Development and cross-species/genera transferability of microsatellite markers discovered using 454 genome sequencing in chokecherry (Prunus virginiana L.)

Chokecherry (Prunus virginiana L.) (2n?=?4x?=?32) is a unique Prunus species for both genetics and disease-resistance research due to its tetraploid nature and X-disease resistance. However, no genetic and genomic information on chokecherry is available. A partial chokecherry genome was sequenced using Roche 454 sequencing technology. A total of 145,094 reads covering 4.8?Mbp of the chokecherry genome were generated and 15,113 contigs were assembled, of which 11,675 contigs were larger than 100?bp in size. A total of 481 SSR loci were identified from 234 (out of 11,675) contigs and 246 polymerase chain reaction (PCR) primer pairs were designed. Of 246 primers, 212 (86.2?%) effectively produced amplification from the genomic DNA of chokecherry. All 212 amplifiable chokecherry primers were used to amplify genomic DNA from 11 other rosaceous species (sour cherry, sweet cherry, black cherry, peach, apricot, plum, apple, crabapple, pear, juneberry, and raspberry). Thus, chokecherry SSR primers can be transferable across Prunus species and other rosaceous species. An average of 63.2 and 58.7?% of amplifiable chokecherry primers amplified DNA from cherry and other Prunus species, respectively, while 47.2?% of amplifiable chokecherry primers amplified DNA from other rosaceous species. Using random genome sequence data generated from next-generation sequencing technology to identify microsatellite loci appears to be rapid and cost-efficient, particularly for species with no sequence information available. Sequence information and confirmed transferability of the identified chokecherry SSRs among species will be valuable for genetic research in Prunus and other rosaceous species. Key message A total of 246 SSR primers were identified from chokecherry genome sequences. Of which, 212 were confirmed amplifiable both in chokecherry and other 11 other rosaceous species.     

De novo assembly of a chromosome‐level reference genome of red‐spotted grouper (Epinephelus akaara) using nanopore sequencing and Hi‐C

The red‐spotted grouper Epinephelus akaara (E. akaara) is one of the most economically important marine fish in China, Japan and South‐East Asia and is a threatened species. The species is also considered a good model for studies of sex inversion, development, genetic diversity and immunity. Despite its importance, molecular resources for E. akaara remain limited and no reference genome has been published to date. In this study, we constructed a chromosome‐level reference genome of E. akaara by taking advantage of long‐read single‐molecule sequencing and de novo assembly by Oxford Nanopore Technology (ONT) and Hi‐C. A red‐spotted grouper genome of 1.135 Gb was assembled from a total of 106.29 Gb polished Nanopore sequence (GridION, ONT), equivalent to 96‐fold genome coverage. The assembled genome represents 96.8% completeness (BUSCO) with a contig N50 length of 5.25 Mb and a longest contig of 25.75 Mb. The contigs were clustered and ordered onto 24 pseudochromosomes covering approximately 95.55% of the genome assembly with Hi‐C data, with a scaffold N50 length of 46.03 Mb. The genome contained 43.02% repeat sequences and 5,480 noncoding RNAs. Furthermore, combined with several RNA‐seq data sets, 23,808 (99.5%) genes were functionally annotated from a total of 23,923 predicted protein‐coding sequences. The high‐quality chromosome‐level reference genome of E. akaara was assembled for the first time and will be a valuable resource for molecular breeding and functional genomics studies of red‐spotted grouper in the future.     

Self-compatibility and incompatibility in tetraploid sour cherry (Prunus cerasus L.)

. Gametophytic self-incompatibility (GSI) typically "breaks down" due to polyploidy in many Solanaceous species, resulting in self-compatible (SC) tetraploid individuals. However, sour cherry (Prunus cerasus L.), a tetraploid species resulting from hybridization of the diploid sweet cherry (P. avium L.) and the tetraploid ground cherry (P. fruticosa Pall.), is an exception, consisting of both self-incompatible (SI) and SC individuals. Since sweet cherry exhibits GSI with 13 S-ribonucleases (S-RNases) identified as the stylar S-locus product, the objectives were to compare sweet and sour cherry S-allele function, S-RNase sequences and linkage map location as initial steps towards understanding the genetic basis of SI and SC in sour cherry. S-RNases from two sour cherry cultivars that were the parents of a linkage mapping population were cloned and sequenced. The sequences of two S-RNases were identical to those of sweet cherry S-RNases, whereas three other S-RNases had unique sequences. One of the S-RNases mapped to the Prunus linkage group 6, similar to its location in sweet cherry and almond, whereas two other S-RNases were linked to each other but were unlinked to any other markers. Interspecific crosses between sweet and sour cherry demonstrated that GSI exists in sour cherry and that the recognition of common S-alleles has been maintained in spite of polyploidization. It is hypothesized that self-compatibility in sour cherry is caused by the existence of non-functional S-RNases and pollen S-genes that may have arisen from natural mutations.     

Genome sequencing and analysis of two early-flowering cherry (Cerasus × kanzakura) varieties, ‘Kawazu-zakura’ and ‘Atami-zakura’

To gain genetic insights into the early-flowering phenotype of ornamental cherry, also known as sakura, we determined the genome sequences of two early-flowering cherry (Cerasus × kanzakura) varieties, ‘Kawazu-zakura’ and ‘Atami-zakura’. Because the two varieties are interspecific hybrids, likely derived from crosses between Cerasus campanulata (early-flowering species) and Cerasus speciosa, we employed the haplotype-resolved sequence assembly strategy. Genome sequence reads obtained from each variety by single-molecule real-time sequencing (SMRT) were split into two subsets, based on the genome sequence information of the two probable ancestors, and assembled to obtain haplotype-phased genome sequences. The resultant genome assembly of ‘Kawazu-zakura’ spanned 519.8 Mb with 1,544 contigs and an N50 value of 1,220.5 kb, while that of ‘Atami-zakura’ totalled 509.6 Mb with 2,180 contigs and an N50 value of 709.1 kb. A total of 72,702 and 69,528 potential protein-coding genes were predicted in the genome assemblies of ‘Kawazu-zakura’ and ‘Atami-zakura’, respectively. Gene clustering analysis identified 2,634 clusters uniquely presented in the C. campanulata haplotype sequences, which might contribute to its early-flowering phenotype. Genome sequences determined in this study provide fundamental information for elucidating the molecular and genetic mechanisms underlying the early-flowering phenotype of ornamental cherry tree varieties and their relatives.     

Complete chloroplast genome sequences of Dipterygium glaucum and Cleome chrysantha and other Cleomaceae Species,comparative analysis and phylogenetic relationships

This current study presents, for the first time, the complete chloroplast genome of two Cleomaceae species: Dipterygium glaucum and Cleome chrysantha in order to evaluate the evolutionary relationship. The cp genome is 158,576 bp in length with 35.74% GC content in D. glaucum and 158,111 bp with 35.96% GC in C. chrysantha. Inverted repeats IR 26,209 bp, 26,251 bp each, LSC of 87,738 bp, 87,184 bp and SSC of 18,420 bp, 18,425 bp respectively. There are 136 genes in the genome, which includes 80 protein coding genes, 31 tRNA genes and four rRNA genes were observed in both chloroplast genomes. 117 genes are unique while the remaining 19 genes are duplicated in IR regions. The analysis of repeats shows that the cp genome includes all types of repeats with more frequent occurrences of palindromic; Also, this analysis indicates that the total number of simple sequence repeats (SSR) were 323 in D. glaucum, and 313 in C. chrysantha, of which the majority of the SSRs in these plastid genomes were mononucleotide repeats A/T which are located in the intergenic spacer. Moreover, the comparative analysis of the four cp sequences revealed four hotspot genes (atpF, rpoC2, rps19, and ycf1), these variable regions could be used as molecular makers for the species authentication as well as resources for inferring phylogenetic relationships of the species. All the relationships in the phylogenetic tree are with high support, this indicate that the complete chloroplast genome is a useful data for inferring phylogenetic relationship within the Cleomaceae and other families. The simple sequence repeats identified will be useful for identification, genetic diversity, and other evolutionary studies of the species. This study reported the first cp genome of the genus Dipterygium and Cleome. The finding of this study will be beneficial for biological disciplines such as evolutionary and genetic diversity studies of the species within the core Cleomaceae.     

A chromosome‐level genome assembly of the parasitoid wasp Pteromalus puparum

Parasitoid wasps represent a large proportion of hymenopteran species. They have complex evolutionary histories and are important biocontrol agents. To advance parasitoid research, a combination of Illumina short‐read, PacBio long‐read and Hi‐C scaffolding technologies was used to develop a high‐quality chromosome‐level genome assembly for Pteromalus puparum, which is an important pupal endoparasitoid of caterpillar pests. The chromosome‐level assembly has aided in studies of venom and detoxification genes. The assembled genome size is 338 Mb with a contig N50 of 38.7 kb and a scaffold N50 of 1.16 Mb. Hi‐C analysis assembled scaffolds onto five chromosomes and raised the scaffold N50 to 65.8 Mb, with more than 96% of assembled bases located on chromosomes. Gene annotation was assisted by RNA sequencing for the two sexes and four different life stages. Analysis detected 98% of the BUSCO (Benchmarking Universal Single‐Copy Orthologs) gene set, supporting a high‐quality assembly and annotation. In total, 40.1% (135.6 Mb) of the assembly is composed of repetitive sequences, and 14,946 protein‐coding genes were identified. Although venom genes play important roles in parasitoid biology, their spatial distribution on chromosomes was poorly understood. Mapping has revealed venom gene tandem arrays for serine proteases, pancreatic lipase‐related proteins and kynurenine–oxoglutarate transaminases, which have amplified in the P. puparum lineage after divergence from its common ancestor with Nasonia vitripennis. In addition, there is a large expansion of P450 genes in P. puparum. These examples illustrate how chromosome‐level genome assembly can provide a valuable resource for molecular, evolutionary and biocontrol studies of parasitoid wasps.     

Construction of an intra-specific sweet cherry (<Emphasis Type="Italic">Prunus avium</Emphasis> L.) genetic linkage map and synteny analysis with the <Emphasis Type="Italic">Prunus</Emphasis> reference map

Linkage maps of the sweet cherry cultivar ‘Emperor Francis’ (EF) and the wild forest cherry ‘New York 54’ (NY) were constructed using primarily simple sequence repeat (SSR) markers and gene-derived markers with known positions on the Prunus reference map. The success rate for identifying SSR markers that could be placed on either the EF or NY maps was only 26% due to two factors: a reduced transferability of other Prunus-species-derived markers and a low level of polymorphism in the mapping parents. To increase marker density, we developed four cleaved amplified polymorphic sequence markers (CAPS), 19 derived CAPS markers, and four insertion–deletion markers for cherry based on 101 Prunus expressed sequence tags. In addition, four gene-derived markers representing orthologs of a tomato vacuolar invertase and fruit size gene and two sour cherry sorbitol transporters were developed. To complete the linkage analysis, 61 amplified fragment length polymorphism and seven sequence-related amplified polymorphism markers were also used for map construction. This analysis resulted in the expected eight linkage groups for both parents. The EF and NY maps were 711.1 cM and 565.8 cM, respectively, with the average distance between markers of 4.94 cM and 6.22 cM. A total of 82 shared markers between the EF and NY maps and the Prunus reference map showed that the majority of the marker orders were the same with the Prunus reference map suggesting that the cherry genome is colinear with that of the other diploid Prunus species. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Complete Plastid Genome Sequencing of Four Tilia Species (Malvaceae): A Comparative Analysis and Phylogenetic Implications

Tilia is an ecologically and economically important genus in the family Malvaceae. However, there is no complete plastid genome of Tilia sequenced to date, and the taxonomy of Tilia is difficult owing to frequent hybridization and polyploidization. A well-supported interspecific relationships of this genus is not available due to limited informative sites from the commonly used molecular markers. We report here the complete plastid genome sequences of four Tilia species determined by the Illumina technology. The Tilia plastid genome is 162,653 bp to 162,796 bp in length, encoding 113 unique genes and a total number of 130 genes. The gene order and organization of the Tilia plastid genome exhibits the general structure of angiosperms and is very similar to other published plastid genomes of Malvaceae. As other long-lived tree genera, the sequence divergence among the four Tilia plastid genomes is very low. And we analyzed the nucleotide substitution patterns and the evolution of insertions and deletions in the Tilia plastid genomes. Finally, we build a phylogeny of the four sampled Tilia species with high supports using plastid phylogenomics, suggesting that it is an efficient way to resolve the phylogenetic relationships of this genus.     

Comprehensive whole genome survey analyses of male and female brown-spotted flathead fish Platycephalus sp.1

The flathead fish Platycephalus sp.1 is an ecologically and commercially important marine fish in the northwestern Pacific with notable sexual differences in growth and development. Yet the genomic data of this species is lacking. In the present study, whole genome sequencing of two individuals (one male and one female) of Platycephalus sp.1 were conducted to provide fundamental genomic information. The genome sizes were estimated to be 674.96 Mb (male) and 684.15 Mb (female) by using k-mer analyses. The heterozygosity and repeat ratios suggested possible male heterogamety of this species. The draft genome sequences were initially assembled and genome-wide microsatellite motifs were identified. Besides, the complete mitochondrial genome sequences were assembled and the phylogenetic analyses genetically supported the validation of Platycephalus sp.1. The reported genomic data and genetic markers in this study could be useful in future comparative genomics and evolutionary biology studies.     

Genotypic and Phenotypic Diversity of Cherry Species Collected in Serbia

Genetic diversity of cherry species collected in Serbia has been investigated using 26 simple sequence repeat (SSR) markers developed in Prunus. This material consisted of 77 cherry accessions corresponding to the five following species, Prunus cerasus, Prunus avium, Prunus fruticosa, Prunus mahaleb, and Prunus serrulata. A total of 98 alleles were detected, with an average of 3.7 putative alleles per primer combination. Sixteen unique, species-specific, alleles were detected with nine primer pairs in four species, P. avium, P. cerasus, P. mahaleb, and P. serrulata. The highest number of unique alleles, 8, was observed in P. mahaleb and no species-specific alleles were detected in P. fruticosa. SSR markers generated unique fingerprints for all cherry accessions. Cluster analysis classified accessions into four groups according to their taxonomy, where P. avium and P. cerasus were grouped together, supporting P. avium as one of the progenitors of sour cherry. The highest genetic variability and potential value in rootstock breeding was observed in P. mahaleb and P. serrulata material. Principal component (PC) analysis explained more than 50 % of the total observed phenotypic variability using the first two components. The most important characteristics of PC1 were leaf length and width, fruit taste, color of leaf nectaries, fruit weight, leaf blade margin incisions, petiole length, size of vegetative buds, and length of internode. The most important characteristics of PC2 were shape of leaf blade at base, fruit skin color, and leaf blade length and tip angle. The investigated germplasm proved to be sufficiently genetically diverse for use in breeding programs and development of new cherry cultivars and rootstocks.     

Discovery and experimental analysis of microsatellites in an oil woody plant Camellia chekiangoleosa

Oil camellia trees are important woody plants for the production of high-quality cooking oil. On the contrary to their economic importance, their genetic and genomic resources are very limited, which greatly hamper the genetic studies on oil camellia trees. Microsatellites or simple sequence repeats (SSRs) have great value in many aspects of genetic analyses due to their high polymorphism and codominant inheritance. In this study, we report the large-scale development and characterization of SSR markers derived from genomic sequences of Camellia chekiangoleosa by high-throughput pyrosequencing technology. A total of 1,091,393 genomic shotgun reads were generated using Roche 454 FLX sequencer, the average read length was 319 bp, and the total sequence throughput was 347.9 Mb. These sequences were assembled into 35,315 contigs with total length of 14.8 Mb and the N50 contig size of 770 bp. By analyzing with microsatellite (MISA), a total of 5,844 perfect microsatellites were detected from the assembled sequences. Among them, tetranucleotide repeats were found to be the most frequent microsatellites in the genome of C. chekiangoleosa, and all the dominant repeat motifs for different types of SSRs were detected to be rich in A/T. Experimental analysis with 900 SSR primer pairs revealed that 66 % of them succeeded in PCR amplification. Further investigation with 345 SSR primer pairs showed that a relatively high percentage of primers amplified polymorphic loci (31.9 %). Experimental data also revealed that, overall, long microsatellite repeats (>20 bp) were more variable than the short ones (<20 bp) in the genome of oil camellia tree.     

High-quality Arabidopsis thaliana Genome Assembly with Nanopore and HiFi Long Reads

Arabidopsis thaliana is an important and long-established model species for plant molecular biology, genetics, epigenetics, and genomics. However, the latest version of reference genome still contains a significant number of missing segments. Here, we reported a high-quality and almost complete Col-0 genome assembly with two gaps (named Col-XJTU) by combining the Oxford Nanopore Technologies ultra-long reads, Pacific Biosciences high-fidelity long reads, and Hi-C data. The total genome assembly size is 133,725,193 bp, introducing 14.6 Mb of novel sequences compared to the TAIR10.1 reference genome. All five chromosomes of the Col-XJTU assembly are highly accurate with consensus quality (QV) scores > 60 (ranging from 62 to 68), which are higher than those of the TAIR10.1 reference (ranging from 45 to 52). We completely resolved chromosome (Chr) 3 and Chr5 in a telomere-to-telomere manner. Chr4 was completely resolved except the nucleolar organizing regions, which comprise long repetitive DNA fragments. The Chr1 centromere (CEN1), reportedly around 9 Mb in length, is particularly challenging to assemble due to the presence of tens of thousands of CEN180 satellite repeats. Using the cutting-edge sequencing data and novel computational approaches, we assembled a 3.8-Mb-long CEN1 and a 3.5-Mb-long CEN2. We also investigated the structure and epigenetics of centromeres. Four clusters of CEN180 monomers were detected, and the centromere-specific histone H3-like protein (CENH3) exhibited a strong preference for CEN180 Cluster 3. Moreover, we observed hypomethylation patterns in CENH3-enriched regions. We believe that this high-quality genome assembly, Col-XJTU, would serve as a valuable reference to better understand the global pattern of centromeric polymorphisms, as well as the genetic and epigenetic features in plants.     

Editor's choice: Dissection of the Octoploid Strawberry Genome by Deep Sequencing of the Genomes of Fragaria Species

Cultivated strawberry (Fragaria x ananassa) is octoploid and shows allogamous behaviour. The present study aims at dissecting this octoploid genome through comparison with its wild relatives, F. iinumae, F. nipponica, F. nubicola, and F. orientalis by de novo whole-genome sequencing on an Illumina and Roche 454 platforms. The total length of the assembled Illumina genome sequences obtained was 698 Mb for F. x ananassa, and ∼200 Mb each for the four wild species. Subsequently, a virtual reference genome termed FANhybrid_r1.2 was constructed by integrating the sequences of the four homoeologous subgenomes of F. x ananassa, from which heterozygous regions in the Roche 454 and Illumina genome sequences were eliminated. The total length of FANhybrid_r1.2 thus created was 173.2 Mb with the N50 length of 5137 bp. The Illumina-assembled genome sequences of F. x ananassa and the four wild species were then mapped onto the reference genome, along with the previously published F. vesca genome sequence to establish the subgenomic structure of F. x ananassa. The strategy adopted in this study has turned out to be successful in dissecting the genome of octoploid F. x ananassa and appears promising when applied to the analysis of other polyploid plant species.     

Development and Evaluation of a Genome-Wide 6K SNP Array for Diploid Sweet Cherry and Tetraploid Sour Cherry

High-throughput genome scans are important tools for genetic studies and breeding applications. Here, a 6K SNP array for use with the Illumina Infinium® system was developed for diploid sweet cherry (Prunus avium) and allotetraploid sour cherry (P. cerasus). This effort was led by RosBREED, a community initiative to enable marker-assisted breeding for rosaceous crops. Next-generation sequencing in diverse breeding germplasm provided 25 billion basepairs (Gb) of cherry DNA sequence from which were identified genome-wide SNPs for sweet cherry and for the two sour cherry subgenomes derived from sweet cherry (avium subgenome) and P. fruticosa (fruticosa subgenome). Anchoring to the peach genome sequence, recently released by the International Peach Genome Initiative, predicted relative physical locations of the 1.9 million putative SNPs detected, preliminarily filtered to 368,943 SNPs. Further filtering was guided by results of a 144-SNP subset examined with the Illumina GoldenGate® assay on 160 accessions. A 6K Infinium® II array was designed with SNPs evenly spaced genetically across the sweet and sour cherry genomes. SNPs were developed for each sour cherry subgenome by using minor allele frequency in the sour cherry detection panel to enrich for subgenome-specific SNPs followed by targeting to either subgenome according to alleles observed in sweet cherry. The array was evaluated using panels of sweet (n = 269) and sour (n = 330) cherry breeding germplasm. Approximately one third of array SNPs were informative for each crop. A total of 1825 polymorphic SNPs were verified in sweet cherry, 13% of these originally developed for sour cherry. Allele dosage was resolved for 2058 polymorphic SNPs in sour cherry, one third of these being originally developed for sweet cherry. This publicly available genomics resource represents a significant advance in cherry genome-scanning capability that will accelerate marker-locus-trait association discovery, genome structure investigation, and genetic diversity assessment in this diploid-tetraploid crop group.     

Chromosome‐level genome assembly of a cyprinid fish Onychostoma macrolepis by integration of nanopore sequencing,Bionano and Hi‐C technology

Onychostoma macrolepis is an emerging commercial cyprinid fish species. It is a model system for studies of sexual dimorphism and genome evolution. Here, we report the chromosome‐level assembly of the O.macrolepis genome obtained from the integration of nanopore long‐read sequencing with physical maps produced using Bionano and Hi‐C technology. A total of 87.9 Gb of nanopore sequence provided approximately 100‐fold coverage of the genome. The preliminary genome assembly was 883.2 Mb in size with a contig N50 size of 11.2 Mb. The 969 corrected contigs obtained from Bionano optical mapping were assembled into 853 scaffolds and produced an assembly of 886.5 Mb with a scaffold N50 of 16.5 Mb. Finally, using the Hi‐C data, 881.3 Mb (99.4% of genome) in 526 scaffolds were anchored and oriented in 25 chromosomes ranging in size from 25.27 to 56.49 Mb. In total, 24,770 protein‐coding genes were predicted in the genome, and ~96.85% of the genes were functionally annotated. The annotated assembly contains 93.3% complete genes from the BUSCO reference set. In addition, we identified 409 Mb (46.23% of the genome) of repetitive sequence, and 11,213 non‐coding RNAs, in the genome. Evolutionary analysis revealed that O. macrolepis diverged from common carp approximately 24.25 million years ago. The chromosomes of O. macrolepis showed an unambiguous correspondence to the chromosomes of zebrafish. The high‐quality genome assembled in this work provides a valuable genomic resource for further biological and evolutionary studies of O. macrolepis.     

Description of complete mitochondrial genome of the black-veined white,Aporia crataegi (Lepidoptera: Papilionoidea), and comparison to papilionoid species

The black-veined white, Aporia crataegi (Lepidoptera: Papilionoidea) is nearly extinct in South Korea, although substantial numbers of dried specimens are available. One of the common practices used to rescue such endangered species is to launch a re-introduction program after a proper amount of genetic information is analyzed from donor and donee populations. In this study, we sequenced the complete mitochondrial genome (mitogenome) of A. crataegi to accumulate genetic information for subsequent population studies and to further understand the mitogenome evolution in true butterflies, Papilionoidea. The 15,140-bp long A. crataegi mitogenome has typical sets of 37 genes and is the smallest among the true butterfly species, with overall slightly smaller size genes and regions throughout the genome. The A/T content of the genome (81.3%) is the highest in Pieridae, where A. crataegi belongs, but lower than that of the lycaenid species (81.7%–82.7%). Unlike the diversified or modified usage of an anticodon for tRNA^Ser(AGN), the species of Pieridae including A. crataegi all contain GCT that has been hypothesized as being ancestral for Lepidoptera. A total of 111 bp of non-coding sequences are interspersed in 13 regions, ranging in size from 1–49 bp. Among these sequences, relatively longer ones (≥ 16 bp) all have relatively higher sequence identity to other regions of the genome, suggesting partial duplication of the sequences during A. crataegi evolution.