| Pygmy mole crickets,or pygmy sand crickets,are the common names of species in Tridactyloidea.Normally,they are small in body size,good at jumping and widely distributed all around the globe.Unlike other orthopteran insects,pygmy mole crickets often feed on moss and occasionally on crops such as corns and rice.Because of its small body size,hidden living environment and being little harmful to crops,up to now,people have not paid enough attention to this group of species.However,as an important group of orthoptera,the study on them will provide important supplementary information on the physiology and biochemistry,species origin as well as evolutionary adaptation of this group and even orthopteran insects.Current studies on pygmy mole crickets are primarily confined to morphological classification,while molecular biology and genomics analysis have rarely been reported.Therefore,this study adopted high-throughput sequencing technology to analyze the genome and transcriptome characteristics of Xya riparia,a commonly seen species of pygmole cricket,and conducted a comparative genomics study based on the currently available genomic data of orthopteran insects.The main results are as follows:(1)By using flow cytometry(FCM)and genome survey technology,the preliminary analyses on genome size,heterozygosity and CG content of Xya riparia were detected.Based on the results from FCM and genome survey,we combined the third-generation Nanopore sequencing technology with the second-generation Illumina sequencing technology,and used Hi-C technology as assistance to assemble the genome of Xya riparia.A total of 184.71 Gb of raw data was obtained.After data filtering,a total of 164.01 Gb of clean data was obtained,with the average length of reads being27.64 Kb.After data assembly and error correction,the genome size obtained was 1.67 Gb,with the Contig N50 size being 4.33 Mb,and the Scaffold N50 size being 319.09 Mb.With the help of HI-C genome assembly,a total of 1.66 Gb genome data was mounted to 6 chromosomes,accounting for 99.44% of the total genome size,and 1.58 Gb of sequences were correctly ordered and orientated,accounting for 95.63% of the total mounted genome size.A total of 714.02 Mb of repeats and 18,733 protein-coding genes were predicted,of which 16,468 genes were functionally annotated.43 mi RNA,60,317 t RNA,282 sn RNA and 205 r RNA were predicted.Meanwhile,2,691 pseudogenes were predicted.The assembly results shows a high quality in genome assembly,and can be used as a reference genome for downstream analysis such as referenced transcriptome analysis.(2)Transcriptome sequencing was performed on female and male adults of Xya riparia using the second-generation Illumina Novaseq 6000 platform,and 37.76 Gb of clean data was obtained,with Q30 values being no less than 93.7%.By aligning clean data with the reference genome,more than 91.51% of reads were aligned to the reference genome.69.99% and 6% of reads were aligned to the exon region and intron region,respectively,and 19.88% of reads were aligned to the intergene region.Reads aligned to intergenic regions may be caused by species-specific genes that have not yet been annotated.The transcripts were aligned against Nr,COG,egg NOG,GO,KOG,Pfam and Swissprot databases,17,266,6,818,14,728,15,096,12,069,15,502 and 8,695 annotations were obtained,respectively.4,013 and 807 transcripts with unknown functions were annotated against the egg NOG database and KOG database respectively,which may be because the databases are not comprehensive,or these transcripts are species-specific to Xya riparia but have not been annotated yet.6,064 new genes were identified,of which 1,628 were annotated functionally.The average number of SNP sites in the 6 groups was 162,050,and the average number of SNP sites was reversed in41.47%.Heterozygous SNP sites accounted for 68.08% of the total number of heterozygous sites.We also found that the frequency of transformation was much higher than that of inversion.Totally 13,968 In Del were annotated to intergenic regions,454 In Del annotated to genetic regions,and 26,367 In Del annotated to non-genetic regions.According to statistics of alternative splicing events,the results showed that the first exon on the 5’-terminal and the last exon on the 3’-terminal occurred the most frequently.The frequency of single exon jump event and variable 5’-or 3’-end splicing event ranked second.Multiple intron retention occurred the least.A total of 3,365 differentially expressed transcripts(DETs)were obtained from female and male samples,of which 2,552 were annotated,with an annotation efficiency being 75.84%.Compared with male adults,a total of 2,486 up-regulated transcripts were detected in female samples and were mainly related to metabolic process,including carbon metabolism,amino carbohydrate and ribose metabolism,oxidative phosphorylation,starch and sucrose metabolism,and glutathione metabolism.There were 879 up-regulated transcripts in male adults,which were mainly related to toxin metabolism,m TOR signaling pathway,Fo XO signaling pathway and other related processes.It is speculated that female adults are larger than male adults in body size,so their energy expenditure is relatively higher.Mature females also need more energy input to complete the reproduction process.For males,the up-regulated transcripts are mainly related to environmental adaptation,mainly to guarantee the individual survival under environmental stimulations or in adverse environments.(3)Genome data and gff files of Schistocerca gregaria,Locusta migratoria,Gryllus bimaculatus and Laupala kohalensis were downloaded from NCBI for comparative genomic analysis.Gene family clustering showed that 65% of the 18,733 protein sequences of Xya riparia were involved,and 545 specific gene families with 1,649 genes were found.Phylogenetic results showed that G.bimaculatus and L.Kohalensis clustered into one clade,S.Gregaria and L.Migratoria clustered into one clade,and X.Riparia clustered into a single clade and formed a sister group with S.Gregaria and L.Migratoria.The results of divergence time showed that the orthoptera divergented 383-439 MYA,caelifera and ensifera divergented 218-319 MYA,and acridoidea and Tridactylidea divergented 159-284 MYA.The gene family expansion and contraction analysis of Xya riparia showed that 612 genes in 63 gene families expanded.The expanded genes are mainly related to growth and development.Meanwhile,heat shock protein and cytochrome P450 related gene families also expanded.The contracted genes involved sugar metabolism and energy production,and may have something to do with their small body size and weak flying ability,thus the energy demand was relatively low.A total of 34 positive selection genes were screened and 16 were functionally annotated.1 gene related to contractin assembly,muscle tissue formation and actin cytoskeleton tissue regulation showed positive selection,which may be becase of the conspicuous jumping ablitiy of Xya riparia.1 gene involved in the development of sensory organ of touch was also positively selected,presumably because of the need to be sensitive to vibrations in the environment to avoid predators.In addition,no genome-wide replication from the analysis. |
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