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The Neuroscience Monoclonal Antibody Sequencing Initiative (NeuroMabSeq) is a concerted effort to determine and make publicly available hybridoma-derived sequences of monoclonal antibodies (mAbs) valuable to neuroscience research. Over 30 years of research and development efforts including those at the UC Davis/NIH NeuroMab Facility have resulted in the generation of a large collection of mouse mAbs validated for neuroscience research. To enhance dissemination and increase the utility of this valuable resource, we applied a high-throughput DNA sequencing approach to determine immunoglobulin heavy and light chain variable domain sequences from source hybridoma cells. The resultant set of sequences was made publicly available as a searchable DNA sequence database (neuromabseq.ucdavis.edu) for sharing, analysis and use in downstream applications. We enhanced the utility, transparency, and reproducibility of the existing mAb collection by using these sequences to develop recombinant mAbs. This enabled their subsequent engineering into alternate forms with distinct utility, including alternate modes of detection in multiplexed labeling, and as miniaturized single chain variable fragments or scFvs. The NeuroMabSeq website and database and the corresponding recombinant antibody collection together serve as a public DNA sequence repository of mouse mAb heavy and light chain variable domain sequences and as an open resource for enhancing dissemination and utility of this valuable collection of validated mAbs.

Formation of highly unique and complex facial structures is controlled by genetic programs that are responsible for the precise coordination of three-dimensional tissue morphogenesis. However, the underlying mechanisms governing these processes remain poorly understood. We combined mouse genetic and genomic approaches to define the mechanisms underlying normal and defective midfacial morphogenesis. Conditional inactivation of the Wnt secretion protein Wls in Pax3-expressing lineage cells disrupted frontonasal primordial patterning, cell survival and directional outgrowth, resulting in altered facial structures, including midfacial hypoplasia and midline facial clefts. Single-cell RNA sequencing revealed unique transcriptomic atlases of mesenchymal subpopulations in the midfacial primordia, which are disrupted in the conditional Wls mutants. Differentially expressed genes and cis-regulatory sequence analyses uncovered that Wls modulates and integrates a core gene regulatory network, consisting of key midfacial regulatory transcription factors (including Msx1, Pax3 and Pax7) and their downstream targets (including Wnt, Shh, Tgfβ and retinoic acid signaling components), in a mesenchymal subpopulation of the medial nasal prominences that is responsible for midline facial formation and fusion. These results reveal fundamental mechanisms underlying mammalian midfacial morphogenesis and related defects at single-cell resolution., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2022. Published by The Company of Biologists Ltd.)

The wild tomato relative Solanum sitiens is a xerophyte endemic to the Atacama Desert of Chile and a potential source of genes for tolerance to drought, salinity and low-temperature stresses. However, until recently, strong breeding barriers prevented its hybridization and introgression with cultivated tomato, Solanum lycopersicum L. We overcame these barriers using embryo rescue, bridging lines and allopolyploid hybrids, and synthesized a library of introgression lines (ILs) that captures the genome of S. sitiens in the background of cultivated tomato. The IL library consists of 56 overlapping introgressions that together represent about 93% of the S. sitiens genome: 65% in homozygous and 28% in heterozygous (segregating) ILs. The breakpoints of each segment and the gaps in genome coverage were mapped by single nucleotide polymorphism (SNP) genotyping using the SolCAP SNP array. Marker-assisted selection was used to backcross selected introgressions into tomato, to recover a uniform genetic background, to isolate recombinant sub-lines with shorter introgressions and to select homozygous genotypes. Each IL contains a single S. sitiens chromosome segment, defined by markers, in the genetic background of cv. NC 84173, a fresh market inbred line. Large differences were observed between the lines for both qualitative and quantitative morphological traits, suggesting that the ILs contain highly divergent allelic variation. Several loci contributing to unilateral incompatibility or hybrid necrosis were mapped with the lines. This IL population will facilitate studies of the S. sitiens genome and expands the range of genetic variation available for tomato breeding and research., (© 2019 The Authors The Plant Journal © 2019 John Wiley & Sons Ltd.)

Genetic markers are essential when developing or working with genetically variable populations. Indel Group in Genomes (IGG) markers are primer pairs that amplify single-locus sequences that differ in size for two or more alleles. They are attractive for their ease of use for rapid genotyping and their codominant nature. Here, we describe a heuristic algorithm that uses a k-mer-based approach to search two or more genome sequences to locate polymorphic regions suitable for designing candidate IGG marker primers. As input to the IGG pipeline software, the user provides genome sequences and the desired amplicon sizes and size differences. Primer sequences flanking polymorphic insertions/deletions are produced as output. IGG marker files for three sets of genomes, Solanum lycopersicum/Solanum pennellii, Arabidopsis (Arabidopsis thaliana) Columbia-0/Landsberg erecta-0 accessions, and S. lycopersicum/S. pennellii/Solanum tuberosum (three-way polymorphic) are included., (© 2016 American Society of Plant Biologists. All rights reserved.)

Interspecific hybridization often induces epigenetic remodeling that leads to transposon activation, gene expression changes, and loss of imprinting. These genomic changes can be deleterious and contribute to postzygotic hybrid incompatibility. In Arabidopsis, loss of genomic imprinting of PHERES1 and presumed failure of Polycomb Repressive Complex contributes to seed inviability observed in A. thaliana X A. arenosa interspecific hybrids. We used this species pair to further analyze the relationship between parentally biased gene expression and postzygotic hybrid incompatibility using two A. thaliana accessions, Col-0 and C24, with differential seed survival. We found that parentally biased expression was perturbed to a similar degree in both A. thaliana hybrids for PHERES1, HDG3, and six other normally paternally expressed genes. We propose that early genome remodeling and loss of imprinting of seed development genes induces lethality in both compatible and incompatible hybrids.

Seed death resulting from hybridization between Arabidopsis thaliana and Arabidopsis arenosa has complex genetic determination and involves deregulation 5 to 8 d after pollination (DAP) of agamous-like genes and retroelements. To identify causal mechanisms, we compared transcriptomes of compatible and incompatible hybrids and parents at 3 DAP. Hybrids misexpressed endosperm and seed coat regulators and hyperactivated genes encoding ribosomal, photosynthetic, stress-related, and immune response proteins. Regulatory disruption was more severe in Columbia-0 hybrids than in C24 hybrids, consistent with the degree of incompatibility. Maternal loss-of-function alleles for endosperm growth factor transparent testa glabra2 and HAIKU1 and defense response regulators non-expressor of pathogenesis related1 and salicylic acid induction-deficient2 increased hybrid seed survival. The activation of presumed polycomb repressive complex (PRC) targets, together with a 20-fold reduction in expression of fertilization independent seed2, indicated a PRC role. Proximity to transposable elements affected natural variation for gene regulation, but transposon activation did not differ from controls. Collectively, this investigation provides candidates for multigenic orchestration of the incompatibility response through disruption of endosperm development, a novel role for communication between endosperm and maternal tissues and for pathways previously connected to immunity, but, surprisingly, does not identify a role for transposons.

The cross between Arabidopsis thaliana and the closely related species Arabidopsis arenosa results in postzygotic hybrid incompatibility, manifested as seed death. Ecotypes of A. thaliana were tested for their ability to produce live seed when crossed to A. arenosa. The identified genetic variation was used to map quantitative trait loci (QTLs) encoded by the A. thaliana genome that affect the frequency of postzygotic lethality and the phenotypes of surviving seeds. Seven QTLs affecting the A. thaliana component of this hybrid incompatibility were identified by crossing a Columbia × C24 recombinant inbred line population to diploid A. arenosa pollen donors. Additional epistatic loci were identified based on their pairwise interaction with one or several of these QTLs. Epistatic interactions were detected for all seven QTLs. The two largest additive QTLs were subjected to fine-mapping, indicating the action of at least two genes in each. The topology of this network reveals a large set of minor-effect loci from the maternal genome controlling hybrid growth and viability at different developmental stages. Our study establishes a framework that will enable the identification and characterization of genes and pathways in A. thaliana responsible for hybrid lethality in the A. thaliana × A. arenosa interspecific cross.

Aneuploid cells are characterized by incomplete chromosome sets. The resulting imbalance in gene dosage has phenotypic consequences that are specific to each karyotype. Even in the case of Down syndrome, the most viable and studied form of human aneuploidy, the mechanisms underlying the connected phenotypes remain mostly unclear. Because of their tolerance to aneuploidy, plants provide a powerful system for a genome-wide investigation of aneuploid syndromes, an approach that is not feasible in animal systems. Indeed, in many plant species, populations of aneuploid individuals can be easily obtained from triploid individuals. We phenotyped a population of Arabidopsis thaliana aneuploid individuals containing 25 different karyotypes. Even in this highly heterogeneous population, we demonstrate that certain traits are strongly associated with the dosage of specific chromosome types and that chromosomal effects can be additive. Further, we identified subtle developmental phenotypes expressed in the diploid progeny of aneuploid parent(s) but not in euploid controls from diploid lineages. These results indicate long-term phenotypic consequences of aneuploidy that can persist after chromosomal balance has been restored. We verified the diploid nature of these individuals by whole-genome sequencing and discuss the possibility that trans-generational phenotypic effects stem from epigenetic modifications passed from aneuploid parents to their diploid progeny.

The molecular mechanisms underlying lethality of F1 hybrids between diverged parents are one target of speciation research. Crosses between diploid and tetraploid individuals of the same genotype can result in F1 lethality, and this dosage-sensitive incompatibility plays a role in polyploid speciation. We have identified variation in F1 lethality in interploidy crosses of Arabidopsis thaliana and determined the genetic architecture of the maternally expressed variation via QTL mapping. A single large-effect QTL, DR. STRANGELOVE 1 (DSL1), was identified as well as two QTL with epistatic relationships to DSL1. DSL1 affects the rate of postzygotic lethality via expression in the maternal sporophyte. Fine mapping placed DSL1 in an interval encoding the maternal effect transcription factor TTG2. Maternal parents carrying loss-of-function mutations in TTG2 suppressed the F1 lethality caused by paternal excess interploidy crosses. The frequency of cellularization in the endosperm was similarly affected by both natural variation and ttg2 loss-of-function mutants. The simple genetic basis of the natural variation and effects of single-gene mutations suggests that F1 lethality in polyploids could evolve rapidly. Furthermore, the role of the sporophytically active TTG2 gene in interploidy crosses indicates that the developmental programming of the mother regulates the viability of interploidy hybrid offspring.