Study On The Construction Of Detailed Genome-wide Maps Of Three Leeches And The Evolution Of Their Anticoagulant Mechanisms

Leech is the common name for the species from class Hirudinae,phylum Annelida.Leeches parasitic living on the body surface of fish,reptiles and mammals,and feeding on blood or feeding on mollusks.The blood sucking leeches are widely used in clinical treatment of thrombotic diseases by Chinese and western medicine because of the production of highly effective anticoagulant active substances in the process of blood sucking.Besides hirudin,a variety of anticoagulant active substances have been reported.However,the genetic synthesis pathway of these anticoagulant active substances and their generation process in the evolution of leeches are not clear.The genome contains all the genetic information of a species,which is the basis of biological life activity and evolution research.The complete genome is often referred to as the complete genome map,which includes all the gene sequences of a species,as well as the number,location,function,correlation and other information of these genes.The construction of the whole gene map of a species not only lays a foundation for studying the expression mechanism and evolution model of genes,but also reveals the pathways and mechanisms of the formation and physiological evolution of biological species by comparing with the genomes of other species.However,due to the high cost of sequencing technology at present and the difficulty of map construction and analysis,only a few model organisms or those closely related to humans have carried out whole genome sequencing.Among the leeches,only the entire genome Helobdella robusta,which is a species distributed in the United States feeding on the mollusk's,was reported.There were three medical leeches included in the 'Chinese pharmacopoeia':Hirudo nipponica,Whitmania pigra and Hirudinaria manillensis.The whole genome mapping and comparative genomic analysis of these species will help to discover new anticoagulant substance,elucidate their working mechanism,and explore the formation process of leech blood-sucking habit,providing data for the development of evolutionary biology and medicine.This study applied the current advanced 3-generation long segment Pacbio sequel sequencing technology and the second-generation Illumina sequencing technology to obtain DNA and RNA data,and conducted assembly,annotation and comparative genome analysis of the genome of Hirudo nipponica,Whitmania pigra and Hirudinaria manidllensis.The results are as follows:1.Data acquisition,assembly and annotation of leech genomeIn this study,the second-generation short-read Illumina sequencing was used to obtain the DNA sequence data of H.nipponica,W.pigra and H.manillensis and retrieved 12.43 Gb,12.34Gb and 12.23Gb,respectively.These data were applied in estimation based on the k-mer distribution genome size and heterozygosity.Third generations long-read Pacbio SMRT cell sequencing were used to obtain 17.46 Gb,13.50 Gb and 14.98 Gb DNA sequences from of H.nipponica,W.pigra and H.manillensis,respectively.After quality control,the assembly was completed in WTDBG after falcon correction.Repeatmodeler and repeatMasker were combined to annotate the repeat sequencse,and software such as Augustus,PASA,GeMoMa and EVM was used to identify the number and location of genes.Gene functions were obtained by blast comparison with GO,KEGG and interproscan databases.The anticoagulant protein sequences were downloaded from the swiss-prot library,and the genome assembled was screened to look for all similar gene sequences using tBlastn.RNA sequence data of H.nipponica,W.pigra and H.manillensis of 43.05 Gb,43.29 Gb and 41.80 Gb were obtained by second-generation short-read Illumina sequencing.Each species consists of two groups:before and after feeding.These transcriptome reads were compared to corresponding reference genomes using Hisat2+stringtie,and the most efficient samples were used for differential gene expression analysis.Identification of homologous genes were conducted in Orthomcl.Single copy homologous genes were aligned in MAFFT,and Timetree was constructed in beast.Pam1X was used to calculate the rate of gene evolution.The gene family list of homologous genes screened by CAFE is used for inter-species comparison.2.The genomes of H.nipponica,W.pigra and H.manillensis.The genome sizes of the H.nipponica,W.pigra and H.manillensis were 170.4 Mb,164.9 Mb and 15.7 Mb,respectively.Their contig\scaffold N50 were 1.78 Mb,2.88 Mb and 2.28 Mb,respectively.The number of scaffolds of the assembly of H.nipponica,W.pigra and H.manillensis was 713,467,455,and the longest reached 3.97 Mb,6.46 Mb and 5.92 Mb,respectively.All these assembly parameters were better than the existing genome of H.robusta,and was comparable to most available invertebrate genomes.In this study,23,131,20,582 and 21,005 genes were identified from the H.nipponica,W.pigra and H.manillensis genome,and the core genes of insect core gene integrity BUSCO detection showed that 93.1%,94.1%and 90.1%were found respectively.All genes were annotated using the existing database,and the results showed that 18,493,16,884 and 17,051 were able to obtain functional annotations,among which 167,155 and 238 were related to anticoagulation.In this study,we also obtained the mitochondrial DNA(mtDNA)genome of H.nipponica,W.pigra and H.manillensis.These mtDNA genomes retain same gene content as in most multicellular animals,but the final assembly obtained by using three-generation Pacbio sequencing long reads show that there are previously undetected noncoding sequences of more than 5,000 bp in H.nipponica and W.pigra.3.Analysis of the expression pattern of ancoagulant gene based on transcriptomeThe differences in expression genes of three kinds of leeches before and after feeding were mapped and compared based on the annotated genome.The results showed that 14,356,13,549 and 13,360 genes were expressed before and after feeding in the H.nipponica,W.pigra and H.manillensis,and there were 104,155 and 128 genes related to anticoagulation,respectively.The functional genes related to anticoagulant and promote blood flow,including Hirudin,were differentially expressed in H.nipponica and H.manillensis.The anticoagulant genes expressed by the W.pigra was similar to that of the medical leech,but the difference in expression level was not obvious before and after feeding.4.Evolution of anticoagulant mechanisms in leeches.By comparing the genomes of H.nipponica,W.pigra and H.manillensis,the number of homologous anticoagulant genes represented by Hirudin/Antistasin gene family was found increased during the evolution of the leeches.The presence of anticoagulant genes is not the result of rapid evolution.Based on the results of the construction of single-copy homologous sequences,we speculated that the occurrence of these genes was accompanied by species differentiation,with time nodes around about 65 Mya.At this time,mammals began to replace reptiles on the surface of earth,and the anticoagulant properties of leeches could due to the coevolution of mammalian,driven by feeding.The genetic mechanism of blood-sucking anticoagulant gene of W.pigra degenerates,although sequences are still retained in the genome.The strong evolutionary adaptation of this species occurred in lipid metabolism and other aspects.