A k-mer-based bulked segregant analysis approach to map seed traits in unphased heterozygous potato genomes.

High-throughput sequencing-based methods for bulked segregant analysis (BSA) allow for the rapid identification of genetic markers associated with traits of interest. BSA studies have successfully identified qualitative (binary) and quantitative trait loci (QTLs) using QTL mapping. However, most require population structures that fit the models available and a reference genome. Instead, high-throughput short-read sequencing can be combined with BSA of k-mers (BSA-k-mer) to map traits that appear refractory to standard approaches. This method can be applied to any organism and is particularly useful for species with genomes diverged from the closest sequenced genome. It is also instrumental when dealing with highly heterozygous and potentially polyploid genomes without phased haplotype assemblies and for which a single haplotype can control a trait. Finally, it is flexible in terms of population structure. Here, we apply the BSA-k-mer method for the rapid identification of candidate regions related to seed spot and seed size in diploid potato. Using a mixture of F1 and F2 individuals from a cross between 2 highly heterozygous parents, candidate sequences were identified for each trait using the BSA-k-mer approach. Using parental reads, we were able to determine the parental origin of the loci. Finally, we mapped the identified k-mers to a closely related potato genome to validate the method and determine the genomic loci underlying these sequences. The location identified for the seed spot matches with previously identified loci associated with pigmentation in potato. The loci associated with seed size are novel. Both loci are relevant in future breeding toward true seeds in potato.

Recurrent neo-sex chromosome evolution in kiwifruit.

Sex chromosome evolution is thought to be tightly associated with the acquisition and maintenance of sexual dimorphisms. Plant sex chromosomes have evolved independently in many lineages1,2 and can provide a powerful comparative framework to study this. We assembled and annotated genome sequences of three kiwifruit species (genus Actinidia) and uncovered recurrent sex chromosome turnovers in multiple lineages. Specifically, we observed structural evolution of the neo-Y chromosomes, which was driven via rapid bursts of transposable element insertions. Surprisingly, sexual dimorphisms were conserved in the different species studied, despite the fact that the partially sex-linked genes differ between them. Using gene editing in kiwifruit, we demonstrated that one of the two Y-chromosome-encoded sex-determining genes, Shy Girl, shows pleiotropic effects that can explain the conserved sexual dimorphisms. These plant sex chromosomes therefore maintain sexual dimorphisms through the conservation of a single gene, without a process involving interactions between separate sex-determining genes and genes for sexually dimorphic traits.

The Arabidopsis Hop1 homolog ASY1 mediates cross-over assurance and interference.

The chromosome axis plays a crucial role in meiotic recombination. Here, we study the function of ASY1, the Arabidopsis homolog of the yeast chromosome axis-associated component Hop1. Specifically, we characterized cross-over (CO) distribution in female and male meiosis by deep sequencing of the progeny of an allelic series of asy1 mutants. Combining data from nearly 1,000 individual plants, we find that reduced ASY1 functionality leads to genomic instability and sometimes drastic genomic rearrangements. We further observed that COs are less frequent and appear in more distal chromosomal regions in plants with no or reduced ASY1 functionality, consistent with previous analyses. However, our sequencing approach revealed that the reduction in CO number is not as dramatic as suggested by cytological analyses. Analysis of double mutants of asy1 with mutants with three other CO factors, MUS81, MSH4, and MSH5, as well as the determination of foci number of the CO regulator MLH1 demonstrates that the majority of the COs in asy1, similar to the situation in the wildtype (WT), largely belong to the class I, which are subject to interference. However, these COs are redistributed in asy1 mutants and typically appear much closer than in the WT. Hence, ASY1 plays a key role in CO interference that spaces COs along a chromosome. Conversely, since a large proportion of chromosomes do not receive any CO, we conclude that CO assurance, the process that ensures the obligatory assignment of one CO per chromosome, is also affected in asy1 mutants.

Chromoanagenesis in plants: triggers, mechanisms, and potential impact.

Chromoanagenesis is a single catastrophic event that involves, in most cases, localized chromosomal shattering and reorganization, resulting in a dramatically restructured chromosome. First discovered in cancer cells, it has since been observed in various other systems, including plants. In this review, we discuss the origin, characteristics, and potential mechanisms underlying chromoanagenesis in plants. We report that multiple processes, including mutagenesis and genetic engineering, can trigger chromoanagenesis via a variety of mechanisms such as micronucleation, breakage-fusion-bridge (BFB) cycles, or chain-like translocations. The resulting rearranged chromosomes can be preserved during subsequent plant growth, and sometimes inherited to the next generation. Because of their high tolerance to genome restructuring, plants offer a unique system for investigating the evolutionary consequences and potential practical applications of chromoanagenesis.

Chromoanagenesis in the asy1 meiotic mutant of Arabidopsis.

Chromoanagenesis is a catastrophic event that involves localized chromosomal shattering and reorganization. In this study, we report a case of chromoanagenesis resulting from defective meiosis in the MEIOTIC ASYNAPTIC MUTANT 1 (asy1) background in Arabidopsis thaliana. We provide a detailed characterization of the genomic structure of this individual with a severely shattered segment of chromosome 1. We identified 260 novel DNA junctions in the affected region, most of which affect gene sequence on 1 or both sides of the junction. Our results confirm that asy1-related defective meiosis is a potential trigger for chromoanagenesis. This is the first example of chromoanagenesis associated with female meiosis and indicates the potential for genome evolution during oogenesis. PLAIN LANGUAGE SUMMARY: Chromoanagenesis is a complex and catastrophic event that results in severely restructured chromosomes. It has been identified in cancer cells and in some plant samples, after specific triggering events. Here, we identified this kind of genome restructuring in a mutant that exhibits defective meiosis in the model plant system Arabidopsis thaliana.

Reinvention of hermaphroditism via activation of a RADIALIS-like gene in hexaploid persimmon.

In flowering plants, different lineages have independently transitioned from the ancestral hermaphroditic state into and out of various sexual systems1. Polyploidizations are often associated with this plasticity in sexual systems2,3. Persimmons (the genus Diospyros) have evolved dioecy via lineage-specific palaeoploidizations. More recently, hexaploid D. kaki has established monoecy and also exhibits reversions from male to hermaphrodite flowers in response to natural environmental signals (natural hermaphroditism, NH), or to artificial cytokinin treatment (artificial hermaphroditism, AH). We sought to identify the molecular pathways underlying these polyploid-specific reversions to hermaphroditism. Co-expression network analyses identified regulatory pathways specific to NH or AH transitions. Surprisingly, the two pathways appeared to be antagonistic, with abscisic acid and cytokinin signalling for NH and AH, respectively. Among the genes common to both pathways leading to hermaphroditic flowers, we identified a small-Myb RADIALIS-like gene, named DkRAD, which is specifically activated in hexaploid D. kaki. Consistently, ectopic overexpression of DkRAD in two model plants resulted in hypergrowth of the gynoecium. These results suggest that production of hermaphrodite flowers via polyploidization depends on DkRAD activation, which is not associated with a loss-of-function within the existing sex determination pathway, but rather represents a new path to (or reinvention of) hermaphroditism.

Diploid mint (M. longifolia) can produce spearmint type oil with a high yield potential.

Mint oil is a key source of natural flavors with wide industrial applications. Two unbalanced polyploid cultivars named Native (Mentha Spicata L) and Scotch (M. × gracilis Sole) are the main producers of spearmint type oil, which is characterized by high levels of the monoterpenes (-)-carvone and (-)-limonene. These cultivars have been the backbone of spearmint oil production for decades, while breeding and improvement remained largely unexplored, in part, due to sterility in cultivated lines. Here we show that sexual breeding at the diploid level can be leveraged to develop new varieties that produce spearmint type oil, along with the improvement of other important traits. Using field trials and GC-FID oil analysis we characterized plant materials from a public germplasm repository and identified a diploid accession that exhibited 89.5% increase in oil yield, compared to the industry standard, and another that produces spearmint type oil. Spearmint-type oil was present at high frequency in a segregating F2 population (32/160) produced from these two accessions. Field-testing of ten of these F2 lines showed segregation for oil yield and confirmed the production of spearmint-type oil profiles. Two of these lines combined high yield and spearmint-type oil with acceptable analytic and sensory profiles. These results demonstrate that spearmint-type oil can be produced in a diploid background with high yield potential, providing a simpler genetic system for the development of improved spearmint varieties.

LD-CNV: rapid and simple discovery of chromosomal translocations using linkage disequilibrium between copy number variable loci.

Large-scale structural variations, such as chromosomal translocations, can have profound effects on fitness and phenotype, but are difficult to identify and characterize. Here, we describe a simple and effective method aimed at identifying translocations using only the dosage of sequence reads mapped on the reference genome. We binned reads on genomic segments sized according to sequencing coverage and identified instances when copy number segregated in populations. For each dosage-polymorphic 1 Mb bin, we tested independence, effectively an apparent linkage disequilibrium (LD), with other variable bins. In nine potato (Solanum tuberosum) dihaploid families translocations affecting pericentromeric regions were common and in two cases were due to genomic misassembly. In two populations, we found evidence for translocation affecting euchromatic arms. In cv. PI 310467, a nonreciprocal translocation between chromosomes (chr.) 7 and 8 resulted in a 5-3 copy number change affecting several Mb at the respective chromosome tips. In cv. "Alca Tarma," the terminal arm of chr. 4 translocated to the tip of chr. 1. Using oligonucleotide-based fluorescent in situ hybridization painting probes (oligo-FISH), we tested and confirmed the predicted arrangement in PI 310467. In 192 natural accessions of Arabidopsis thaliana, dosage haplotypes tended to vary continuously and resulted in higher noise, while apparent LD between pericentromeric regions suggested the effect of repeats. This method, LD-CNV, should be useful in species where translocations are suspected because it tests linkage without the need for genotyping.

Efficient construction of a linkage map and haplotypes for Mentha suaveolens using sequence capture.

The sustainability of many crops is hindered by the lack of genomic resources and a poor understanding of natural genetic diversity. Particularly, application of modern breeding requires high-density linkage maps that are integrated into a highly contiguous reference genome. Here, we present a rapid method for deriving haplotypes and developing linkage maps, and its application to Mentha suaveolens, one of the diploid progenitors of cultivated mints. Using sequence-capture via DNA hybridization to target single nucleotide polymorphisms (SNPs), we successfully genotyped ∼5000 SNPs within the genome of >400 individuals derived from a self cross. After stringent quality control, and identification of nonredundant SNPs, 1919 informative SNPs were retained for linkage map construction. The resulting linkage map defined a total genetic space of 942.17 cM divided among 12 linkage groups, ranging from 56.32 to 122.61 cM in length. The linkage map is in good agreement with pseudomolecules from our preliminary genome assembly, proving this resource effective for the correction and validation of the reference genome. We discuss the advantages of this method for the rapid creation of linkage maps.

Genetic Regulation of Vessel Morphology in Populus.

During secondary growth, forest trees can modify the anatomy of the wood produced by the vascular cambium in response to environmental conditions. Notably, the trees of the model angiosperm genus, Populus, reduce the risk of cavitation and hydraulic failure under water stress by producing water-conducting vessel elements with narrow lumens, which are more numerous and more interconnected with each other. Here, we determined the genetic architecture of vessel traits affecting hydraulic physiology and resilience to water stress. Vessel traits were measured for clonally replicated genotypes of a unique Populus deltoides x nigra population carrying genomically defined insertions and deletions that create gene dosage variation. We found significant phenotypic variation for all traits measured (mean vessel diameter, height-corrected mean vessel diameter, vessel frequency, height-corrected vessel frequency, vessel grouping index, and mean vessel circularity), and that all traits were under genetic control and showed moderate heritability values, ranging from 0.32 to 0.53. Whole-genome scans of correlations between gene dosage and phenotypic traits identified quantitative trait loci for tree height, mean vessel diameter, height-corrected mean vessel diameter, height-corrected vessel frequency, and vessel grouping index. Our results demonstrate that vessel traits affecting hydraulic physiology are under genetic control, and both pleiotropic and trait-specific quantitative trait loci are found for these traits.

Doses delivered by portal imaging quality assurance in routine practice of adjuvant breast radiotherapy worth to by monitored and compensated in some cases.

BACKGROUND: Imaging, in radiotherapy, has become a routine tool for repositioning of the target volume at each session. The repositioning precision, currently infracentimetric, evolves along with the irradiation techniques. This retrospective study aimed to identify practices and doses resulting from the use of high energy planar imaging (portal imaging) in daily practice. METHODS: A retrospective survey of portal images (PIs) was carried out over 10 years for 2,403 patients and for three linacs (1 Elekta SLi, 2 Varian Clinac) for postoperative mammary irradiations. Images were taken using a standardized number of monitor units (MU) for all patients. Due to the variable sensitivities of the detectors and the possibility of adjustment of the detector-patient distance, the number of MU were 3; 2 and 1 respectively, for Elekta SLi®, Clinac 600® and Clinac 2100®. Then, a representative cumulated dose was calculated in simplified reference conditions (5 cm depth, beam of 10 cm × 10 cm, 6 MV), considering the total number of images taken during the whole treatment course. The consistency between the representative doses and the actual absorbed doses received by the patients was verified by simulating a series of typical cases with the treatment plan dose calculation system. RESULTS: The delivered doses differ significantly between the three linacs. The mean representative dose values by complete treatment were 0.695; 0.241 and 0.216 Gy, respectively, for SLi, Clinac 600 and Clinac 2100. However, 15 patients were exposed to a dose >2 Gy with a maximum dose of 5.05 Gy. The simulated doses were very similar to the representative doses. CONCLUSIONS: A significant dose delivery was highlighted by this study. These representative doses are presently communicated weekly to the radiation oncologist for the radiation protection of their patients. Moreover, they should be taken into account in a possible study of long-term stochastic risks.

Chromoanagenesis from radiation-induced genome damage in Populus.

Chromoanagenesis is a genomic catastrophe that results in chromosomal shattering and reassembly. These extreme single chromosome events were first identified in cancer, and have since been observed in other systems, but have so far only been formally documented in plants in the context of haploid induction crosses. The frequency, origins, consequences, and evolutionary impact of such major chromosomal remodeling in other situations remain obscure. Here, we demonstrate the occurrence of chromoanagenesis in poplar (Populus sp.) trees produced from gamma-irradiated pollen. Specifically, in this population of siblings carrying indel mutations, two individuals exhibited highly frequent copy number variation (CNV) clustered on a single chromosome, one of the hallmarks of chromoanagenesis. Using short-read sequencing, we confirmed the presence of clustered segmental rearrangement. Independently, we identified and validated novel DNA junctions and confirmed that they were clustered and corresponded to these rearrangements. Our reconstruction of the novel sequences suggests that the chromosomal segments have reorganized randomly to produce a novel rearranged chromosome but that two different mechanisms might be at play. Our results indicate that gamma irradiation can trigger chromoanagenesis, suggesting that this may also occur when natural or induced mutagens cause DNA breaks. We further demonstrate that such events can be tolerated in poplar, and even replicated clonally, providing an attractive system for more in-depth investigations of their consequences.

Rare instances of haploid inducer DNA in potato dihaploids and ploidy-dependent genome instability.

In cultivated tetraploid potato (Solanum tuberosum), reduction to diploidy (dihaploidy) allows for hybridization to diploids and introgression breeding and may facilitate the production of inbreds. Pollination with haploid inducers (HIs) yields maternal dihaploids, as well as triploid and tetraploid hybrids. Dihaploids may result from parthenogenesis, entailing the development of embryos from unfertilized eggs, or genome elimination, entailing missegregation and the loss of paternal chromosomes. A sign of genome elimination is the occasional persistence of HI DNA in some dihaploids. We characterized the genomes of 919 putative dihaploids and 134 hybrids produced by pollinating tetraploid clones with three HIs: IVP35, IVP101, and PL-4. Whole-chromosome or segmental aneuploidy was observed in 76 dihaploids, with karyotypes ranging from 2n = 2x - 1 = 23 to 2n = 2x + 3 = 27. Of the additional chromosomes in 74 aneuploids, 66 were from the non-inducer parent and 8 from the inducer parent. Overall, we detected full or partial chromosomes from the HI parent in 0.87% of the dihaploids, irrespective of parental genotypes. Chromosomal breaks commonly affected the paternal genome in the dihaploid and tetraploid progeny, but not in the triploid progeny, correlating instability to sperm ploidy and to haploid induction. The residual HI DNA discovered in the progeny is consistent with genome elimination as the mechanism of haploid induction.

A systems genetics approach to deciphering the effect of dosage variation on leaf morphology in Populus.

Gene copy number variation is frequent in plant genomes of various species, but the impact of such gene dosage variation on morphological traits is poorly understood. We used a large population of Populus carrying genomically characterized insertions and deletions across the genome to systematically assay the effect of gene dosage variation on a suite of leaf morphology traits. A systems genetics approach was used to integrate insertion and deletion locations, leaf morphology phenotypes, gene expression, and transcriptional network data, to provide an overview of how gene dosage influences morphology. Dosage-sensitive genomic regions were identified that influenced individual or pleiotropic morphological traits. We also identified cis-expression quantitative trait loci (QTL) within these dosage QTL regions, a subset of which modulated trans-expression QTL as well. Integration of data types within a gene co-expression framework identified co-expressed gene modules that are dosage sensitive, enriched for dosage expression QTL, and associated with morphological traits. Functional description of these modules linked dosage-sensitive morphological variation to specific cellular processes, as well as candidate regulatory genes. Together, these results show that gene dosage variation can influence morphological variation through complex changes in gene expression, and suggest that frequently occurring gene dosage variation has the potential to likewise influence quantitative traits in nature.

Early steroid withdrawal has a positive effect on bone in kidney transplant recipients: a propensity score study with inverse probability-of-treatment weighting.

BACKGROUND: Long-term corticosteroid use after kidney transplantation is associated with a decrease in bone mineral density (BMD) and a high fracture risk. We hypothesized that patients with early steroid withdrawal (ESW) would display a gain in BMD in the year following kidney transplantation, when compared with patients on long-term corticosteroid therapy. METHODS: In a cohort of kidney transplant recipients, 356 patients were included between 2012 and 2019. Dual-energy X-ray absorptiometry was performed 1 and 12 months after transplantation. The data were analyzed using linear regression with inverse probability-of-treatment weighting (based on a propensity score). RESULTS: At 1 year after transplantation, the gain in BMD was significantly greater in recipients with ESW than in recipients on long-term corticosteroid therapy for the lumbar spine (+0.036 g/cm2, p < 0.001) and the femoral neck (+0.020 g/cm2, p = 0.035). Among patients with ESW, (i) none had osteoporosis, (ii) the percentage with normal BMD increased from 33.3% at month 1 to 54.4% at month 12, and (iii) the percentage with osteopenia fell from 56.2% to 45.6%. In patients undergoing long-term corticosteroid therapy, the fracture incidence was 13.5 per 1000 person-years. None of the patients in the ESW group experienced a fracture. CONCLUSION: ESW has a positive effect on bone in kidney transplant recipients.

Association between Uremic Toxin Concentrations and Bone Mineral Density after Kidney Transplantation.

Although uremic osteoporosis is a component of mineral and bone disorder in chronic kidney disease, uremic toxin (UT) concentrations in patients with end-stage kidney disease and bone mineral density (BMD) changes after kidney transplantation have not previously been described. We hypothesized that elevated UT concentrations at the time of transplantation could have a negative impact on bone during the early post-transplantation period. Hence, we sought to determine whether concentrations of UTs (trimethylamine-N-oxide, indoxylsulfate, p-cresylsulfate, p-cresylglucuronide, indole-3-acetic acid, hippuric acid, and 3-carboxy-4-methyl-5-propyl-furanpropionic acid) upon transplantation are predictive markers for (i) osteoporosis one month after transplantation, and (ii) a BMD decrease and the occurrence of fractures 12 and 24 months after kidney transplantation. Between 2012 and 2018, 310 kidney transplant recipients were included, and dual-energy X-ray absorptiometry was performed 1, 12, and 24 months after transplantation. The UT concentrations upon transplantation were determined by reverse-phase high-performance liquid chromatography. Indoxylsulfate concentrations upon transplantation were positively correlated with BMD one month after transplantation for the femoral neck but were not associated with osteoporosis status upon transplantation. Concentrations of the other UTs upon transplantation were not associated with osteoporosis or BMD one month after transplantation. None of the UT concentrations were associated with BMD changes and the occurrence of osteoporotic fractures 12 and 24 months after transplantation. Hence, UT concentrations at the time of kidney transplantation were not predictive markers of osteoporosis or fractures.

Non-Vocal Behaviors Are More Frequent During the Decisive Negotiation Phases in Barn Owl Siblings.

Animals produce vibrations or noises by means of body movements, which can play a role in communication. These behaviors enhance signal transmission or receiver attention and could be specifically used during turn-taking phases of a reciprocal exchange of signals. In the barn owl Tyto alba, nestlings vocalize one after the other to negotiate which individual will have priority access to the impending prey item to be delivered by the parents. Owlets adjust their vocalization to their own hunger level and to their siblings' vocalization, withdrawing from the contest in front of highly vocal, and hence hungry, motivated nestmates. As sibling negotiation is a multicomponent display, we examined whether body movements could also be part of the negotiation process. To this end, we analyzed whether the vocalizations of one nestling affected its nestmate's movements in three separate experiments: in natural nests, in the lab, and using a playback procedure. Nestling barn owls move in a variety of ways, such as repeated tapping of the floor with a foot, scratching the floor with claws, or flapping wings. Body movements were more frequent during the turn-taking phases of vocal interactions, when siblings emitted longer calls and at a greater rate. Once an individual monopolized vocal activity, siblings became less vocal and less active. Moreover, owlets produced more noisy body movements during the phases of vocal interactions which are crucial to prevail in negotiation. Non-vocal physical activities might reinforce vocal signals during sibling to sibling (sib-sib) interactions, or reflect owlets' arousal, in the critical period during which they vocally settle which individual will dominate the competition.

Genome-wide study on the polysomic genetic factors conferring plasticity of flower sexuality in hexaploid persimmon.

Sexuality is one of the fundamental mechanisms that work towards maintaining genetic diversity within a species. In diploid persimmons (Diospyros spp.), separated sexuality, the presence of separate male and female individuals (dioecy), is controlled by the Y chromosome-encoded small-RNA gene, OGI. On the other hand, sexuality in hexaploid Oriental persimmon (Diospyros kaki) is more plastic, with OGI-bearing genetically male individuals, able to produce both male and female flowers (monoecy). This is thought to be linked to the partial inactivation of OGI by a retrotransposon insertion, resulting in DNA methylation of the OGI promoter region. To identify the genetic factors regulating branch sexual conversion, genome-wide correlation/association analyses were conducted using ddRAD-Seq data from an F1 segregating population, and using both quantitative and diploidized genotypes, respectively. We found that allelic ratio at the Y-chromosomal region, including OGI, was correlated with male conversion based on quantitative genotypes, suggesting that OGI can be activated in cis in a dosage-dependent manner. Genome-wide association analysis based on diploidized genotypes, normalized for the effect of OGI allele dosage, detected three fundamental loci associated with male conversion. These loci underlie candidate genes, which could potentially act epigenetically for the activation of OGI expression.

Correction: The persimmon genome reveals clues to the evolution of a lineage-specific sex determination system in plants.

[This corrects the article DOI: 10.1371/journal.pgen.1008566.].