Genome Survey Sequences Research Articles

Genome variations in sea cucumbers: Insights from genome survey sequencing and comparative analysis of mitochondrial genomes

Sea cucumbers, marine benthic invertebrates, play crucial roles in maintaining the stability of marine ecosystems and hold key evolutionary positions. However, information regarding their genomes remains limited. Here, we conducted genome survey analyses on seven species from four orders. Results indicated that Colochirus anceps, Colochirus quadrangularis, and Pseudocolochirus violaceus within the order Dendrochirotida have significantly larger genomes (2238–3754 Mbp) compared to conventional sea cucumber genomes, accompanied by a very high proportion of repeat sequences (69.39–72.52 %). While Holothuria edulis and Holothuria atra exhibited similar genome sizes comparable to those of other species within the order Holothuriida, heterozygosity and repeat content varied among all the six species in this order. The representative species Apostichopus californicus of the order Synallactida possesses the smallest genome size (573.45Mbp) within its order, but its heterozygosity (2.24 %) is significantly higher than that of other species. The representative species Synapta maculata of the order Apodida exhibited a normal genome size (900.97 Mbp), lower proportion of repeat sequences (42.19 %), and lower heterozygosity (0.84 %), making it the species with the least challenges for genome sequencing and assembly in the future among all surveyed species. Subsequently, we compiled genomic information from a total of 19 sea cucumber genomes, both newly sequenced and previously reported, revealing a significant linear relationship (P = 0.0001) between genome size and the proportion of repeat sequences in sea cucumbers. Additionally, phylogenetic and comparative analysis of mitochondrial genomes among them indicated extensive rearrangements within the order Apodida, leading to significant discrepancies between mitochondrial and nuclear genome phylogenies.

Genome Survey Sequencing of the Medical Plant Dioscorea futschauensis: Genomic Characteristics Estimation and Genomic Resources Development

Dioscorea futschauensis Uline ex R. Knuth, a perennial climbing herb belonging to the Stenophora section of the genus Dioscorea L. (Dioscoreaceae), originates from Southeast China and is highly regarded for its medicinal properties. Despite its medicinal significance, the absence of genomic information has impeded the effective utilization and conservation of this species. Here, we conducted genome survey sequencing of D. futschauensis to unveil its genomic characteristics and identify species-specific genomic markers, including whole plastome sequence, plastome-divergent hotspots, single-copy nuclear genes and polymorphic nuclear SSRs. The genome of D. futschauensis was approximated to be 1392.76 Mb in length, accompanied by a substantial heterozygosity rate of 1.75% and a repeat content of 67.43%. Comparative plastome analysis illuminated the conserved genome structure and gene content within D. futschauensis, and revealed four highly divergent hotspots (atpF intron, psbA-matK, matK -rps16, and rrn16-trnI) that could serve as molecular markers for D. futschauensis and its closely related species. Furthermore, more than 200 single-copy nuclear genes and 78 polymorphic nuclear SSRs were identified within D. futschauensis. Overall, the comprehensive genomic information and abundant genetic resources provided herein will not only facilitate extended research in the realms of population genetics, phylogenetics and conservation biology of D. futschauensis, but also hold considerable value for its cultivation and molecular breeding applications.

Insights into the nuclear and mitochondrial genome of the Lemon shark Negaprion brevirostris using low-coverage sequencing: Genome size, repetitive elements, mitochondrial genome, and phylogenetic placement

The Lemon shark Negaprion brevirostris is an important species experiencing conservation issues that is in need of genomic resources. Herein, we conducted a genome survey sequencing in N. brevirostris and determined genome size, explored repetitive elements, assembled and annotated the 45S rRNA DNA operon, and assembled and described in detail the mitochondrial genome. Lastly, the phylogenetic position of N. brevirostris in the family Carcharhinidae was examined using translated protein coding genes. The estimated haploid genome size ranged between 2.29 and 2.58 Gbp using a k-mer analysis, which is slightly below the genome size estimated for other sharks belonging to the family Carcharhinidae. Using a k-mer analysis, approx. 64–71 % of the genome of N. brevirostris was composed of repetitive elements. A relatively large proportion of the ‘repeatome’ could not be annotated. Taking into account only annotated repetitive elements, Class I - Long Interspersed Nuclear Element (LINE) were the most abundant repetitive elements followed by Class I – Penelope and Satellite DNA. The nuclear ribosomal operon was fully assembled. The AT-rich complete mitochondrial genome was 16,703 bp long and encoded 13 protein coding genes, 2 ribosomal RNA genes, and 22 transfer RNA genes. Negaprion brevirostris is closely related to the genera Carcharhinus, Glyphis and Lamiopsis in the family Carcharinidae. This new genomic resources will aid with the development of conservation plans for this large coastal shark.

The nature and organization of satellite DNAs in Petunia hybrida, related, and ancestral genomes.

The garden petunia, Petunia hybrida (Solanaceae) is a fertile, diploid, annual hybrid species (2n=14) originating from P. axillaris and P. inflata 200 years ago. To understand the recent evolution of the P. hybrida genome, we examined tandemly repeated or satellite sequences using bioinformatic and molecular cytogenetic analysis. Raw reads from available genomic assemblies and survey sequences of P. axillaris N (PaxiN), P. inflata S6, (PinfS6), P. hybrida (PhybR27) and the here sequenced P. parodii S7 (PparS7) were used for graph and k-mer based cluster analysis of TAREAN and RepeatExplorer. Analysis of repeat specific monomer lengths and sequence heterogeneity of the major tandem repeat families with more than 0.01% genome proportion were complemented by fluorescent in situ hybridization (FISH) using consensus sequences as probes to chromosomes of all four species. Seven repeat families, PSAT1, PSAT3, PSAT4, PSAT5 PSAT6, PSAT7 and PSAT8, shared high consensus sequence similarity and organisation between the four genomes. Additionally, many degenerate copies were present. FISH in P. hybrida and in the three wild petunias confirmed the bioinformatics data and gave corresponding signals on all or some chromosomes. PSAT1 is located at the ends of all chromosomes except the 45S rDNA bearing short arms of chromosomes II and III, and we classify it as a telomere associated sequence (TAS). It is the most abundant satellite repeat with over 300,000 copies, 0.2% of the genomes. PSAT3 and the variant PSAT7 are located adjacent to the centromere or mid-arm of one to three chromosome pairs. PSAT5 has a strong signal at the end of the short arm of chromosome III in P. axillaris and P.inflata, while in P. hybrida additional interstitial sites were present. PSAT6 is located at the centromeres of chromosomes II and III. PSAT4 and PSAT8 were found with only short arrays. These results demonstrate that (i) repeat families occupy distinct niches within chromosomes, (ii) they differ in the copy number, cluster organization and homogenization events, and that (iii) the recent genome hybridization in breeding P. hybrida preserved the chromosomal position of repeats but affected the copy number of repetitive DNA.

A first insight into the genomic background of Ilex pubescens (Aquifoliaceae) by flow cytometry and genome survey sequencing

BackgroundIlex pubescens is an important traditional Chinese medicinal plant with many naturally occurring compounds and multiple pharmacological effects. However, the lack of reference genomic information has led to tardiness in molecular biology research and breeding programs of this plant.ResultsTo obtain knowledge on the genomic information of I. pubescens, a genome survey was performed for the first time by next generation sequencing (NGS) together with genome size estimation using flow cytometry. The whole genome survey of I. pubescens generated 46.472 Gb of sequence data with approximately 82.2 × coverage. K-mer analysis indicated that I. pubescens has a small genome of approximately 553 Mb with 1.93% heterozygosity rate and 39.1% repeat rate. Meanwhile, the genome size was estimated to be 722 Mb using flow cytometry, which was possibly more precise for assessment of genome size than k-mer analysis. A total of 45.842 Gb clean reads were assembled into 808,938 scaffolds with a relatively short N50 of 760 bp. The average guanine and cytosine (GC) content was 37.52%. In total, 197,429 microsatellite motifs were detected with a frequency of 2.8 kb, among which mononucleotide motifs were the most abundant (up to 62.47% of the total microsatellite motifs), followed by dinucleotide and trinucleotide motifs.ConclusionIn summary, the genome of I. pubescens is small but complex with a high level of heterozygosity. Even though not successfully applied for estimation of genome size due to its complex genome, the survey sequences will help to design whole genome sequencing strategies and provide genetic information support for resource protection, genetic diversity analysis, genetic improvement and artificial breeding of I. pubescens.

Genome survey sequencing-based SSR marker development and their validation in Dendrocalamus longispathus.

Bamboo is an important genetic resource in India, supporting rural livelihood and industries. Unfortunately, most Indian bamboo taxa are devoid of basic genomic or marker information required to comprehend the genetic processes for further conservation and management. In this study, we perform genome survey sequencing for development of de novo genomic SSRs in Dendrocalamus longispathus, a socioeconomically important bamboo species of northeast India. Using Illumina platform, 69.49 million raw reads were generated and assembled into 1,145,321 contig with GC content 43% and N50 1228bp. In total, 46,984 microsatellite repeats were mined-out wherein di-nucleotide repeats were most abundant (54.71%) followed by mono- (31.91%) and tri-repeats (9.85%). Overall, AT-rich repeats were predominant in the genome, but GC-rich motifs were more frequent in tri-repeats. Afterwards, 21,596 SSR loci were successfully tagged with the primer pairs, and a subset of 50 were validated through polymerase chain reaction amplification. Of these, 36 SSR loci were successfully amplified, and 16 demonstrated polymorphism. Using 13 polymorphic SSRs, a moderate level of gene diversity (He = 0.480; Ar = 3.52) was recorded in the analysed populations of D. longispathus. Despite the high gene flow (Nm = 4.928) and low genetic differentiation (FST = 0.119), severe inbreeding (FIS = 0.407) was detected. Further, genetic clustering and STRUCTURE analysis revealed that the entire genetic variability is captured under two major gene pools. Conclusively, we present a comprehensive set of novel SSR markers in D. longispathus as well as other taxa of tropical woody bamboos.

Genome survey sequencing and mining of genome-wide microsatellite markers in yellow-billed babbler (Turdoides affinis)

Turdoides affinis is a species of group dwelling old world passerine of family Leiothrichidae. Unavailability of genome-wide sequence and species-specific molecular markers have hindered comprehensive understanding of cooperative breeding behaviour in T. affinis. Therefore, we generated genome-wide microsatellite markers through whole genome short read sequencing of T. affinis. A total of 68.8 gigabytes of paired-end raw data were sequenced containing 195,067,054 reads. Total sequenced reads spanned a coverage of 17X with genome size of 1.18 Gb. A large number of microsatellite markers (265,297) were mined in the T. affinis genome using Krait, and 50 most informative markers were identified and validated further. In-silico PCR results validated 47 markers. Of these 47 markers, five were randomly selected and validated in-vitro in twelve individuals of T. affinis. Genotyping data on these five loci estimated observed heterozygosity (H0) and expected heterozygosity (He) ratios between 0.333 – 0.833 and 0.851–0.906, respectively. Effective allele size ranged from 6.698 to 10.667, inbreeding coefficient of the population ranged from 0.080 to 0.631 and null allele frequency was calculated at 0.055 to 0.303. Polymorphic information content of all the five loci varied between 0.850 and 0.906. Probabilities of exclusion and identity across 5 loci was estimated to be 0.95 and 0.0036, respectively. All the loci showed significant adherence to Hardy-Weinberg equilibrium. The microsatellite markers reported in this study will facilitate future population genetics studies on T. affinis and other congeneric species.

A first genome survey sequencing of alvinocaridid shrimp Shinkaicaris leurokolos in deep-sea hydrothermal vent environment

Abstract The alvinocaridid shrimp Shinkaicaris leurokolos Kikuchi and Hashimoto, 2000, is an evolutionarily important deep-sea species in hydrothermal vents of north-western Pacific. A genome survey of S. leurokolos was carried out in order to provide a foundation for its whole-genome sequencing. A total of 599 Gb high-quality sequence data were obtained in the study, representing approximately 118× coverage of the S. leurokolos genome. According to the 17-mer distribution frequency, the estimated genome size was 5.08 Gb, and its heterozygosity ratio and percentage of repeated sequences were 2.85 and 87.03%, respectively, showing a complex genome. The final scaffold assembly accounted for a total size of 9.53 Gb (32,796,062 scaffolds, N50 = 597 bp). Repetitive elements nearly constituted 45% of the nuclear genome, among which the most ubiquitous were long interspersed nuclear elements, DNA transposons and long-terminal repeat elements. A total of 12,121,553 genomic simple sequence repeats were identified, with the most frequent repeat motif being di-nucleotide (70.27%), followed by tri-nucleotide and tetra-nucleotide. From the genome survey sequences, the mitochondrial genome of S. leurokolos was also constructed and 71 single nucleotide polymorphisms were identified by comparison with previous published reference. This is the first report of de novo whole-genome sequencing and assembly of S. leurokolos. These newly developed genomic data contribute to a better understanding of genomic characteristics of shrimps from deep-sea chemosynthetic ecosystems, and provides valuable resources for further molecular marker development.

Profile of the genomic characteristics and comparative studies of five Trichiuridae species by genome survey sequencing

Trichiuridae fish are economically important species and are widely distributed across the nearshore to the open ocean. In the present study, the genomic survey sequencing method was used to analyze the genomic characteristics of five Trichiuridae fish. The calculated genome size was 913 Mb, 868 Mb, 871 Mb, 747 Mb, and 670 Mb for Trichiurus japonicus, Trichiurus nanhaiensis, Trichiurus brevis, Lepturacanthus savala, and Eupleurogrammus muticus, respectively. The average GC content of the five Trichiuridae fish ranged from 39.59% to 42.05%, and the repeat sequence content ranged from 33.21% to 45.87%. The heterozygous ratio of E. muticus was the highest, and that of L. savala was the smallest. The proportion of microsatellite motifs showed a decreasing trend with the increase in repeat numbers: the dinucleotide repeats were dominant, followed by the trinucleotide repeats, tetranucleotide repeats, pentanucleotide repeats, and hexanucleotide repeats. The mitochondrial genomes of five Trichiuridae species were excavated from the genome data, and the ML tree revealed that T. japonicus, T. nanhaiensis, T. brevis, L. savala, and E. muticus formed into one clade. E. muticus showed earlier expansion than the other four species and had a significant population decline at the Last Glacial period by pairwise sequentially Markovian coalescent (PSMC) analysis. This is the first report to sequence and characterize the whole genomes of five Trichiuridae species.

Genome Survey and SSR Analysis of Camellia nitidissima Chi (Theaceae).

Camellia nitidissima Chi (CNC), a species of golden Camellia, is well known as "the queen of camellias." It is an ornamental, medicinal, and edible plant grown in China. In this study, we conducted a genome survey sequencing analysis and simple sequence repeat (SSR) identification of CNC using the Illumina sequencing platform. The 21-mer analysis predicted its genome size to be 2,778.82 Mb, with heterozygosity and repetition rates of 1.42% and 65.27%, respectively. The CNC genome sequences were assembled into 9,399,197 scaffolds, covering ∼2,910 Mb and an N50 of 869 base pair. Its genomic characteristics were found to be similar to those of Camellia oleifera. In addition, 1,940,616 SSRs were identified from the genome data, including mono-(61.85%), di-(28.71%), tri-(6.51%), tetra-(1.85%), penta-(0.57%), and hexanucleotide motifs (0.51%). We believe these data will provide a useful foundation for the development of novel molecular markers for CNC as well as for further whole-genome sequencing of CNC.

A Genome Sequence Assembly of the Phototactic and Optogenetic Model Fungus Blastocladiella emersonii Reveals a Diversified Nucleotide-Cyclase Repertoire.

The chytrid fungus Blastocladiella emersonii produces spores with swimming tails (zoospores); these cells can sense and swim toward light. Interest in this species stems from ongoing efforts to develop B. emersonii as a model for understanding the evolution of phototaxis and the molecular cell biology of the associated optogenetic circuits. Here, we report a highly contiguous genome assembly and gene annotation of the B. emersonii American Type Culture Collection 22665 strain. We integrate a PacBio long-read library with an Illumina paired-end genomic sequence survey leading to an assembly of 21 contigs totaling 34.27 Mb. Using these data, we assess the diversity of sensory system encoding genes. These analyses identify a rich complement of G-protein-coupled receptors, ion transporters, and nucleotide cyclases, all of which have been diversified by domain recombination and tandem duplication. In many cases, these domain combinations have led to the fusion of a protein domain to a transmembrane domain, tying a putative signaling function to the cell membrane. This pattern is consistent with the diversification of the B. emersonii sensory-signaling systems, which likely plays a varied role in the complex life cycle of this fungus.

Identification of polyketide synthase genes required for aspinolide biosynthesis in Trichoderma arundinaceum

The fungus Trichoderma arundinaceum exhibits biological control activity against crop diseases caused by other fungi. Two mechanisms that likely contribute to this activity are upregulation of plant defenses and production of two types of antifungal secondary metabolites: the sesquiterpenoid harzianum A (HA) and the polyketide-derived aspinolides. The goal of the current study was to identify aspinolide biosynthetic genes as part of an effort to understand how these metabolites contribute to the biological control activity of T. arundinaceum. Comparative genomics identified two polyketide synthase genes (asp1 and asp2) that occur in T. arundinaceum and Aspergillus ochraceus, which also produces aspinolides. Gene deletion and biochemical analyses in T. arundinaceum indicated that both genes are required for aspinolide production: asp2 for formation of a 10-member lactone ring and asp1 for formation of a butenoyl subsituent at position 8 of the lactone ring. Gene expression and comparative genomics analyses indicated that asp1 and asp2 are located within a gene cluster that occurs in both T. arundinaceum and A. ochraceus. A survey of genome sequences representing 35 phylogenetically diverse Trichoderma species revealed that intact homologs of the cluster occurred in only two other species, which also produced aspinolides. An asp2 mutant inhibited fungal growth more than the wild type, but an asp1 mutant did not, and the greater inhibition by the asp2 mutant coincided with increased HA production. These findings indicate that asp1 and asp2 are aspinolide biosynthetic genes and that loss of either aspinolide or HA production in T. arundinaceum can be accompanied by increased production of the other metabolite(s).Key points• Two polyketide synthase genes are required for aspinolide biosynthesis.• Blocking aspinolide production increases production of the terpenoid harzianum A.• Aspinolides and harzianum A act redundantly in antibiosis of T. arundinaceum.

Genome-wide survey and genetic characteristics of Ophichthus evermanni based on Illumina sequencing platform.

Ophichthidae fishes limit to continental shelf of all tropical and subtropical oceans and contain more than 350 species, representing the greatest specialization diversity in the order Anguiliformes. In the present study, we conducted a genome survey sequencing (GSS) analysis of Ophichthus evermanni by Illumina sequencing platform to briefly reveal its genomic characteristics and phylogenetic relationship. The first de novo assembled 1.97 Gb draft genome of O. evermanni was predicted based on K-mer analysis without obvious nucleotide bias. The heterozygosity ratio was 0.70%, and the sequence repeat ratio was calculated to be 43.30%. A total of 9016 putative coding genes were successfully predicted, in which 3587 unigenes were identified by gene ontology (GO) analysis and 4375 unigenes were classified into cluster of orthologous groups for enkaryotic complete genomes (KOG) functional categories. About 2,812,813 microsatellite motifs including mono-, di-, tri-, tetra-, penta- and hexanucleotide motifs were identified, with an occurrence frequency of 23.32%. The most abundant type was dinucleotide repeat motifs, accounting for 49.19% of the total repeat types. The mitochondrial genome, as a byproduct of GSS, was assembled to investigate the evolutionary relationships between O. evermanni and its relatives. Bayesian inference (BI) phylogenetic tree inferring from concatenated 12 protein-coding genes (PCGs) showed complicated relationships among Ophichthidae species, indicating a polyphyletic origin of the family. The results would achieve more thorough genetic information of snake eels and provide a theoretical basis and reference for further genome-wide analysis of O. evermanni.

Genome survey sequencing of the long-legged spiny lobsterPanulirus longipes(A. Milne-Edwards, 1868) (Decapoda: Achelata: Palinuridae): improved mitochondrial genome annotation, nuclear repetitive elements classification, and SSR marker discovery

AbstractThe long-legged spiny lobster Panulirus longipes (A. Milne-Edwards, 1868) inhabits shallow water rock and coral reefs in the Indo-West Pacific region and is currently targeted by local fisheries along most of its range of distribution. This study conducted, for the first time, a genome survey in P. longipes. Using low coverage short Illumina 150 bp pair-end reads sequencing, we assembled, annotated, and manually curated the mitochondrial genome, described nuclear repetitive elements, and discovered microsatellites (SRR). The pipeline NOVOPlasty assembled the complete mitochondrial genome of P. longipes (coverage = 551×). The mitochondrial genome of P. longipes is 15,739 bp in length and comprised of 13 protein coding genes (PCGs), 2 ribosomal RNA genes, and 22 transfer RNA genes. A single 804 bp long intergenic space is assumed to be the control region. The secondary structures of the tRNA genes exhibit a standard “cloverleaf” structure, with the exception of trnS1 that is missing the DHU arm. A large portion of repetitive sequences (~56%) was detected in the nuclear genome of P. longipes and many repetitive elements could not be assigned to known families. Taking into account only annotated repetitive elements, the most common belong to Class I-Long Interspersed Nuclear Element (LINE) and Satellite DNA which are considerably more abundant than Class I-Penelope and Class I-LTR Ty3-gypsy elements. Three clusters were classified as 45S rDNA. A total of 18 SSRs were identified. These newly developed genomic resources will contribute to clarifying the putative existence of cryptic species or subspecies, developing conservation plans, and managing the fishery of the long-legged spiny lobster Panulirus longipes.

Genome survey sequencing of wild cotton (Gossypium robinsonii) reveals insights into proteomic responses of pollen to extreme heat.

Heat stress specifically affects fertility by impairing pollen viability but cotton wild relatives successfully reproduce in hot savannas where they evolved. An Australian arid‐zone cotton (Gossypium robinsonii) was exposed to heat events during pollen development then mature pollen was subjected to deep proteomic analysis using 57 023 predicted genes from a genomic database we assembled for the same species. Three stages of pollen development, including tetrads (TEs), uninucleate microspores (UNs) and binucleate microspores (BNs) were exposed to 36°C or 40°C for 5 days and the resulting mature pollen was collected at anthesis (p‐TE, p‐UN and p‐BN, respectively). Using the sequential windowed acquisition of all theoretical mass spectra proteomic analysis, 2704 proteins were identified and quantified across all pollen samples analysed. Proteins predominantly decreased in abundance at all stages in response to heat, particularly after exposure of TEs to 40°C. Functional enrichment analyses demonstrated that extreme heat increased the abundance of proteins that contributed to increased messenger RNA splicing via spliceosome, initiation of cytoplasmic translation and protein refolding in p‐TE40. However, other functional categories that contributed to intercellular transport were inhibited in p‐TE40, linked potentially to Rab proteins. We ascribe the resilience of reproductive processes in G. robinsonii at temperatures up to 40°C, relative to commercial cotton, to a targeted reduction in protein transport.

Genome survey sequence of black carp provides insights into development‐related gene duplications

AbstractAs one of the traditional "Four Domesticated Fishes" in China, black carp (Mylopharyngodon piceus) is an important species in freshwater aquaculture, an important fishery resource in China, and an invasive species in the United Sates. Here, we reported the draft genome of black carp with an estimated size of 975 Mb and 27,541 predicted protein‐coding genes. The genome of black carp exhibited a high synteny with those of grass carp and zebrafish. Phylogenetic estimation suggested that black carp likely diverged from grass carp and zebrafish around 13 and 57 million years ago, respectively. The comparison of expanded gene families among eight fish species identified functional genes involved in skeletal growth, hormone regulation and rhythmic control, cellular differentiation and other development‐related pathways underwent the duplications in the large body fishes such as black carp and grass carp. Further analysis of functional interaction networks of the proteins encoded by these duplicated genes revealed their important role associated with regulation of development pathways. In addition, Sox and TLR gene families were found to have diverse subfamily evolution among different fishes. The first draft genome of black carp offers insights into its genomic structure, function, and evolution and provides an additional resource for improved molecular selection and breeding.

Genome survey of Misgurnus anguillicaudatus to identify genomic information, simple sequence repeat (SSR) markers, and mitochondrial genome.

The dojo loach Misgurnus anguillicaudatus is an important economic species in Asia because of its nutritional value and broad environmental adaptability. Despite its economic importance, genomic data for M. anguillicaudatus is currently unavailable. In the present study, we conducted a genome survey of M. anguillicaudatus using next-generation sequencing technology. Its genome size was estimated to be 1105.97Mb by using K-mer analysis, and its heterozygosity ratio, repeat sequence content, GC content were 1.45%, 58.98%, and 38.03%, respectively. A total of 376,357 microsatellite motifs were identified, and mononucleotides, with a frequency of 42.57%, were the most frequently repeated motifs, followed by 40.83% dinucleotide, 7.49% trinucleotide, 8.09% tetranucleotide, and 0.91% pentanucleotide motifs. The AC/GT, AAT/ATT, and ACAG/CTGT repeats were the most abundant motifs among dinucleotide, trinucleotide, and tetranucleotide motifs, respectively. Besides,the complete mitochondrial genome was sequenced. Based on the Maximum Likelihood and Bayesian inference analyses, M. anguillicaudatus yingde in this study was the "introgressed" mitochondrial type. Seventy microsatellite loci were randomly selected from detected SSR loci to test polymorphic, of which, 20 microsatellite loci were assessed in 30 individuals from a wild population. The number of alleles (Na), observed heterozygosity (Ho), and expected heterozygosity (He) per locus ranged from 7 to 19, 0.400 to 0.933, and 0.752 to 0.938, respectively. All 20 loci were highly informative (PIC > 0.700). Eight loci deviated from Hardy-Weinberg equilibrium after Bonferroni correction (P < 0.05). This is the first report of genome survey sequencing in M. anguillicaudatus, genome information, mitochondrial genome, andmicrosatellite markers will be valuable for further studies on population genetic analysis, natural resource conservation, and molecular marker-assisted selective breeding.

Genome survey sequencing and characterization of simple sequence repeat (SSR) markers in Platostoma palustre (Blume) A.J.Paton (Chinese mesona)

Platostoma palustre (Blume) A.J.Paton is an annual herbaceous persistent plant of the Labiatae family. However, there is a lack of genomic data for this plant, which severely restricts its genetic improvement. In this study, we performed genome survey sequencing of P. palustre and developed simple sequence repeat (SSR) markers based on the resulting sequence. K-mer analysis revealed that the assembled genome size was approximately 1.21 Gb. A total of 15,498 SSR motifs were identified and characterized in this study; among them, dinucleotide, and hexanucleotide repeats had the highest and lowest, respectively. Among the dinucleotide repeat motifs, AT/TA repeat motifs were the most abundant, and GC/CG repeat motifs were rather rare, accounting for 44.28% and 0.63%, respectively. Genetic similarity coefficient analysis by the UPMGA methods clustered 12 clones, of P. palustre and related species into two subgroups. These results provide helpful information for further research on P. palustre resources and variety improvements.

Genome survey sequencing of Ailanthus altissima and identification of simple sequence repeat (SSR) markers

Abstract Ailanthus altissima is a deciduous tree native to China and introduced to other parts of the world as an ornamental plant. It exhibits resistance to both abiotic and biotic stress factors and has various pharmacological effects and strong allelopathy, generating significant research interests. However, the genome sequence of this species has not been reported, limiting its research development. The purpose of the study was to determine the genome size and characteristics of A. altissima to conduct its genomic survey. Next-generation sequencing and K-mer analysis were employed to measure the genome size of A. altissima. Overall, a total of 61.93 Gb high-quality clean data were acquired, representing approximately 64.09× coverage of the A. altissima genome. The genomic characteristics of A. altissima include a genome size of 966.38 Mbp, a heterozygosis rate of 0.78 %, and a repeat rate of 41.22 %. A total of 735,179 genomic SSRs markers were identified based on genome survey sequences. Alignment analysis showed that A. altissima was closely related to Citrus sinensis and Leitneria florida-na. This study provides basic information for future whole-genomic sequencing of A. altissima. This will facilitate a knowledge of the population structure, genetic diversity, long distance-gene transfer, and pollen-based gene flow analyses of A. altissima populations from its known distribution ranges in China, focusing on planted and natural forest stands.

A Comprehensive Plant microRNA Simple Sequence Repeat Marker Database to Accelerate Genetic Improvements in Crops

Microsatellites, or simple sequences repeat (SSRs), are distributed in genes, intergenic regions and transposable elements in the genome. SSRs were identified for developing markers from draft genome assemblies, transcriptome sequences and genome survey sequences in plant and animals. The identification, distribution, and density of microsatellites in pre-microRNAs (miRNAs) are not well documented in plants. In this study, SSRs were identified in 16,892 pre-miRNA sequences from 292 plant species in six taxonomic groups (algae to dicots). Fifty-one percent of pre-miRNA sequences contained SSRs. Mononucleotide repeats were the most abundant, followed by di- and trinucleotide repeats. Tetra-, penta-, and hexarepeats were rare. A total of 9,498 (57.46%) microsatellite loci had potential as pre-miRNA SSR markers. Of the markers, 3,573 (37.62%) were non-redundant, and 2,341 (65.51%) primer pairs could be transferred to at least one of the plant taxonomic groups. All data and primer pairs were deposited in a user-friendly, freely accessible plant miRNA SSR marker database. The data presented in this study, accelerate the understanding of pre-miRNA evolution and serve as valuable genomic treasure for genetic improvements in a wide range of crops, including legumes, cereals, and cruciferous crops.