Genome design of hybrid potato.

Reinventing potato from a clonally propagated tetraploid into a seed-propagated diploid, hybrid potato, is an important innovation in agriculture. Due to deleterious mutations, it has remained a challenge to develop highly homozygous inbred lines, a prerequisite to breed hybrid potato. Here, we employed genome design to develop a generation of pure and fertile potato lines and thereby the uniform, vigorous F1s. The metrics we applied in genome design included the percentage of genome homozygosity and the number of deleterious mutations in the starting material, the number of segregation distortions in the S1 population, the haplotype information to infer the break of tight linkage between beneficial and deleterious alleles, and the genome complementarity of the parental lines. This study transforms potato breeding from a slow, non-accumulative mode into a fast-iterative one, thereby potentiating a broad spectrum of benefits to farmers and consumers.

De novo deletions and duplications at recombination hotspots in mouse germlines.

Numerous DNA double-strand breaks (DSBs) arise during meiosis to initiate homologous recombination. These DSBs are usually repaired faithfully, but here, we uncover a distinct type of mutational event in which deletions form via joining of ends from two closely spaced DSBs (double cuts) within a single hotspot or at adjacent hotspots on the same or different chromatids. Deletions occur in normal meiosis but are much more frequent when DSB formation is dysregulated in the absence of the ATM kinase. Events between chromosome homologs point to multi-chromatid damage and aborted gap repair. Some deletions contain DNA from other hotspots, indicating that double cutting at distant sites creates substrates for insertional mutagenesis. End joining at double cuts can also yield tandem duplications or extrachromosomal circles. Our findings highlight the importance of DSB regulation and reveal a previously hidden potential for meiotic mutagenesis that is likely to affect human health and genome evolution.

Mendel, mechanism, models, marketing, and more.

This year marks the 150(th) anniversary of the presentation by Gregor Mendel of his studies of plant hybridization to the Brunn Natural History Society. Their nature and meaning have been discussed many times. However, on this occasion, we reflect on the scientific enterprise and the perception of new discoveries.

Identification and specification of the mouse skeletal stem cell.

How are skeletal tissues derived from skeletal stem cells? Here, we map bone, cartilage, and stromal development from a population of highly pure, postnatal skeletal stem cells (mouse skeletal stem cells, mSSCs) to their downstream progenitors of bone, cartilage, and stromal tissue. We then investigated the transcriptome of the stem/progenitor cells for unique gene-expression patterns that would indicate potential regulators of mSSC lineage commitment. We demonstrate that mSSC niche factors can be potent inducers of osteogenesis, and several specific combinations of recombinant mSSC niche factors can activate mSSC genetic programs in situ, even in nonskeletal tissues, resulting in de novo formation of cartilage or bone and bone marrow stroma. Inducing mSSC formation with soluble factors and subsequently regulating the mSSC niche to specify its differentiation toward bone, cartilage, or stromal cells could represent a paradigm shift in the therapeutic regeneration of skeletal tissues.

Selfish conflict underlies RNA-mediated parent-of-origin effects.

Genomic imprinting-the non-equivalence of maternal and paternal genomes-is a critical process that has evolved independently in many plant and mammalian species1,2. According to kinship theory, imprinting is the inevitable consequence of conflictive selective forces acting on differentially expressed parental alleles3,4. Yet, how these epigenetic differences evolve in the first place is poorly understood3,5,6. Here we report the identification and molecular dissection of a parent-of-origin effect on gene expression that might help to clarify this fundamental question. Toxin-antidote elements (TAs) are selfish elements that spread in populations by poisoning non-carrier individuals7-9. In reciprocal crosses between two Caenorhabditis tropicalis wild isolates, we found that the slow-1/grow-1 TA is specifically inactive when paternally inherited. This parent-of-origin effect stems from transcriptional repression of the slow-1 toxin by the PIWI-interacting RNA (piRNA) host defence pathway. The repression requires PIWI Argonaute and SET-32 histone methyltransferase activities and is transgenerationally inherited via small RNAs. Remarkably, when slow-1/grow-1 is maternally inherited, slow-1 repression is halted by a translation-independent role of its maternal mRNA. That is, slow-1 transcripts loaded into eggs-but not SLOW-1 protein-are necessary and sufficient to counteract piRNA-mediated repression. Our findings show that parent-of-origin effects can evolve by co-option of the piRNA pathway and hinder the spread of selfish genes that require sex for their propagation.

Species-wide genetic incompatibility analysis identifies immune genes as hot spots of deleterious epistasis.

Intraspecific genetic incompatibilities prevent the assembly of specific alleles into single genotypes and influence genome- and species-wide patterns of sequence variation. A common incompatibility in plants is hybrid necrosis, characterized by autoimmune responses due to epistatic interactions between natural genetic variants. By systematically testing thousands of F1 hybrids of Arabidopsis thaliana strains, we identified a small number of incompatibility hot spots in the genome, often in regions densely populated by nucleotide-binding domain and leucine-rich repeat (NLR) immune receptor genes. In several cases, these immune receptor loci interact with each other, suggestive of conflict within the immune system. A particularly dangerous locus is a highly variable cluster of NLR genes, DM2, which causes multiple independent incompatibilities with genes that encode a range of biochemical functions, including NLRs. Our findings suggest that deleterious interactions of immune receptors limit the combinations of favorable disease resistance alleles accessible to plant genomes.

Apoptotic caspases suppress mtDNA-induced STING-mediated type I IFN production.

Activated caspases are a hallmark of apoptosis induced by the intrinsic pathway, but they are dispensable for cell death and the apoptotic clearance of cells in vivo. This has led to the suggestion that caspases are activated not just to kill but to prevent dying cells from triggering a host immune response. Here, we show that the caspase cascade suppresses type I interferon (IFN) production by cells undergoing Bak/Bax-mediated apoptosis. Bak and Bax trigger the release of mitochondrial DNA. This is recognized by the cGAS/STING-dependent DNA sensing pathway, which initiates IFN production. Activated caspases attenuate this response. Pharmacological caspase inhibition or genetic deletion of caspase-9, Apaf-1, or caspase-3/7 causes dying cells to secrete IFN-ß. In vivo, this precipitates an elevation in IFN-ß levels and consequent hematopoietic stem cell dysfunction, which is corrected by loss of Bak and Bax. Thus, the apoptotic caspase cascade functions to render mitochondrial apoptosis immunologically silent.

Competing E3 ubiquitin ligases govern circadian periodicity by degradation of CRY in nucleus and cytoplasm.

Period determination in the mammalian circadian clock involves the turnover rate of the repressors CRY and PER. We show that CRY ubiquitination engages two competing E3 ligase complexes that either lengthen or shorten circadian period in mice. Cloning of a short-period circadian mutant, Past-time, revealed a glycine to glutamate missense mutation in Fbxl21, an F-box protein gene that is a paralog of Fbxl3 that targets the CRY proteins for degradation. While loss of function of FBXL3 leads to period lengthening, mutation of Fbxl21 causes period shortening. FBXL21 forms an SCF E3 ligase complex that slowly degrades CRY in the cytoplasm but antagonizes the stronger E3 ligase activity of FBXL3 in the nucleus. FBXL21 plays a dual role: protecting CRY from FBXL3 degradation in the nucleus and promoting CRY degradation within the cytoplasm. Thus, the balance and cellular compartmentalization of competing E3 ligases for CRY determine circadian period of the clock in mammals.

Mutation in folate metabolism causes epigenetic instability and transgenerational effects on development.

The importance of maternal folate consumption for normal development is well established, yet the molecular mechanism linking folate metabolism to development remains poorly understood. The enzyme methionine synthase reductase (Mtrr) is necessary for utilization of methyl groups from the folate cycle. We found that a hypomorphic mutation of the mouse Mtrr gene results in intrauterine growth restriction, developmental delay, and congenital malformations, including neural tube, heart, and placental defects. Importantly, these defects were dependent upon the Mtrr genotypes of the maternal grandparents. Furthermore, we observed widespread epigenetic instability associated with altered gene expression in the placentas of wild-type grandprogeny of Mtrr-deficient maternal grandparents. Embryo transfer experiments revealed that Mtrr deficiency in mice lead to two distinct, separable phenotypes: adverse effects on their wild-type daughters' uterine environment, leading to growth defects in wild-type grandprogeny, and the appearance of congenital malformations independent of maternal environment that persist for five generations, likely through transgenerational epigenetic inheritance.

Systems genetics of metabolism: the use of the BXD murine reference panel for multiscalar integration of traits.

Metabolic homeostasis is achieved by complex molecular and cellular networks that differ significantly among individuals and are difficult to model with genetically engineered lines of mice optimized to study single gene function. Here, we systematically acquired metabolic phenotypes by using the EUMODIC EMPReSS protocols across a large panel of isogenic but diverse strains of mice (BXD type) to study the genetic control of metabolism. We generated and analyzed 140 classical phenotypes and deposited these in an open-access web service for systems genetics (www.genenetwork.org). Heritability, influence of sex, and genetic modifiers of traits were examined singly and jointly by using quantitative-trait locus (QTL) and expression QTL-mapping methods. Traits and networks were linked to loci encompassing both known variants and novel candidate genes, including alkaline phosphatase (ALPL), here linked to hypophosphatasia. The assembled and curated phenotypes provide key resources and exemplars that can be used to dissect complex metabolic traits and disorders.

Telomerase reactivation following telomere dysfunction yields murine prostate tumors with bone metastases.

To determine the role of telomere dysfunction and telomerase reactivation in generating pro-oncogenic genomic events and in carcinoma progression, an inducible telomerase reverse transcriptase (mTert) allele was crossed onto a prostate cancer-prone mouse model null for Pten and p53 tumor suppressors. Constitutive telomerase deficiency and associated telomere dysfunction constrained cancer progression. In contrast, telomerase reactivation in the setting of telomere dysfunction alleviated intratumoral DNA-damage signaling and generated aggressive cancers with rearranged genomes and new tumor biological properties (bone metastases). Comparative oncogenomic analysis revealed numerous recurrent amplifications and deletions of relevance to human prostate cancer. Murine tumors show enrichment of the TGF-ß/SMAD4 network, and genetic validation studies confirmed the cooperative roles of Pten, p53, and Smad4 deficiencies in prostate cancer progression, including skeletal metastases. Thus, telomerase reactivation in tumor cells experiencing telomere dysfunction enables full malignant progression and provides a mechanism for acquisition of cancer-relevant genomic events endowing new tumor biological capabilities.

Genetic crosses within and between species of Cryptosporidium.

Parasites and their hosts are engaged in reciprocal coevolution that balances competing mechanisms of virulence, resistance, and evasion. This often leads to host specificity, but genomic reassortment between different strains can enable parasites to jump host barriers and conquer new niches. In the apicomplexan parasite Cryptosporidium, genetic exchange has been hypothesized to play a prominent role in adaptation to humans. The sexual lifecycle of the parasite provides a potential mechanism for such exchange; however, the boundaries of Cryptosporidium sex are currently undefined. To explore this experimentally, we established a model for genetic crosses. Drug resistance was engineered using a mutated phenylalanyl tRNA synthetase gene and marking strains with this and the previously used Neo transgene enabled selection of recombinant progeny. This is highly efficient, and genomic recombination is evident and can be continuously monitored in real time by drug resistance, flow cytometry, and PCR mapping. Using this approach, multiple loci can now be modified with ease. We demonstrate that essential genes can be ablated by crossing a Cre recombinase driver strain with floxed strains. We further find that genetic crosses are also feasible between species. Crossing Cryptosporidium parvum, a parasite of cattle and humans, and Cryptosporidium tyzzeri a mouse parasite resulted in progeny with a recombinant genome derived from both species that continues to vigorously replicate sexually. These experiments have important fundamental and translational implications for the evolution of Cryptosporidium and open the door to reverse- and forward-genetic analysis of parasite biology and host specificity.

Phylogenetic tree building in the genomic age.

Knowing phylogenetic relationships among species is fundamental for many studies in biology. An accurate phylogenetic tree underpins our understanding of the major transitions in evolution, such as the emergence of new body plans or metabolism, and is key to inferring the origin of new genes, detecting molecular adaptation, understanding morphological character evolution and reconstructing demographic changes in recently diverged species. Although data are ever more plentiful and powerful analysis methods are available, there remain many challenges to reliable tree building. Here, we discuss the major steps of phylogenetic analysis, including identification of orthologous genes or proteins, multiple sequence alignment, and choice of substitution models and inference methodologies. Understanding the different sources of errors and the strategies to mitigate them is essential for assembling an accurate tree of life.

Malaria parasite clag3 genes determine channel-mediated nutrient uptake by infected red blood cells.

Development of malaria parasites within vertebrate erythrocytes requires nutrient uptake at the host cell membrane. The plasmodial surface anion channel (PSAC) mediates this transport and is an antimalarial target, but its molecular basis is unknown. We report a parasite gene family responsible for PSAC activity. We used high-throughput screening for nutrient uptake inhibitors to identify a compound highly specific for channels from the Dd2 line of the human pathogen P. falciparum. Inheritance of this compound's affinity in a Dd2 × HB3 genetic cross maps to a single parasite locus on chromosome 3. DNA transfection and in vitro selections indicate that PSAC-inhibitor interactions are encoded by two clag3 genes previously assumed to function in cytoadherence. These genes are conserved in plasmodia, exhibit expression switching, and encode an integral protein on the host membrane, as predicted by functional studies. This protein increases host cell permeability to diverse solutes.

Sickle hemoglobin confers tolerance to Plasmodium infection.

Sickle human hemoglobin (Hb) confers a survival advantage to individuals living in endemic areas of malaria, the disease caused by Plasmodium infection. As demonstrated hereby, mice expressing sickle Hb do not succumb to experimental cerebral malaria (ECM). This protective effect is exerted irrespectively of parasite load, revealing that sickle Hb confers host tolerance to Plasmodium infection. Sickle Hb induces the expression of heme oxygenase-1 (HO-1) in hematopoietic cells, via a mechanism involving the transcription factor NF-E2-related factor 2 (Nrf2). Carbon monoxide (CO), a byproduct of heme catabolism by HO-1, prevents further accumulation of circulating free heme after Plasmodium infection, suppressing the pathogenesis of ECM. Moreover, sickle Hb inhibits activation and/or expansion of pathogenic CD8(+) T cells recognizing antigens expressed by Plasmodium, an immunoregulatory effect that does not involve Nrf2 and/or HO-1. Our findings provide insight into molecular mechanisms via which sickle Hb confers host tolerance to severe forms of malaria.

A yeast living ancestor reveals the origin of genomic introgressions.

Genome introgressions drive evolution across the animal1, plant2 and fungal3 kingdoms. Introgressions initiate from archaic admixtures followed by repeated backcrossing to one parental species. However, how introgressions arise in reproductively isolated species, such as yeast4, has remained unclear. Here we identify a clonal descendant of the ancestral yeast hybrid that founded the extant Saccharomyces cerevisiae Alpechin lineage5, which carries abundant Saccharomyces paradoxus introgressions. We show that this clonal descendant, hereafter defined as a 'living ancestor', retained the ancestral genome structure of the first-generation hybrid with contiguous S. cerevisiae and S. paradoxus subgenomes. The ancestral first-generation hybrid underwent catastrophic genomic instability through more than a hundred mitotic recombination events, mainly manifesting as homozygous genome blocks generated by loss of heterozygosity. These homozygous sequence blocks rescue hybrid fertility by restoring meiotic recombination and are the direct origins of the introgressions present in the Alpechin lineage. We suggest a plausible route for introgression evolution through the reconstruction of extinct stages and propose that genome instability allows hybrids to overcome reproductive isolation and enables introgressions to emerge.

Solanum pennellii (LA5240) backcross inbred lines (BILs) for high resolution mapping in tomato.

Wild species are an invaluable source of new traits for crop improvement. Over the years, the tomato community bred cultivated lines that carry introgressions from different species of the tomato tribe to facilitate trait discovery and mapping. The next phase in such projects is to find the genes that drive the identified phenotypes. This can be achieved by genotyping a few thousand individuals resulting in fine mapping that can potentially identify the causative gene. To couple trait discovery and fine mapping, we are presenting large, recombination-rich, Backcross Inbred Line (BIL) populations involving an unexplored accession of the wild, green-fruited species Solanum pennellii (LA5240; the 'Lost' Accession) with two modern tomato inbreds: LEA, determinate, and TOP, indeterminate. The LEA and TOP BILs are in BC2F6-8 generation and include 1400 and 500 lines, respectively. The BILs were genotyped with 5000 SPET markers, showing that in the euchromatic regions there was one recombinant every 17-18 Kb while in the heterochromatin a recombinant every 600-700 Kb (TOP and LEA respectively). To gain perspective on the topography of recombination we compared five independent members of the Self-pruning gene family with their respective neighboring genes; based on PCR markers, in all cases we found recombinants. Further mapping analysis of two known morphological mutations that segregated in the BILs (self-pruning and hairless) showed that the maximal delimited intervals were 73 Kb and 210 Kb, respectively, and included the known causative genes. The 'Lost'_BILs provide a solid framework to study traits derived from a drought-tolerant wild tomato.

Development and application of an efficient genomic mating method to maximize the production performances of three-way crossbred pigs.

Creating synthetic lines is the standard mating mode for commercial pig production. Traditional mating performance was evaluated through a strictly designed cross-combination test at the 'breed level' to maximize the benefits of production. The Duroc-Landrace-Yorkshire (DLY) three-way crossbred production system became the most widely used breeding scheme for pigs. Here, we proposed an 'individual level' genomic mating procedure that can be applied to commercial pig production with efficient algorithms for estimating marker effects and for allocating the appropriate boar-sow pairs, which can be freely accessed to public in our developed HIBLUP software at https://www.hiblup.com/tutorials#genomic-mating. A total of 875 Duroc boars, 350 Landrace-Yorkshire sows and 3573 DLY pigs were used to carry out the genomic mating to assess the production benefits theoretically. The results showed that genomic mating significantly improved the performances of progeny across different traits compared with random mating, such as the feed conversion rate, days from 30 to 120 kg and eye muscle area could be improved by -0.12, -4.64 d and 2.65 cm2, respectively, which were consistent with the real experimental validations. Overall, our findings indicated that genomic mating is an effective strategy to improve the performances of progeny by maximizing their total genetic merit with consideration of both additive and dominant effects. Also, a herd of boars from a richer genetic source will increase the effectiveness of genomic mating further.

Predictability and parallelism in the contemporary evolution of hybrid genomes.

Hybridization between species is widespread across the tree of life. As a result, many species, including our own, harbor regions of their genome derived from hybridization. Despite the recognition that this process is widespread, we understand little about how the genome stabilizes following hybridization, and whether the mechanisms driving this stabilization tend to be shared across species. Here, we dissect the drivers of variation in local ancestry across the genome in replicated hybridization events between two species pairs of swordtail fish: Xiphophorus birchmanni × X. cortezi and X. birchmanni × X. malinche. We find unexpectedly high levels of repeatability in local ancestry across the two types of hybrid populations. This repeatability is attributable in part to the fact that the recombination landscape and locations of functionally important elements play a major role in driving variation in local ancestry in both types of hybrid populations. Beyond these broad scale patterns, we identify dozens of regions of the genome where minor parent ancestry is unusually low or high across species pairs. Analysis of these regions points to shared sites under selection across species pairs, and in some cases, shared mechanisms of selection. We show that one such region is a previously unknown hybrid incompatibility that is shared across X. birchmanni × X. cortezi and X. birchmanni × X. malinche hybrid populations.

Genome-wide identification of quantitative trait loci and candidate genes for seven carcass traits in a four-way intercross porcine population.

BACKGROUND: Carcass traits are essential economic traits in the commercial pig industry. However, the genetic mechanism of carcass traits is still unclear. In this study, we performed a genome-wide association study (GWAS) based on the specific-locus amplified fragment sequencing (SLAF-seq) to study seven carcass traits on 223 four-way intercross pigs, including dressing percentage (DP), number of ribs (RIB), skin thinkness (ST), carcass straight length (CSL), carcass diagonal length (CDL), loin eye width (LEW), and loin eye thickness (LET). RESULTS: A total of 227,921 high-quality single nucleotide polymorphisms (SNPs) were detected to perform GWAS. A total of 30 SNPs were identified for seven carcass traits using the mixed linear model (MLM) (p < 1.0 × 10- 5), of which 9 SNPs were located in previously reported quantitative trait loci (QTL) regions. The phenotypic variation explained (PVE) by the significant SNPs was from 2.43 to 16.32%. Furthermore, 11 candidate genes (LYPLAL1, EPC1, MATN2, ZFAT, ZBTB10, ZNF704, INHBA, SMYD3, PAK1, SPTBN2, and ACTN3) were found for carcass traits in pigs. CONCLUSIONS: The GWAS results will improve our understanding of the genetic basis of carcass traits. We hypothesized that the candidate genes associated with these discovered SNPs would offer a biological basis for enhancing the carcass quality of pigs in swine breeding.