A chromosome-level,haplotype-phased Vanilla planifolia genome highlights the challenge of partial endoreplication for accurate wholegenome assembly

Vanilla planifolia, the species cultivated to produce one of the world’s most popular flavors, is highly proneto partial genome endoreplication, which leads to highly unbalanced DNA content in cells. We report herethe first molecular evidence of partial endoreplication at the chromosome scale by the assembly and annotation of an accurate haplotype-phased genome of V. planifolia. Cytogenetic data demonstrated that thediploid genome size is 4.09 Gb, with 16 chromosome pairs, although aneuploid cells are frequentlyobserved. Using PacBio HiFi and optical mapping, we assembled and phased a diploid genome of 3.4 Gbwith a scaffold N50 of 1.2 Mb and 59 128 predicted protein-coding genes. The atypical k-mer frequenciesand the uneven sequencing depth observed agreed with our expectation of unbalanced genome representation. Sixty-seven percent of the genes were scattered over only 30% of the genome, putatively linkinggene-rich regions and the endoreplication phenomenon. By contrast, low-coverage regions (non-endoreplicated) were rich in repeated elements but also contained 33% of the annotated genes. Furthermore, this assembly showed distinct haplotype-specific sequencing depth variation patterns, suggesting complexmolecular regulation of endoreplication along the chromosomes. This high-quality, anchored assemblyrepresents 83% of the estimated V. planifolia genome. It provides a significant step toward the elucidationof this complex genome. To support post-genomics efforts, we developed the Vanilla Genome Hub, a userfriendly integrated web portal that enables centralized access to high-throughput genomic and other omicsdata and interoperable use of bioinformatics tools.

Dual role of FGF in proliferation and endoreplication of Drosophila tracheal adult progenitor cells

Adult progenitor cells activation is a key event in the formation of adult organs.In Drosophilat formation of abdominal adult trachea depends on the specific activation of tracheal adult progenitors(tracheoblasts)at the Tr4 and Tr5 spiracular branches.Proliferation of these tracheoblasts generates a pool of tracheal cells that migrate toward the posterior part of the trachea by the activation of the branchless/fibroblast growth factor(Bnl/FGF)signaling to form the abdominal adult trachea.Here,we show that,in addition to migration,Bnl/FGF signaling,mediated by the transcription factor Pointed,is also required for tracheoblast proliferation.This tracheoblast activation relies on the expression of the FGF ligand bnl in their nearby branches.Finally,we show that,in the absence of the transcription factor Cut(Ct),Bnl/FGF signaling induces endoreplication of tracheoblasts partially by promoting fizzy-related expression.Altogether,our results suggest a dual role of Bnl/FGF signaling in tracheoblasts,inducing both proliferation and endoreplication,depending on the presence or absence of the transcription factor Ct,respectively.

Haploidization of Human Diploid Metaphase Cells: Is This Genome Reductive Mechanism Opperational in Near-Haploid Leukemia?

The present study presents cytogenetics/cytology of haploidization in the origin of a new, fast growing diploid, small cell-type (F-dPCs). The sequence of events was haploid groupings of the chromosomes in normal, human metaphase cells, followed by genomic doubling to homozygousdiploidy. These events were responses to DNA replication stress fromamino acid glutamine deprivation. Importantly, these homozygous cells outgrew normal fibroblasts in 2 - 3 passages—they had gained proliferative advantage (GPA), presumably from loss (LOH) of tumor suppressor genes. They were morphologically changed cells with rounded nuclei that grew in a “streaming” growth pattern and with changed form and size of mitosis, similar to some hyperplasias. The grouping of the chromosomes in metaphase cells was asymmetric with a narrow range around the median (23) (no micro-nuclei), suggesting genetic control. The root-origin of haploidization was evidenced by maternal and paternal genomes occupying separate territories in metaphase cells, which assumedly permitted independent segregations of bichromatid chromosomes. In near-haploid ALL-L1 leukemia the loss of virtually, whole chromosomal complements was judged by SNP array analyses, as a primary event before genomic doubling to hyperdiploidy with LOH. From the present data such specific, non-random loss of chromosomes strongly suggested, a haploidization process capable of genomic doubling, as observed for the “birth” of the small, F-dPCs. This suggestion was supported by this type of leukemia being the L1-type, where L1 signifies small cells. The possibility now exists that a tumorigenic process can be initiated directly from diploid cells through haploid (near-haploid) distributed chromosomes in normal metaphase cells. This event followed by monosomic doublings to UPDs would lead to massive LOH and a return to para-diploidy, a frequent occurrence in many types of tumors. The present simple, cultural derivations of the extraordinary F-dPCs allow GPA-identification and experimental manipulations, perhaps relevant in a vaccine program.

Tissue-specific genome editing of laminA/C in the posterior silk glands of Bombyx mori

The RNA-guided CRISPR/Cas9 system has been shown to be a powerful tool for genome editing in various organisms. A comprehensive toolbox for multiplex genome editing has been developed for the silkworm,Bombyx mori, a lepidopteran model insect of economic importance. However, as previous methods mainly relied on delivery of transient Cas9/guide RNA（gRNA）, they could not be used in loss-of-function studies of essential genes. Here, we report a simple and versatile tissue-specific genome editing strategy.We perform a proof-of-principle demonstration by establishing and crossing two transgenic B. mori lines,one expressing Cas9 protein in the posterior silk glands（PSGs） and the other constitutively expressing BmlaminA/C（BmLMN） gRNA. All BmLMN alleles in the PSG cells were edited precisely at the target genome region, resulting in diverse mutations. mRNA expression of BmLMN was reduced by up to 75%,and only very low levels of BmLaminA/C protein were detected. Knockout of BmLMN produced obvious defects in gland cell development and cocoon production. In this study, we developed an efficient strategy for spatially controlled genome editing, providing unprecedented opportunities for investigating the function of essential/lethal genes in B. mori, with potential application for other insects.