Precise Characterization and Tracking of Stably Inherited Artificial Minichromosomes Made by Telomere-Mediated Chromosome Truncation in Brassica napus

Plant artificial minichromosomes are the next-generation technology for plant genetic engineering and represent an independent platform for expressing foreign genes and the tools for studying the structure and function of chromosomes. Minichromosomes have been successfully produced by telomere-mediated chromosome truncation in several plants. However, previous studies have primarily focused on the construction and rough characterization of minichromosomes, while the development of stably inherited minichromosomes and their precise characterization and tracking over different generations have rarely been demonstrated. In this study, a 0.35-kb direct repeat of the Arabidopsis telomeric sequence was transformed into Brassica napus to produce artificial minichromosomes, which were analyzed by multifluorescence in situ hybridization (multi-FISH), Southern hybridization, and primer extension telomere rapid amplification (PETRA). The stably inherited minichromosomes C2 and C4 were developed by crossing transgenic plants with wild-type plants and then selfing the hybrids. Notably, two truncation sites on chromosomes C2 and C4, respectively, were identified by resequencing; thus, the artificial minichromosomes were tracked over different generations with insertion site-specific PCR. This study provided two stably inherited minichromosomes in oilseed rape and describes approaches to precisely characterize the truncation position and track the minichromosomes in offspring through multi-FISH, genome resequencing, and insertion site-specific PCR.

Molecular cytogenetics and St-chromosome-specific molecular markers development of novel stripe rust resistant wheat–Thinopyrum intermedium as well as wheat–Thinopyrum ponticum substitution lines

Background Owing to the excellent resistance to abiotic and biotic stress, Thionpyrum intermedium (2n = 6x = 42, JJJsJsStSt) and Thinopyrum ponticum (2n = 10x = 70) are both widely utilized in wheat germplasm innovation programs. Disomic substitution lines (DSLs) carrying one pair of alien chromosomes are valuable bridge materials for novel genes transmission, FISH karyotype construction and specific molecular marker development. Results Six wheat–Thinopyrum DSLs derived from crosses between Abbondanza nullisomic lines (2n = 40) and two octoploid Trititrigia lines (2n = 8x = 56), were characterized by a sequential fluorescence in situ hybridization (FISH)–genome in situ hybridization (GISH), a multicolor GISH (mc-GISH), and an analysis of wheat 15K SNP array combined with molecular marker selection. ES-9 (DS2St (2A)) and ES-10 (DS3St (3D)) are wheat–Th. ponticum DSLs, while ES-23 (DS2St (2A)), ES-24 (DS3St (3D)), ES-25(DS2St (2B)), and ES-26 (DS2St (2D)) are wheat–Th. intermedium DSLs. ES-9, ES-23, ES-25 and ES-26 conferred higher thousand-kernel weight and stripe rust resistance at adult stages, while ES-10 and ES-24 performed highly resistant to stripe rust at all stages. Furthermore, cytological analysis showed that the alien chromosomes (2St/3St) belonging to the same homoeologous group derived from different donors carried the same FISH karyotype and could normally form a bivalent. Based on specific-locus amplified fragment sequencing (SLAF-seq), two 2St-chromosome-specific markers (PTH-005 and PTH-013) and two 3St-chromosome-specific markers (PTH-113 and PTH-135) were developed. Conclusions The six wheat–Thinopyrum disomic substitution lines conferring stripe rust resistance will be used as bridging parents for valuable resistant genes transmission. And the utility of PTH-113 and PTH-135 in a BC1F2 population showed the newly developed markers could be useful tools for efficient identification of St chromosomes in a common wheat background.

A Chromosome-Level Genome Assembly of the Mandarin Fish (Siniperca chuatsi)

The mandarin fish, Siniperca chuatsi, is an economically important perciform species with widespread aquaculture practices in China. Its special feeding habit, acceptance of only live prey fishes, contributes to its delicious meat. However, little is currently known about related genetic mechanisms. Here, we performed whole-genome sequencing and assembled a 758.78 Mb genome assembly of the mandarin fish, with the scaffold and contig N50 values reaching 2.64 Mb and 46.11 Kb, respectively. Approximately 92.8% of the scaffolds were ordered onto 24 chromosomes (Chrs) with the assistance of a previously established genetic linkage map. The chromosome-level genome contained 19,904 protein-coding genes, of which 19,059 (95.75%) genes were functionally annotated. The special feeding behavior of mandarin fish could be attributable to the interaction of a variety of sense organs (such as vision, smell, and endocrine organs). Through comparative genomics analysis, some interesting results were found. For example, olfactory receptor (OR) genes (especially the beta and delta types) underwent a significant expansion, and endocrinology/vision related npy, spexin, and opsin genes presented various functional mutations. These may contribute to the special feeding habit of the mandarin fish by strengthening the olfactory and visual systems. Meanwhile, previously identified sex-related genes and quantitative trait locis (QTLs) were localized on the Chr14 and Chr17, respectively. 155 toxin proteins were predicted from mandarin fish genome. In summary, the high-quality genome assembly of the mandarin fish provides novel insights into the feeding habit of live prey and offers a valuable genetic resource for the quality improvement of this freshwater fish.

Molecular Cytogenetics of Chromosome 2St as Well as Chromosome 3St Derived from Thinopyrum Intermedium and Thinopyrum Ponticum

Owing to the excellent resistance to abiotic and biotic stress, Thionpyrum intermedium (2n = 6x = 42, JJJsJsStSt) and Thinopyrum ponticum (2n = 10x = 70) are both widely utilized in wheat germplasm innovation programs. Disomic substitution lines (DSLs) carrying one pair of alien chromosomes are valuable bridge materials for novel genes transmission. In this study, six wheat-Thinopyrum DSLs were derived from crosses between Abbondanza nullisomic lines (2n = 40) and two octoploid Trititrigia lines (2n = 8x = 56), characterized by a sequential fluorescence in situ hybridization (FISH)-genome in situ hybridization (GISH), a multicolor GISH (mc-GISH), and an analysis of wheat 15K SNP array combined with molecular marker selection. ES-9 and ES-10 were two wheat- Th. ponticum disomic substitution lines, DS2St (2A) and DS3St (3D). While ES-23, ES-24, ES-25, and ES-26 were four wheat- Th. intermedium disomic substitution lines, DS2St (2A), DS3St (3D), DS2St (2B), DS2St (2D). The FISH karyotypes of Th. ponticum 2St/3St chromosomes were well coincident with the ones of Th. intermedium. The chromosome configurations of F1 hybrids derived from crosses between ES-23 and ES-9, as well as ES-24 and ES-10 were mostly formed 21Ⅱ. Four St-chromosome-specific markers were developed by specific-locus amplified fragment sequencing (SLAF-seq). Additionally, the substitution lines containing chromosome 2St conferred higher thousand-kernel weight and stripe rust resistance at adult stages, while the substitution lines containing chromosome 3St were highly resistant to stripe rust at all stages. Therefore, these six substitution lines could serve as useful bridging parents for wheat genetic improvement.

A chromosome-level genome of Astyanax mexicanus surface fish for comparing population-specific genetic differences contributing to trait evolution

Identifying the genetic factors that underlie complex traits is central to understanding the mechanistic underpinnings of evolution. Cave-dwelling Astyanax mexicanus populations are well adapted to subterranean life and many populations appear to have evolved troglomorphic traits independently, while the surface-dwelling populations can be used as a proxy for the ancestral form. Here we present a high-resolution, chromosome-level surface fish genome, enabling the first genome-wide comparison between surface fish and cavefish populations. Using this resource, we performed quantitative trait locus (QTL) mapping analyses and found new candidate genes for eye loss such as dusp26. We used CRISPR gene editing in A. mexicanus to confirm the essential role of a gene within an eye size QTL, rx3, in eye formation. We also generated the first genome-wide evaluation of deletion variability across cavefish populations to gain insight into this potential source of cave adaptation. The surface fish genome reference now provides a more complete resource for comparative, functional and genetic studies of drastic trait differences within a species.

Genetic and Epigenetic Changes Are Rapid Responses of the Genome to the Newly Synthesized Autotetraploid Carassius auratus

Whole genome duplication events have occurred frequently during the course of vertebrate evolution. To better understand the influence of polyploidization on the fish genome, we herein used the autotetraploid Carassius auratus (4n = 200, RRRR) (4nRR) resulting from the whole genome duplication of Carassius auratus (2n = 100, RR) (RCC) to explore the genomic and epigenetic alterations after polyploidization. We subsequently performed analyses of full-length transcriptome dataset, amplified fragment length polymorphism (AFLP) and methylation sensitive amplification polymorphism (MSAP) on 4nRR and RCC. By matching the results of 4nRR and RCC isoforms with reference genome in full-length transcriptome dataset, 649 and 1,971 novel genes were found in the RCC and 4nRR full-length geneset, respectively. Compared to Carassius auratus and Megalobrama amblycephala, 4nRR presented 3,661 unexpressed genes and 2,743 expressed genes. Furthermore, GO enrichment analysis of expressed genes in 4nRR revealed that they were enriched in meiosis I, whereas KEGG enrichment analysis displayed that they were mainly enriched in proteasome. Using AFLP analysis, we noted that 32.61% of RCC fragments had disappeared, while 32.79% of new bands were uncovered in 4nRR. Concerning DNA methylation, 4nRR exhibited a lower level of global DNA methylation than RCC. Additionally, 60.31% of methylation patterns in 4nRR were altered compared to RCC. These observations indicated that transcriptome alterations, genomic changes and regulation of DNA methylation levels and patterns had occurred in the newly established autotetraploid genomes, suggesting that genetic and epigenetic alterations were influenced by autotetraploidization. In summary, this study provides valuable novel insights into vertebrate genome evolution and generates relevant information for fish breeding.

A Major QTL for Resistance to Vibrio anguillarum in Rainbow Trout

Genetic selection of disease resistant fish is a major strategy to improve health, welfare and sustainability in aquaculture. Mapping of single nucleotide polymorphisms (SNP) in the fish genome may be a fruitful tool to define relevant quantitative trait loci (QTL) and we here show its use for characterization of Vibrio anguillarum resistant rainbow trout (Oncorhynchus mykiss). Fingerlings were exposed to the pathogen V. anguillarum serotype O1 in a solution of 1.5 × 107 cfu/ml and observed for 14 days. Disease signs appeared 3 days post exposure (dpe) whereafter mortality progressed exponentially until 6 dpe reaching a total mortality of 55% within 11 days. DNA was sampled from all fish – including survivors – and analyzed on a 57 k Affymetrix SNP platform whereby it was shown that disease resistance was associated with a major QTL on chromosome 21 (Omy 21). Gene expression analyses showed that diseased fish activated genes associated with innate and adaptive immune responses. The possible genes associated with resistance are discussed.

Comparative Homology Modeling and Physicochemical Characterization of Cyprinus carpio Hsp70 Protein

Background: Expression of the heat shock proteins (Hsp) is responsible for the protection from adverse climatic changes particularly heat stress in Common carp (Cyprinus carpio). Although, with advancement of molecular techniques, Hsp70 protein has been isolated but this protein needs to be characterized by both physicochemically and structurally for the functional annotation of fish genome. So this current study was undertaken with aim of generation of various protein models and also for thorough physiochemical characterization of this protein.Methods: In this study, Hsp70 protein of common carp was characterized by both physiochemical and structurally through insilco platform and as the crystal structure of this protein is not available, protein models were created though homology modelling upon Modeller version 9.21, Phyre2 and Swiss-model and then the generated predicted models were evaluated through Rampage, Errat, Verify 3D, ProQ and ProSA analysis.Result: Our investigation showed that this protein is very stable, hydrophilic with no disulphide bonds and the protein models which were generated from this study, are of good quality with z value of - 9.58, -9.48 and -10.93 and quality factor of 82.56, 59.43 and 95.27 respectively. So this study was concluded that the generated Hsp70 protein models would provide an avenue for the other researchers for development of high-throughput gene function assignment in fish.

Genome of the four-finger threadfin Eleutheronema tetradactylum (Perciforms: Polynemidae)

Background Teleost fish play important roles in aquatic ecosystems and aquaculture. Threadfins (Perciformes: Polynemidae) show a range of interesting biology, and are of considerable importance for both wild fisheries and aquaculture. Additionally, the four-finger threadfin Eleutheronema tetradactylum is of conservation relevance since its populations are considered to be in rapid decline and it is classified as endangered. However, no genomic resources are currently available for the threadfin family Polynemidae. Results We sequenced and assembled the first threadfin fish genome, the four-finger threadfin E. tetradactylum. We provide a genome assembly for E. tetradactylum with high contiguity (scaffold N50 = 56.3 kb) and high BUSCO completeness at 96.5%. The assembled genome size of E. tetradactylum is just 610.5 Mb, making it the second smallest perciform genome assembled to date. Just 9.07–10.91% of the genome sequence was found to consist of repetitive elements (standard RepeatMasker analysis vs custom analysis), making this the lowest repeat content identified to date for any perciform fish. A total of 37,683 protein-coding genes were annotated, and we include analyses of developmental transcription factors, including the Hox, ParaHox, and Sox families. MicroRNA genes were also annotated and compared with other chordate lineages, elucidating the gains and losses of chordate microRNAs. Conclusions The four-finger threadfin E. tetradactylum genome presented here represents the first available genome sequence for the ecologically, biologically, and commercially important clade of threadfin fish. Our findings provide a useful genomic resource for future research into the interesting biology and evolution of this valuable group of food fish.