of each miRNA.3.Bio information analysis shows that 41 miRNAs were significantly upregulated and 34 miRNAs were significantly downregulated in TCC compared with RCC,and 53 miRNAs were significantly upregulated and 62 miRNAs were significantly downregulated in TCC compared with AT.Among them,simultaneously,17 miRNAs were downregulated and 30 miRNAs were upregulated in TCC compared with RCC and AT;while 2 miRNAs' expressions were increased as ploidy increasing.To comfirm the expression differences between the three different ploidy Cyprinids,9 differently expressed miRNAs were selected randomly from TCC compared with RCC and AT to analyze by qRT-PCR.Results show that there were 8 miRNAs had the same expressed pattern in the 3 species as that from sequencing data,but there were differences in relative expressed level.4.According to the target prediction and expression profile,some testified miRNAs may be involved in the expression of a series of genes associated with testis cell differentiation,sperm flagellar assembly and motility.What's more,the expressiontrends ofthese target genes were totally contrary to the expression trend of the testified miRNAs,further proving the reliability of the libraries.These miRNAs could bind the 3'-UTRs of mRNAs of the target genes,influencing miRNA-mediated mRNA decay or translational repression levels,causing the restriction of flagellar assembly,movement abnormalities of sperm,and the failure of cell differentiation in spermary,resulting in the sterility of TCC ultimately.5.We obtained partial cDNA sequences of kiss1 and kiss2 of RCC,TCC,and AT,and we also analysed and explored the expression difference of 9 kinds of tissues in different ploidy Cyprinids.