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Host genetic variation can shape the diversity and composition of associated microbiomes, which may reciprocally influence host traits and performance. While the genetic basis of phenotypic diversity of plant populations in nature has been studied, comparatively little research has investigated the genetics of host effects on their associated microbiomes. Switchgrass (Panicum virgatum) is a highly outcrossing, perennial, grass species with substantial locally adaptive diversity across its native North American range. Here, we compared 383 switchgrass accessions in a common garden to determine the host genotypic influence on rhizosphere bacterial composition. We hypothesized that the composition and diversity of rhizosphere bacterial assemblages would differentiate due to genotypic differences between hosts (potentially due to root phenotypes and associated life history variation). We observed higher alpha diversity of bacteria associated with upland ecotypes and tetraploids, compared to lowland ecotypes and octoploids, respectively. Alpha diversity correlated negatively with flowering time and plant height, indicating that bacterial composition varies along switchgrass life history axes. Narrow-sense heritability (h2) of the relative abundance of twenty-one core bacterial families was observed. Overall compositional differences among tetraploids, due to genetic variation, supports wide-spread genotypic influence on the rhizosphere microbiome. Lastly, a genome-wide association study identified 1,861 single-nucleotide polymorphisms associated with 110 families and genes containing them related to potential regulatory functions. Our findings suggest that switchgrass genomic and life-history variation influences bacterial composition in the rhizosphere, potentially due to host adaptation to local environments.

Author(s): Yu, Shu; Li, Michelle; Dubcovsky, Jorge; Tian, Li | Abstract: Grains of tetraploid wheat (Triticum turgidum L.) mainly accumulate the non-provitamin A carotenoid lutein-with low natural variation in provitamin A β-carotene in wheat accessions necessitating alternative strategies for provitamin A biofortification. Lycopene e-cyclase (LCYe) and β-carotene hydroxylase (HYD) function in diverting carbons from β-carotene to lutein biosynthesis and catalyzing the turnover of β-carotene to xanthophylls, respectively. However, the contribution of LCYe and HYD gene homoeologs to carotenoid metabolism and how they can be manipulated to increase β-carotene in tetraploid wheat endosperm (flour) is currently unclear. We isolated loss-of-function Targeting Induced Local Lesions in Genomes (TILLING) mutants of LCYe and HYD2 homoeologs and generated higher order mutant combinations of lcye-A, lcye-B, hyd-A2, and hyd-B2. Hyd-A2 hyd-B2, lcye-A hyd-A2 hyd-B2, lcye-B hyd-A2 hyd-B2, and lcye-A lcye-B hyd-A2 hyd-B2 achieved significantly increased β-carotene in endosperm, with lcye-A hyd-A2 hyd-B2 exhibiting comparable photosynthetic performance and light response to control plants. Comparative analysis of carotenoid profiles suggests that eliminating HYD2 homoeologs is sufficient to prevent β-carotene conversion to xanthophylls in the endosperm without compromising xanthophyll production in leaves, and that β-carotene and its derived xanthophylls are likely subject to differential catalysis mechanisms in vegetative tissues and grains. Carotenoid and gene expression analyses also suggest that the very low LCYe-B expression in endosperm is adequate for lutein production in the absence of LCYe-A. These results demonstrate the success of provitamin A biofortification using TILLING mutants while also providing a roadmap for guiding a gene editing-based approach in hexaploid wheat.

Septins play key roles in mammalian cell division and cytokinesis but have not previously been implicated in a germline human disorder. A male infant with severe neutropenia and progressive dysmyelopoiesis with tetraploid myeloid precursors was identified. No known genetic etiologies for neutropenia or bone marrow failure were found. However, next-generation sequencing of germline samples from the patient revealed a novel, de novo germline stop-loss mutation in the X-linked gene SEPT6 that resulted in reduced SEPT6 staining in bone marrow granulocyte precursors and megakaryocytes. Patient skin fibroblast-derived induced pluripotent stem cells (iPSCs) produced reduced myeloid colonies, particularly of the granulocyte lineage. CRISPR/Cas9 knock-in of the patient's mutation or complete knock-out of SEPT6 was not tolerated in non-patient-derived iPSCs or human myeloid cell lines, but SEPT6 knock-out was successful in an erythroid cell line and resulting clones revealed a propensity to multinucleation. In silico analysis predicts that the mutated protein hinders the dimerization of SEPT6 coiled-coils in both parallel and antiparallel arrangements, which could in turn impair filament formation. These data demonstrate a critical role for SEPT6 in chromosomal segregation in myeloid progenitors that can account for the unusual predisposition to aneuploidy and dysmyelopoiesis.

Cytochimera potato plants, which mixed with diploid and tetraploid cells, could cause the highest and significantly increased biomass yield than the polyploid and diploid potato plants. Polyploidization is an important approach in crop breeding for agronomic trait improvement, especially for biomass production. Cytochimera contains two or more mixed cells with different levels of ploidy, which is considered a failure in whole genome duplication. Using colchicine treatment with diploid (Dip) potato (Solanum chacoense) plantlets, this study generated tetraploid (Tet) and cytochimera (Cyt) lines, which, respectively, contained complete and partial cells with genome duplication. Compared to the Dip potato, we observed remarkably enhanced plant growth and biomass yields in Tet and Cyt lines. Notably, the Cyt potato straw, which was generated from incomplete genome doubling, was of significantly higher biomass yield than that of the Tet with a distinctively altered cell wall composition. Meanwhile, we observed that one layer of the tetraploid cells (about 30%) in Cyt plants was sufficient to trigger a gene expression pattern similar to that of Tet, suggesting that the biomass dominance of Cyt may be related to the proportion of different ploidy cells. Further genome-wide analyses of co-expression networks indicated that down-regulation (against Dip) of spliceosomal-related transcripts might lead to differential alternative splicing for specifically improved agronomic traits such as plant growth, biomass yield, and lignocellulose composition in Tet and Cyt plants. In addition, this work examined that the genome of Cyt line was relatively stable after years of asexual reproduction. Hence, this study has demonstrated that incomplete genome doubling is a promising strategy to maximize biomass production in potatoes and beyond.

Combination of UBIQUITIN10 promoter-directed CAS9 and tRNA-gRNA complexes in gene-editing assay induces 80% mutant phenotype with a knockout of the four allelic copies in the T0 generation of allotetraploid tobaccos. While gene-editing methodologies, such as CRISPR-Cas9, have been developed and successfully used in many plant species, their use remains challenging, because they most often rely on stable or transient transgene expression. Regrettably, in all plant species, transformation causes epigenetic effects such as gene silencing and variable transgene expression. Here, UBIQUITIN10 promoters from several plant species were characterized and showed their capacity to direct high levels of transgene expression in transient and stable transformation assays, which in turn was used to improve the selection process of regenerated transformants. Furthermore, we compared various sgRNAs delivery systems and showed that the combination of UBIQUITIN10 promoters and tRNA-sgRNA complexes produced 80% mutant phenotype with a complete knockout of the four allelic copies, while the remaining 20% exhibited weaker phenotype, which suggested partial allelic knockout, in the T0 generation of the allotetraploid Nicotiana tabacum. These data provide valuable information to optimize future designs of gene editing constructs for plant research and crop improvement and open the way for valuable gene editing projects in non-model Solanaceae species.

Lipoxygenase (LOX, EC 1.13.11.12) is a non-haeme iron-containing dioxygenase family that catalyzes the oxygenation of polyunsaturated fatty acids into bio-functionally fatty acid diverse (oxylipins) and plays vital role in plant growth and development and responses to abiotic and biotic stresses. Though LOX genes have been studied in many plant species, their roles in Brassicaceae species are still unknown. Here, a set of 14, 18, and 33 putative LOX genes were identified in Brassica rapa, Brassica oleracea and Brassica napus (allotetraploid rapeseed), respectively, which could be divided into 9-LOX (LOX1/5), 13-LOX type I (LOX3/4/6), and type II (LOX2) subgroups. There was an expansion of LOX2 orthologous genes in Brassicaceae. Most of the LOX genes are intron rich and conserved in gene structure, and the LOX proteins all have the conserved lipoxygenase and PLAT/LH2 domain. Ka/Ks ratio revealed that the majority of LOXs underwent purifying selection in Brassicaceae. The light-, ABA-, MeJA-related cis-elements and MYB-binding sites in the promoters of BnaLOXs were the most abundant. BnaLOXs displayed different spatiotemporal expression patterns and various abiotic/biotic stress responsive expression patterns. BnaLOX1/5 were slightly or no response to phytohormones and abiotic stresses. BnaLOX3/4/6 predominantly express in roots and were strongly up-regulated by salinity and PEG treatments, and BnaLOX3/4 were the methyl jasmonate (MeJA) and salicylic acid (SA) early response genes and strongly induced by infection of Sclerotinia sclerotiorum; while the BnaLOX2 members predominantly express in stamens, were MeJA and SA continuous response genes and strongly repressed by cold, heat and waterlogging treatments in leaves. Our results are useful for understanding the biological functions of the BnaLOX genes in allotetraploid rapeseed.

Polyploid plants often show improved resistance against many diseases, but whether they show increased resistance to grey mould, a devastating disease caused by Botrytis spp. fungi, is seldom reported. Stomata and reactive oxygen species (ROS) play dual roles in defence against grey mould, and it is unclear how their roles change after polyploidization. We addressed these questions in diploid and colchicine-induced Lilium rosthornii after B. elliptica infection. Tetraploids were less susceptible to grey mould, with lower morality rates in naturally infected plants. Before the stomata closed in artificially infected leaves, tetraploids, with larger stomatal apertures, were more easily invaded by the pathogen than diploids. However, the lesion area increased more slowly in tetraploids than in diploids, which may be explained by three causes based on histological and physiological characteristics. First, the pathogen required more time to penetrate the epidermis and closed stomata in tetraploids than in diploids. Second, the pathogen penetrated the reopened stomata more easily than the epidermis, and stomatal density was lower in tetraploids than in diploids. Third, tetraploids showed faster ROS accumulation, a more effective ROS-scavenging system and less malondialdehyde (MDA) accumulation than diploids. Stomatal starch and abnormal guard cell nuclei were present in the infected leaves. This phenomenon may be caused by oxalic acid, a pathogenic factor for many pathogens that promotes stomatal starch degradation and stomatal reopening in Sclerotinia spp., a pathogen closely related to Botrytis spp. This suggestion was primarily confirmed by immersing healthy leaves in oxalic acid solution.

How two subgenomes in allo-tetraploids adapt to coexistence and coordinate through structure and expression evolution requires extensive studies. In the present study, we report an improved genome assembly of allo-tetraploid common carp, an updated genome annotation of allo-tetraploid goldfish and the chromosome-scale assemblies of a progenitor-like diploid Puntius tetrazona and an outgroup diploid Paracanthobrama guichenoti. Parallel subgenome structure evolution in the allo-tetraploids was featured with equivalent chromosome components, higher protein identities, similar transposon divergence and contents, homoeologous exchanges, better synteny level, strong sequence compensation and symmetric purifying selection. Furthermore, we observed subgenome expression divergence processes in the allo-tetraploids, including inter-/intrasubgenome trans-splicing events, expression dominance, decreased expression levels, dosage compensation, stronger expression correlation, dynamic functionalization and balancing of differential expression. The potential disorders introduced by different progenitors in the allo-tetraploids were hypothesized to be alleviated by increasing structural homogeneity and performing versatile expression processes. Resequencing three common carp strains revealed two major ecotypes and uncovered candidate genes relevant to growth and survival rate., Genomic analysis of allo-tetraploid common carp and goldfish identifies parallel subgenome structure and divergent expression processes.

Polish wheat (Triticum polonicum) is a unique tetraploid wheat species characterized by an elongated outer glume. The genetic control of the long-glume trait by a single semi-dominant locus, P1 (from P olish wheat), was established more than 100 years ago, but the underlying causal gene and molecular nature remain elusive. Here, we report the isolation of VRT-A2, encoding an SVP-clade MADS-box transcription factor, as the P1 candidate gene. Genetic evidence suggests that in T. polonicum, a naturally occurring sequence rearrangement in the intron-1 region of VRT-A2 leads to ectopic expression of VRT-A2 in floral organs where the long-glume phenotype appears. Interestingly, we found that the intron-1 region is a key ON/OFF molecular switch for VRT-A2 expression, not only because it recruits transcriptional repressors, but also because it confers intron-mediated transcriptional enhancement. Genotypic analyses using wheat accessions indicated that the P1 locus is likely derived from a single natural mutation in tetraploid wheat, which was subsequently inherited by hexaploid T. petropavlovskyi. Taken together, our findings highlight the promoter-proximal intron variation as a molecular basis for phenotypic differentiation, and thus species formation in Triticum plants.

Fig tree (Ficus carica L.) is a precious fruit tree in semi-arid and arid areas worldwide which has difficulties in its conventional breeding programs. This study was carried out to make new genotypes with superior features based on the ploidy induction method. Thus, in vitro tetraploidization in two fig cultivars, namely 'Sabz' and 'Torsh', was successfully established using shoot tip explants and colchicine as the antimitotic agent in MS medium. The flow cytometry and chromosome counting techniques were used to verify tetraploid plants. The results revealed that, in comparison to the original diploid plants of both cultivars, tetraploid plants significantly had taller stems, larger leaves, a greater number of chloroplasts in guard cells, and higher chlorophyll content and photosynthesis rate. UPLC-MS analysis revealed that the level of growth stimulator phytohormones, including ZR, IAA, GA3, SA, and JA in the tetraploid plants of both cultivars were significantly higher than their diploid controls. In contrast, they had less accumulated growth inhibitor phytohormone (ABA) than their diploid explant source. Moreover, tetraploid plants had significantly accumulated a higher content of phenolic compounds, total soluble sugars, and total soluble proteins, but showed a significantly less total antioxidant activity. Consequently, it is concluded that the growth advantages of tetraploid figs created in this study are substantial in terms of phytohormonal, physiological, and phytochemical superiorities, as compared to their corresponding diploid plants. Polyploidization proves as a promising breeding tool for future breeding programs of the fig tree.

SUMMARY The resistance gene Sr13 is one of the most important genes in durum wheat for controlling stem rust caused by Puccinia graminis f. sp. tritici (Pgt). The Sr13 functional gene CNL13 has haplotypes R1, R2 and R3. The R1/R3 and R2 haplotypes were originally designated as alleles Sr13a and Sr13b, respectively. To detect additional Sr13 alleles, we developed Kompetitive allele specific PCR (KASP™) marker KASPSr13 and four semi‐thermal asymmetric reverse PCR markers, rwgsnp37–rwgsnp40, based on the CNL13 sequence. These markers were shown to detect R1, R2 and R3 haplotypes in a panel of diverse tetraploid wheat accessions. We also observed the presence of Sr13 in durum line CAT‐A1, although it lacked any of the known haplotypes. Sequence analysis revealed that CNL13 of CAT‐A1 differed from the susceptible haplotype S1 by a single nucleotide (C2200T) in the leucine‐rich repeat region and differed from the other three R haplotypes by one or two additional nucleotides, confirming that CAT‐A1 carries a new (R4) haplotype. Stem rust tests on the monogenic, transgenic and mutant lines showed that R1 differed from R3 in its susceptibility to races TCMJC and THTSC, whereas R4 differed from all other haplotypes for susceptibility to TTKSK, TPPKC and TCCJC. Based on these differences, we designate the R1, R3 and R4 haplotypes as alleles Sr13a, Sr13c and Sr13d, respectively. This study indicates that Sr13d may be the primitive functional allele originating from the S1 haplotype via a point mutation, with the other three R alleles probably being derived from Sr13d through one or two additional point mutations., Significance Statement A new resistant haplotype (R4) of Sr13, designated as Sr13d, was identified in the genotype CAT‐A1, and based on sequencing studies this allele is hypothesized to be a primitive functional resistant allele of Sr13. We established from extensive rust tests that haplotypes R1 and R3 should be separated, and R3 was designated Sr13c and determined to be preferable to Sr13a for breeding, while Sr13d was undesirable for breeding due to susceptibility to stem rust Ug99.

Key message Genetic modification of spike architecture is essential for improving wheat yield. Newly identified loci for the ‘Miracle wheat’ phenotype on chromosomes 1AS and 2BS have significant effects on spike traits. Abstract The wheat (Triticum ssp.) inflorescence, also known as a spike, forms an unbranched inflorescence in which the inflorescence meristem generates axillary spikelet meristems (SMs) destined to become sessile spikelets. Previously, we identified the putatively causative mutation in the branched headt (bht) gene (TtBH-A1) of tetraploid wheat (T. turgidum convar. compositum (L.f.) Filat.) responsible for the loss of SM identity, converting the non-branching spike to a branched wheat spike. In the current study, we performed whole-genome quantitative trait loci (QTL) analysis using 146 recombinant inbred lines (RILs) derived from a cross between spike-branching wheat (‘Miracle wheat’) and an elite durum wheat cultivar showing broad phenotypic variation for spike architecture. Besides the previously found gene at the bht-A1 locus on the short arm of chromosome 2A, we also mapped two new modifier QTL for spike-branching on the short arm of chromosome 1A, termed bht-A2, and 2BS. Using biparental mapping population and GWAS in 302 diverse accessions, the 2BS locus was highly associated with coding sequence variation found at the homoeo-allele of TtBH-B1 (bht-B1). Thus, RILs that combined both bht-A1 and bht-B1 alleles showed additive genetic effects leading to increased penetrance and expressivity of the supernumerary spikelet and/or mini-spike formation.

Polyploidy has played a crucial role in plant evolution, development and function. Synthetic autopolyploid represents an ideal system to investigate the effects of polyploidization on transcriptional regulation. In this study, we deciphered the impact of genome duplication at phenotypic and molecular levels in watermelon. Overall, 88% of the genes in tetraploid watermelon followed a >1:1 dosage effect, and accordingly, differentially expressed genes were largely upregulated. In addition, a great number of hypomethylated regions (1688) were identified in an isogenic tetraploid watermelon. These differentially methylated regions were localized in promoters and intergenic regions and near transcriptional start sites of the identified upregulated genes, which enhances the importance of methylation in gene regulation. These changes were reflected in sophisticated higher-order chromatin structures. The genome doubling caused switching of 108 A and 626 B compartments that harbored genes associated with growth, development and stress responses.

Within the agamic Pilosella complex, apomixis (asexual reproduction through seed) involves apospory, parthenogenesis, and autonomous endosperm development. Observations of reproductive biology in P. brzovecensis throughout four growing seasons in the garden have shown that both tetraploid and pentaploid plants of this species do not produce viable seeds and reproduce exclusively vegetatively by underground stolons. The reasons for the seed development failure were unknown, therefore our research focused on the analysis of reproductive events in the ovules of this taxon. We found that apospory was initiated in the ovules of both cytotypes. Multiple aposporous initial (AI) cells differentiated in close proximity to the megaspore mother cell (MMC) and suppressed megasporogenesis at the stage of early prophase I. However, none of the AI cells was able to further develop into a multi-nucleate aposporous embryo sac (AES) due to the inhibition of mitotic divisions. It was unusual that callose was accumulated in the walls of AI cells and its synthesis was most likely associated with a response to the dysfunction of these cells. Callose is regarded as the isolating factor and its surprising deposition in the ovules of P. brzovecensis may signal disruption of reproductive processes that cause premature termination of the aposporous development pathway and ultimately lead to ovule sterility. The results of our embryological analysis may be the basis for undertaking advanced molecular studies aimed at fully understanding of the causes of female sterility in P. brzovecensis.

Polyploidy generally provides an advantage in phenotypic variation and growth vigor. However, the underlying mechanisms remain poorly understood. The tetraploid Liriodendron sino-americanum (Liriodendron × sinoamericanum P.C Yieh ex C.B. Shang & Zhang R.Wang) exhibits altered morphology compared with its diploid counterpart, including larger, thicker and deeper green leaves, bigger stomata, thicker stems and increased tree height. Such characteristics can be useful in ornamental and industrial applications. To elucidate the molecular mechanisms behind this variation, we performed a comparative transcriptome and proteome analysis. Our transcriptome data indicated that some photosynthesis genes and pathways were differentially altered and enriched in tetraploid L. sino-americanum, mainly related to F-type ATPase, the cytochrome b6/f complex, photosynthetic electron transport, the light harvesting chlorophyll protein complexes, and photosystem I and II. Most of the differentially expressed proteins we could identify are also involved in photosynthesis. Our physiological results showed that tetraploids have an enhanced photosynthetic capacity, concomitant with great levels of sugar and starch in leaves. This suggests that tetraploid L. sino-americanum might experience comprehensive transcriptome reprogramming of genes related to photosynthesis. This study has especially emphasized molecular changes involved in photosynthesis that accompany polyploidy, and provides a possible explanation for the altered phenotype of polyploidy plants in comparison with their diploid form.

Key message Mapping combined with expression and variant analyses in switchgrass, a crop with complex genetics, identified a cluster of candidate genes for leaf wax in a fast-evolving region of chromosome 7K. Abstract Switchgrass (Panicum virgatum L.) is a promising warm-season candidate energy crop. It occurs in two ecotypes, upland and lowland, which vary in a number of phenotypic traits, including leaf glaucousness. To initiate trait mapping, two F2 mapping populations were developed by crossing two different F1 sibs derived from a cross between the tetraploid lowland genotype AP13 and the tetraploid upland genotype VS16, and high-density linkage maps were generated. Quantitative trait locus (QTL) analyses of visually scored leaf glaucousness and of hydrophobicity of the abaxial leaf surface measured using a drop shape analyzer identified highly significant colocalizing QTL on chromosome 7K (Chr07K). Using a multipronged approach, we identified a cluster of genes including Pavir.7KG077009, which encodes a Type III polyketide synthase-like protein, and Pavir.7KG013754 and Pavir.7KG030500, two highly similar genes that encode putative acyl-acyl carrier protein (ACP) thioesterases, as strong candidates underlying the QTL. The lack of homoeologs for any of the three genes on Chr07N, the relatively low level of identity with other switchgrass KCS proteins and thioesterases, as well as the organization of the surrounding region suggest that Pavir.7KG077009 and Pavir.7KG013754/Pavir.7KG030500 were duplicated into a fast-evolving chromosome region, which led to their neofunctionalization. Furthermore, sequence analyses showed all three genes to be absent in the two upland compared to the two lowland accessions analyzed. This study provides an example of and practical guide for trait mapping and candidate gene identification in a complex genetic system by combining QTL mapping, transcriptomics and variant analysis.

SUMMARY Polyploidization is a well‐known speciation and adaptation mechanism. Traces of former polyploidization events were discovered within many genomes, and especially in plants. Allopolyploidization by interspecific hybridization between two species is common. Among hybrid plants, many are domesticated species of agricultural interest and many of their genomes and of their presumptive parents have been sequenced. Hybrid genomes remain challenging to analyse because of the presence of multiple subgenomes. The genomes of hybrids often undergo rearrangement and degradation over time. Based on 10 hybrid plant genomes from six different genera, with hybridization dating from 10,000 to 5 million years ago, we assessed subgenome degradation, subgenomic intermixing and biased subgenome fractionation. The restructuring of hybrid genomes does not proceed proportionally with the age of the hybrid. The oldest hybrids in our data set display completely different fates: whereas the subgenomes of the tobacco plant Nicotiana benthamiana are in an advanced stage of degradation, the subgenomes of quinoa (Chenopodium quinoa) are exceptionally well conserved by structure and sequence. We observed statistically significant biased subgenome fractionation in seven out of 10 hybrids, which had different ages and subgenomic intermixing levels. Hence, we conclude that no correlation exists between biased fractionation and subgenome intermixing. Lastly, domestication may encourage or hinder subgenome intermixing, depending on the evolutionary context. In summary, comparative analysis of hybrid genomes and their presumptive parents allowed us to determine commonalities and differences between their evolutionary fates. In order to facilitate the future analysis of further hybrid genomes, we automated the analysis steps within manticore, which is publicly available at https://github.com/MatteoSchiavinato/manticore.git., Significance Statement The purpose of this work was to compare the level of subgenomic intermixing in hybrid plants, providing insight into how intermixing levels are influenced by domestication, biased fractionation or hybrid age. A computational framework is presented to evaluate subgenomic conservation and intermixing in other hybrids, as there seems to be no one rule that fits all.

Gene regulatory divergence is thought to play a central role in determining human-specific traits. However, our ability to link divergent regulation to divergent phenotypes is limited. Here, we utilized human-chimpanzee hybrid induced pluripotent stem cells to study gene expression separating these species. The tetraploid hybrid cells allowed us to separate cis- from trans-regulatory effects, and to control for non-genetic confounding factors. We differentiated these cells into cranial neural crest cells (CNCCs), the primary cell type giving rise to the face. We discovered evidence of lineage-specific selection on the hedgehog signaling pathway, including a human-specific 6-fold down-regulation of EVC2 (LIMBIN), a key hedgehog gene. Inducing a similar down-regulation of EVC2 substantially reduced hedgehog signaling output. Mice and humans lacking functional EVC2 show striking phenotypic parallels to human-chimpanzee craniofacial differences, suggesting that the regulatory divergence of hedgehog signaling may have contributed to the unique craniofacial morphology of humans.

Oogenesis involves a series of biochemical and physiological transformations and numerous regulated genes. The autotetraploid Carassius auratus (4nRR) originated from whole-genome duplication of Carassius auratus red var. (RCC), which produces diploid eggs through pairing of diploid-like chromosome during female meiosis. To explore the molecular mechanisms underlying oogenesis in 4nRR, we used the Illumina sequencing platform to characterize the ovaries of 4nRR and RCC. Transcriptome and whole-genome re-sequencing were performed to uncover the key genes and potential genetic mutations related to oogenesis. Each sample produced paired-end reads in the range of 66.97 to 98.36 million via Illumina HiSeq™ 2500. After comparing of the transcriptome profiles between the 4nRR and RCC, we uncovered 8562 differentially expressed genes (DEGs). The DEGs were enriched in oogenesis-related processes, including oogenesis, oocyte development, ubiquitin-mediated proteolysis, the signaling pathways of MAPK and calcium, and oocyte meiosis as investigated by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis. Additionally, whole-genome re-sequencing revealed 34,058,834 SNPs and 6,153,711 InDels, including 6,677,638 non-synonymous variations (SNPs) and 706,210 frame-shift InDels in the 8510 DEGs of 4nRR fish. Subsequently, whole-genome re-sequencing and transcriptomatic analyses revealed the genes that participate in oogenesis associated processes. Specifically, genes involved in ubiquitin-mediated proteolysis (SMURF1, UBE2I), calcium transport (CALM3, CAMK4), and meiosis (MAPK3, GRB2, CPEB1, CCNB2, YWHAE) were related to oogenesis in 4nRR. These findings enrich our understanding of oogenesis in the autopolyploid fish.

The Hylocereus species that are grown as exotic fruit crops are very often farmed under marginal agronomic conditions, which may include exposure to high temperatures. Here we present a pioneering investigation of grafting as an agro-technique to improve heat tolerance in Hylocereus. To this end, we studied the diploid species H. undatus, the tetraploid H. megalanthus and its di-haploid gamete-derived line 2719, and the interspecific-interploid tetraploid Z-10, all grafted onto H. undatus as the rootstock. Self-grafted, grafted and non-grafted plants were acclimated for one week (to obtain baseline values) and then exposed to heat stress (45/35 °C day/night) for three days, followed by a one-week recovery period under optimal temperatures (30/22 °C). A comparison of the physiological, biochemical and molecular performances of the grafted and self-grafted plants under heat stress and during the recovery period vs those of non-stressed plants (control; 30/22 °C) showed that the grafted and self-grafted plants performed better in most of the assessments: grafted and self-grafted plants recovered more rapidly from the heat stress and suffered far less stem damage. An unexpected – but important – finding that may have implications for other crop was that the self-grafted plants showed better performance than non-grafted plants throughout the trial. Our findings provide support for grafting as a strategy for coping with the stress induced by extremely high temperatures. This study thus paves the way for further investigations of grafting in Hylocereus as a valuable technique that will maintain crop productivity in the face of increasing worldwide temperatures.

Summary To enrich our knowledge about gene network of fatty acid biosynthesis in cottonseed, we conducted comparative transcriptome to reveal the differences in gene expression between Gossypium hirsutum and Gossypium barbadense during cottonseed development. The prolonged expression period and increased expression abundance of oil‐related genes are the main reasons for producing high seed oil content (SOC) in G. barbadense, which manifested as the bias of homeologous gene expression in Dt‐subgenome after 25 day postanthesis (DPA). The dynamic expression profile showed that SAD6 and FATA are more important for oil biosynthesis in G. barbadense than that in G. hirsutum. Three key transcription factors, WRI1, NF‐YB6 and DPBF2, showed their elite roles in regulating seed oil in cotton. We observed that sequence variations in the promoter region of BCCP2 genes might contribute to its divergence in expression level between the two species. Based on the quantitative trait loci (QTL) information of the seed oil content and utilizing additional G. barbadense introgression lines (ILs), we propose 21 candidate genes on the basis of their differential expression level, of which the GbSWEET and the GbACBP6 showed the potential functional to improve the oil content. Taken together, studying the different expression of oil‐related genes and their genetic regulation mechanisms between G. hirsutum and G. barbadense provide new insights to understanding the mechanism of fatty acid biosynthesis network and fatty acid genetic improving breeding in cotton.

Meiotic chromosome pairing between homoeologous chromosomes was reported in many nascent allopolyploids. Homoeologous pairing is gradually eliminated and replaced by exclusive homologous pairing in well-established allopolyploids, an evolutionary process referred to as the diploidization of allopolyploids. A fundamental question of the diploidization of allopolyploids is whether and to what extent the DNA sequence variation among homoeologous chromosomes contribute to the establishment of exclusive homologous chromosome pairing. We developed aneuploid tetraploid maize lines that contain three copies of chromosome 10 derived from inbred lines B73 and H99. We were able to identify the parental origin of each copy of chromosome 10 in the materials using oligonucleotide-based haplotype-specific chromosome painting. We demonstrate that the two identical copies of chromosome 10 from H99 pair preferentially over chromosome 10 from B73 in different stages of prophase I and metaphase I during meiosis. Thus, homologous chromosome pairing is favored to partners with the most similar DNA sequences and can be discriminated based on cryptic sequence variation. We propose that innate preference of homologous chromosome pairing exists in nascent allopolyploids and serves as the first layer that would eventually block all homoeologous chromosome pairing in allopolyploids.

The distribution and abundance of polyploids has intrigued biologists since their discovery in the early 20th century. A pattern in nature that may give insight to processes that shape the distribution and abundance of polyploids is that polyploid populations are sometimes associated with peripheral habitats within the range of a species of mixed ploidy. Here, adaptation and competition of a diploid versus an autotetraploid population in a peripheral habitat are examined theoretically. It is shown that a nascent autotetraploid population adapts to and outcompetes a diploid population in the periphery when the rate of gamete dispersal is high, and when the mode of gene action is recessive for moderate to high rates of selfing. With additive or dominant modes of gene action, the conditions for an autotetraploid to outcompete a diploid in the periphery appear determined more by the rate of selfing and less by gamete dispersal. All of these results are based on empirical work that suggests inbreeding depression is higher in diploids versus autotetraploids. Generally, the results indicate that, although autotetraploids incur minority cytotype exclusion, diploids face burdens themselves. In the case of adaptation to a peripheral habitat, this burden is migration load from gamete and propagule dispersal.

Summary: E2F transcription factors control the expression of cell-cycle genes. Cancers often demonstrate enhanced E2F target gene expression, which can be explained by increased percentages of replicating cells. However, we demonstrate in human cancer biopsy specimens that individual neoplastic cells display abnormally high levels of E2F-dependent transcription. To mimic this situation, we delete the atypical E2F repressors (E2F7/8) or overexpress the E2F3 activator in untransformed cells. Cells with elevated E2F activity during S/G2 phase fail to exit the cell cycle after DNA damage and undergo mitosis. In contrast, wild-type cells complete S phase and then exit the cell cycle by activating the APC/CCdh1 via repression of the E2F target Emi1. Many arrested wild-type cells eventually inactivate APC/CCdh1 to execute a second round of DNA replication and mitosis, thereby becoming tetraploid. Cells with elevated E2F transcription fail to exit the cell cycle after DNA damage, which potentially causes genomic instability, promotes malignant progression, and reduces drug sensitivity.

Tetraploidy is an aneuploidy-permissive condition that can fuel tumorgenesis. The tip-over hypothesis of cytotoxic therapy sensitivity proposes that therapy is effective if it pushes a cell's aneuploidy above a viable tipping point. But elevated aneuploidy alone may not account for this tipping point. Tissue microenvironments that lack sufficient resources to support tetraploid cells can explain the fitness cost of aneuploidy. Raw materials needed to generate deoxynucleotides, the building blocks of DNA, are candidate rate-limiting factors for the evolution of high-ploidy cancer cells. Understanding the resource cost of high ploidy is key to uncover its therapeutic vulnerabilities across tissue sites with versatile energy supplies.

The composition of complex microbial communities found in association with plants is influenced in part by host phenotype. Yet, the salient genetic architecture is often unknown. Genome duplication events are common in the evolutionary history of plants, influence many important plant traits, and may affect associated microbial communities. Using experimentally induced whole genome duplication (WGD), we tested the effect of WGD on rhizosphere bacterial communities in Arabidopsis thaliana. Specifically, we performed 16S rRNA amplicon sequencing to characterize differences between microbiomes associated with specific host genotypes (Columbia vs. Landsberg) and ploidy levels (diploid vs. tetraploid). We modeled abundances of individual bacterial taxa by utilizing a hierarchical Bayesian framework, based on the Dirichlet and multinomial distributions. We found that host genotype and host ploidy level affected rhizosphere community composition, for instance, the microbiome of the tetraploid Columbia genotype differed from that of other host genotypes. We then tested to what extent microbiomes derived from a given host genotype or ploidy level affected plant performance by inoculating sterile seedlings of each genotype with microbial communities harvested from a prior generation. We found a negative effect of the tetraploid Columbia microbiome on growth of all four plant genotypes. The findings suggest that while both host genotype and ploidy affect microbial community assembly, bacterial communities found in association with only some host genotypes may affect growth of subsequent plant generations.ImportancePlants influence the composition of their associated microbial communities; yet the underlying host genetic factors are often unknown. Genome duplication events are common in the evolutionary history of plants and affect many plant traits, including the quality and quantity of compounds exuded into the root zone, which can affect root-bound microbes. In Arabidopsis thaliana, we characterized how whole-genome duplication affected the composition of rhizosphere bacterial communities, and how bacterial communities associated with two host plant genotypes and ploidy levels affected subsequent plant growth. We observed an interaction in which ploidy level within one host genotype affected both bacterial community composition and function. This research reveals how genome duplication, a widespread genetic feature of both wild and crop plant species, influences the coexistence of bacterial taxa and affects plant growth.

The physical locations of citrus centromere are revealed by combining genetic and immunological assays for the first time and nine citrus centromere-specific markers for cytogenetics are mined. Centromere localization is challenging, because highly redundant repetitive sequences in centromeric regions make sequence assembly difficult. Although several citrus genomes have been released, the centromeric regions and their characteristics remain to be elucidated. Here, we mapped citrus centromeres through half-tetrad analysis (HTA) that included the genotyping of 54 tetraploid hybrids derived from 2n megagametophytes of Nadorcott tangor with 212 single nucleotide polymorphism (SNP) markers. The sizes of centromeric regions, which estimated based on the heterozygosity restitution rate pattern along the chromosomes, ranged from 1.12 to 18.19 Mb. We also profiled the binding sequences with the centromere-specific histone variant CenH3 by chromatin immunoprecipitation sequencing (ChIP-seq). Based on the positions of the top ten CenH3-enriched contigs, the sizes of centromeric regions were estimated to range from 0.01 to 7.60 Mb and were either adjacent to or included in the centromeric regions identified by HTA. We used DNA probes from two repeats selected from the centromeric regions and seven CenH3-binding centromeric repeats to verify centromeric locations by fluorescence in situ hybridization (FISH). Centromere localization in citrus will contribute to the mining of centromeric/pericentromeric markers, thus to facilitate the rapid identification of mechanisms underlying 2n gamete formation and serve the polyploidy breeding.

Undomesticated wild species, crop wild relatives, and landraces represent sources of variation for wheat improvement to address challenges from climate change and the growing human population. Here, we study 56,342 domesticated hexaploid, 18,946 domesticated tetraploid and 3,903 crop wild relatives in a massive-scale genotyping and diversity analysis. Using DArTseqTM technology, we identify more than 300,000 high-quality SNPs and SilicoDArT markers and align them to three reference maps: the IWGSC RefSeq v1.0 genome assembly, the durum wheat genome assembly (cv. Svevo), and the DArT genetic map. On average, 72% of the markers are uniquely placed on these maps and 50% are linked to genes. The analysis reveals landraces with unexplored diversity and genetic footprints defined by regions under selection. This provides fertile ground to develop wheat varieties of the future by exploring specific gene or chromosome regions and identifying germplasm conserving allelic diversity missing in current breeding programs., Genebanks hold comprehensive collections of wild species, wild relatives, and landraces that are useful for genetic improvement. Here, the authors report the genotype of nearly 80,000 wheat accessions using DArTseq technology to show the less explored genetic diversity.

MdHAL3 has PPCDC activity and is involved in the salt tolerance of autotetraploid apple. Apple (Malus × domestica) is the most widely planted fruit tree species worldwide. However, the growth and development of apple have been increasingly affected by abiotic stress, such as high salinity. In our previous study, RNA sequencing (RNA-seq) analysis revealed that the expression level of the MdHAL3 gene was significantly upregulated in the autotetraploid apple cultivar Hanfu. In the present study, we first isolated HAL3, whose product was shown to exert 4′-phosphopantothenoylcysteine decarboxylase (PPCDC) activity, from apple. MdHAL3 was expressed in all organs of apple, and its expression was rapidly induced by salt stress. The MdHAL3 protein was localized to the cytomembrane and cytoplasm. Five MdHAL3 overexpression (OE) lines and five MdHAL3-RNAi apple lines were obtained. We found that MdHAL3 enhanced the salt stress tolerance of apple and that the OE plants rooted more easily than the wild-type (WT) plants. The coenzyme A (CoA) content in the leaves of the OE plants was greater than that in the leaves of the WT plants, and the CoA content in the MdHAL3-RNAi plants was lower than that in the WT plants. Taken together, our findings indicate that MdHAL3 plays an essential role in the response to salt stress in apple.

Polyploidy promotes morphological, physiological, and reproductive diversity in plants. The imminent effect of chromosome doubling in plants is the enlargement of organs such as flowers and fruits, which increases the commercial value of crops. Flowering plays a vital role in the growth and development of angiosperms. Here, we prepared an isolated microspore culture of 'FT', a doubled haploid (DH) line of Chinese cabbage (Brassica rapa L. ssp. pekinensis), and obtained diploid and autotetraploid plants with the same genetic background. Compared with diploids, the autotetraploids were characterized by large floral organs, dark petals, delayed flowering, and reduced fertility. The indole-3-acetic acid (IAA) and jasmonic acid (JA) levels in autotetraploid petals were significantly higher and the abscisic acid (ABA) level was significantly lower than those in the diploid petals. The lutein level in autotetraploid petals was nearly two times higher than that in the diploid petals. A comparative transcriptome analysis revealed 14,412 differentially expressed genes (DEGs) between the diploids and autotetraploids, and they were enriched in 117 Gene Ontology terms and 110 Kyoto Encyclopedia of Genes and Genomes pathways. We detected 231 DEGs related to phytohormone signal transduction and 29 DEGs involved in carotenoid biosynthesis. An miRNA-target mRNA analysis showed that 32 DEGs regulated by 16 DEMs were associated with flowering timing (BraA03000336, BraA09004319, and BraA09000515), petal development (BraA05002408, BraA01004006, BraA09004069, and BraA04000966), flower opening (BraA07000350), and pollen development (BraA01000720, BraA09005727, and BraA01000253). This study provides information to help elucidate the molecular mechanisms underlying phenotypic variations induced by autopolyploidy in Chinese cabbage.

Hybridization associated with polyploidization and apomixis is a frequent mechanism of speciation.Sorbusis a genus with ongoing hybridization resulting in a polyploid complex with different parental lineage.Triparensis the smallest hybridogenous subgenus ofSorbusso far known to comprise only two taxa,S. intermediaandS. × liljeforsiithat combine the genomes of three taxa (S. ariaagg.,S. aucupariaandS. torminalis). To elucidate the origins ofS. dacica,S. paxianaandS. tauricola, three new trigenomic candidates formerly believed to be of biparental origin with eitherS. ariaagg. × S. aucupariaorS. ariaagg. × S. torminalislineage we combined data from HPLC and chloroplast DNA analysing additional 33 related taxa as well. We concluded that the ‘torminalis-type’ flavonoid profile and the ‘aucuparia-type’ plastid indicate the participation of bothS. torminalisandS. aucupariaresulting in the formation ofS. dacica,S. paxianaandS. tauricola.Sorbus ariaagg. as the third ancestor and as a necessary link to meet genes ofS. torminalisandS. aucupariain one genome is obvious from morphological features (densely tomentose undersides of leaves). The tetraploid cytotypes and obligate pseudogamy ofS. dacicaandS. paxianawere determined by flow cytometry and are published here for the first time. The most probable evolutionary scenario forTriparensspecies is: 1. a diploid sexualS. aucupariaas pollen acceptor hybridized with a tetraploid apomictic taxon from theS. ariaagg. producing a triploid apomictic taxon with ‘aucuparia-type’ plastid inherited maternally; 2. during a second crossing event this subgenusSorariahybrid as maternal progenitor hybridized with the sexual diploidS. torminalis(providing gene(s) of apigenin O-glucuronide synthesis) forming a tetraploidTriparenshybrid with ‘aucuparia-type’ plastid and ‘torminalis-type’ flavonoids.

We previously developed a large-scale genome restructuring technology called the TAQing system. It can induce genomic rearrangements by introducing transient and conditional formation of DNA double-strand breaks (DSBs) via heat activation of a restriction enzyme TaqI, which can cleave DNA at 5'-TCGA-3' sequences in the genome at higher temperatures (37-42°C). Such heat treatment sometimes confers lethal damage in certain plant species and TaqI cannot induce rearrangements in AT-rich regions. To overcome such problems we developed an extended TAQing (Ex-TAQing) system, which enables the use of a wider range of restriction enzymes active at standard plant-growing temperatures. We established the Ex-TAQing system using MseI that can efficiently cleave DNA at room temperature (at temperatures ranging from 22 to 25°C) and the 5'-TTAA-3' sequence which is highly abundant in the Arabidopsis genome. A synthetic intron-spanning MseI gene, which was placed downstream of a heat-shock-inducible promoter, was conditionally expressed upon milder heat treatment (33°C) to generate DSBs in Arabidopsis chromosomes. Genome resequencing revealed various types of genomic rearrangements, including copy number variations, translocation and direct end-joining at MseI cleavage sites. The Ex-TAQing system could induce large-scale rearrangements in diploids more frequently (17.4%, n = 23) than the standard TAQing system. The application of this system to tetraploids generated several strains with chromosomal rearrangements associated with beneficial phenotypes, such as high salinity stress tolerance and hypersensitivity to abscisic acid. We have developed the Ex-TAQing system, allowing more diverse patterns of genomic rearrangements, by employing various types of endonucleases and have opened a way to expand the capacity for artificial genome reorganization.

Naturally occurring autopolyploid species, such as the autotetraploid potato Solanum tuberosum, face a variety of challenges during meiosis. These include proper pairing, recombination and correct segregation of multiple homologous chromosomes, which can form complex multivalent configurations at metaphase I, and in turn alter allelic segregation ratios through double reduction. Here, we present a reference map of meiotic stages in diploid and tetraploid S. tuberosum using fluorescence in situ hybridisation (FISH) to differentiate individual meiotic chromosomes 1 and 2. A diploid-like behaviour at metaphase I involving bivalent configurations was predominant in all three tetraploid varieties. The crossover frequency per bivalent was significantly reduced in the tetraploids compared with a diploid variety, which likely indicates meiotic adaptation to the autotetraploid state. Nevertheless, bivalents were accompanied by a substantial frequency of multivalents, which varied by variety and by chromosome (7–48%). We identified possible sites of synaptic partner switching, leading to multivalent formation, and found potential defects in the polymerisation and/or maintenance of the synaptonemal complex in tetraploids. These findings demonstrate the rise of S. tuberosum as a model for autotetraploid meiotic recombination research and highlight constraints on meiotic chromosome configurations and chiasma frequencies as an important feature of an evolved autotetraploid meiosis.

Summary Dissecting the genetic architecture of quantitative traits in autotetraploid species is a methodologically challenging task, but a pivotally important goal for breeding globally important food crops, including potato and blueberry, and ornamental species such as rose. Mapping quantitative trait loci (QTLs) is now a routine practice in diploid species but is far less advanced in autotetraploids, largely due to a lack of analytical methods that account for the complexities of tetrasomic inheritance.We present a novel likelihood‐based method for QTL mapping in outbred segregating populations of autotetraploid species. The method accounts properly for sophisticated features of gene segregation and recombination in an autotetraploid meiosis. It may model and analyse molecular marker data with or without allele dosage information, such as that from microarray or sequencing experiments.The method developed outperforms existing bivalent‐based methods, which may fail to model and analyse the full spectrum of experimental data, in the statistical power of QTL detection, and accuracy of QTL location, as demonstrated by an intensive simulation study and analysis of data sets collected from a segregating population of potato (Solanum tuberosum).The study enables QTL mapping analysis to be conducted in autotetraploid species under a rigorous tetrasomic inheritance model.

Artificially improving traits of cultivated alfalfa (Medicago sativa L.), one of the most important forage crops, is challenging due to the lack of a reference genome and an efficient genome editing protocol, which mainly result from its autotetraploidy and self-incompatibility. Here, we generate an allele-aware chromosome-level genome assembly for the cultivated alfalfa consisting of 32 allelic chromosomes by integrating high-fidelity single-molecule sequencing and Hi-C data. We further establish an efficient CRISPR/Cas9-based genome editing protocol on the basis of this genome assembly and precisely introduce tetra-allelic mutations into null mutants that display obvious phenotype changes. The mutated alleles and phenotypes of null mutants can be stably inherited in generations in a transgene-free manner by cross pollination, which may help in bypassing the debate about transgenic plants. The presented genome and CRISPR/Cas9-based transgene-free genome editing protocol provide key foundations for accelerating research and molecular breeding of this important forage crop., Alfalfa is an important forage crop, but genetic improvement is challenging due to the lack of a reference genome and an efficient genome editing protocol. Here, the authors report the chromosome-level assembly of the autotetraploid genome and a CRISPR/Cas9-based transgene-free genome editing protocol.

As the gene pool is exposed to both strain on land resources and a lack of diversity in elite allotetraploid cotton, the acquisition and identification of novel alleles has taken on epic importance in facilitating cotton genetic improvement and functional genomics research. Ethyl methanesulfonate (EMS) is an excellent mutagen that induces genome-wide efficient mutations to activate the mutagenic potential of plants with many advantages. The present study established, determined and verified the experimental procedure suitable for EMS-based mutant library construction as the general reference guide in allotetraploid upland cotton. This optimized method and procedure are efficient, and abundant EMS mutant libraries (approximately 12 000) in allotetraploid cotton were successfully obtained. More than 20 mutant phenotypes were observed and screened, including phenotypes of the leaf, flower, fruit, fiber and plant architecture. Through the plants mutant library, high-throughput and high-resolution melting technology-based variation evaluation detected the EMS-induced site mutation. Additionally, based on overall genome-wide mutation analyses by re-sequencing and mutant library assessment, the examination results demonstrated the ideal quality of the cotton EMS-treated mutant library constructed in this study with appropriate high mutation density and saturated genome. What is more, the collection is composed of a broad repertoire of mutants, which is the valuable resource for basic genetic research and functional genomics underlying complex allotetraploid traits, as well as cotton breeding.

Summary Commercialization of Brassica napus. L (oilseed rape) meal as protein diet is gaining more attention due to its well‐balanced amino acid and protein contents. Phytic acid (PA) is a major source of phosphorus in plants but is considered as anti‐nutritive for monogastric animals including humans due to its adverse effects on essential mineral absorption. The undigested PA causes eutrophication, which potentially threatens aquatic life. PA accounts to 2‐5% in mature seeds of oilseed rape and is synthesized by complex pathways involving multiple enzymes. Breeding polyploids for recessive traits is challenging as gene functions are encoded by several paralogs. Gene redundancy often requires to knock out several gene copies to study their underlying effects. Therefore, we adopted CRISPR‐Cas9 mutagenesis to knock out three functional paralogs of BnITPK. We obtained low PA mutants with an increase of free phosphorus in the canola grade spring cultivar Haydn. These mutants could mark an important milestone in rapeseed breeding with an increase in protein value and no adverse effects on oil contents.

Most deletions for the short arm of chromosome 2A (2AS), and the telocentric chromosome for the long arm of chromosome 2A (2AL), are available only in the heterozygous condition in 'Chinese Spring' hexaploid wheat. This is due to the female sterility, and therefore self-sterility, of their homozygotes, caused by the partial or entire loss of the 2AS chromosome arm on which genes for normal synapsis and female fertility are located. On the other hand, a D-genome disomic substitution line 2D(2A) of 'Langdon' tetraploid wheat, in which chromosome 2D is disomically substituted for chromosome 2A, is available (i.e., self-fertile) despite chromosome 2A being missing in this line. This fact indicates that another gene for female fertility must be present in Langdon 2D(2A). We attempted to develop self-fertile 2AS homozygous deletion and ditelosomic 2AL lines by transferring this female fertility gene, through a series of crosses and cytological screening, from Langdon 2D(2A) to the two aneuploid lines. We finally obtained self-fertile 2AS homozygous deletion and ditelosomic 2AL lines. These lines displayed normal meiotic chromosome pairing and lacked all 12 of the 2AS markers used for PCR analysis.

BackgroundHoxtranscription factors are master regulators of animal development. Although highly conserved, they can contribute to the formation of novel biological characteristics when modified, such as during the generation of hybrid species, thus potentially serving as species-specific molecular markers.Here, we systematically studied the evolution of genomic sequences ofHoxloci in an artificial allotetraploid lineage (4nAT, 4n = 200) derived from a red crucian carp (♀, RCC, 2n = 100) × common carp (♂, CC, 2n = 100) cross and its parents (RCC and CC).ResultsPCR amplification yielded 23 distinctHoxgene fragments from 160 clones in 4nAT, 22 fragments from 90 clones in RCC, and 19 fragments from 90 clones in CC. Sequence alignment of theHoxA3aandHoxC10agenes indicated both the inheritance and loss of paternal genomic DNA in 4nAT. TheHoxA5agene from 4nAT consisted of two subtypes from RCC and two subtypes from CC, indicating that homologous recombination occurred in the 4nAT hybrid genome. Moreover, 4nAT carried genomic pseudogenization in theHoxA10bandHoxC13aloci. Interestingly, a new type ofHoxC9agene was found in 4nAT as a hybrid sequence of CC and RCC by recombination in the intronic region.ConclusionThe results revealed the influence ofHoxgenes during polyploidization in hybrid fish. The data provided insight into the evolution of vertebrate genomes and might be benefit for artificial breeding programs.

Background Autotetraploid Carassius auratus (4nRCC, 4n = 200, RRRR) was derived from the whole genome duplication of diploid red crucian carp (Carassius auratus red var.) (2nRCC, 2n = 100, RR). To investigate the genetic effects of tetraploidization, we analyzed DNA variation, epigenetic modification and gene expression changes in the Sox9a-Amh-Cyp19a1a regulatory cascade between 4nRCC and 2nRCC. Results We found that the Sox9a gene contained two variants in 2nRCC and four variants in 4nRCC. Compared with that in 2nRCC, DNA methylation in the promoter regions of the Amh and Cyp19a1a genes in 4nRCC was altered by single nucleotide polymorphism (SNP) mutations, which resulted in the insertions and deletions of CpG sites, and the methylation levels of the Sox9a, Amh and Cyp19a1a genes increased after tetraploidization. The gene expression level of the Sox9a-Amh-Cyp19a1a regulatory cascade was downregulated in 4nRCC compared with that in 2nRCC. Conclusion The above results demonstrate that tetraploidization leads to significant changes in the genome, epigenetic modification and gene expression in the Sox9a-Amh-Cyp19a1a regulatory cascade; these findings increase the extant knowledge regarding the effects of polyploidization.

The rubber tree, Hevea brasiliensis, produces natural rubber that serves as an essential industrial raw material. Here, we present a high-quality reference genome for a rubber tree cultivar GT1 using single-molecule real-time sequencing (SMRT) and Hi-C technologies to anchor the ∼1.47-Gb genome assembly into 18 pseudochromosomes. The chromosome-based genome analysis enabled us to establish a model of spurge chromosome evolution, since the common paleopolyploid event occurred before the split of Hevea and Manihot. We show recent and rapid bursts of the three Hevea-specific LTR-retrotransposon families during the last 10 million years, leading to the massive expansion by ∼65.88% (∼970 Mbp) of the whole rubber tree genome since the divergence from Manihot. We identify large-scale expansion of genes associated with whole rubber biosynthesis processes, such as basal metabolic processes, ethylene biosynthesis, and the activation of polysaccharide and glycoprotein lectin, which are important properties for latex production. A map of genomic variation between the cultivated and wild rubber trees was obtained, which contains ∼15.7 million high-quality single-nucleotide polymorphisms. We identified hundreds of candidate domestication genes with drastically lowered genomic diversity in the cultivated but not wild rubber trees despite a relatively short domestication history of rubber tree, some of which are involved in rubber biosynthesis. This genome assembly represents key resources for future rubber tree research and breeding, providing novel targets for improving plant biotic and abiotic tolerance and rubber production.

Background Neo-tetraploid rice, which is a new germplasm developed from autotetraploid rice, has a powerful biological and yield potential and could be used for commercial utilization. The length of panicle, as a part of rice panicle architecture, contributes greatly to high yield. However, little information about long panicle associated with heterosis or hybrid vigor is available in neo-tetraploid rice. Results In the present study, we developed a neo-tetraploid rice line, Huaduo 8 (H8), with long panicles and harboring wide-compatibility genes for pollen and embryo sac fertility. All the hybrids generated by H8 produced significant high-parent yield heterosis and displayed long panicles similar to H8. RNA-seq analysis detected a total of 4013, 7050, 6787 and 6195 differentially expressed genes uniquely belonging to F1 and specifically (DEGFu-sp) associated with leaf, sheath, main panicle axis and spikelet in the two hybrids, respectively. Of these DEGFu-sp, 279 and 89 genes were involved in kinase and synthase, and 714 cloned genes, such as GW8, OsGA20ox1, Ghd8, GW6a, and LP1, were identified and validated by qRT-PCR. A total of 2925 known QTLs intervals, with an average of 1~100 genes per interval, were detected in both hybrids. Of these, 109 yield-related QTLs were associated with seven important traits in rice. Moreover, 1393 non-additive DEGs, including 766 up-regulated and 627 down-regulated, were detected in both hybrids. Importantly, eight up-regulated genes associated with panicle were detected in young panicles of the two hybrids compared to their parents by qRT-PCR. Re-sequencing analysis depicted that LP (a gene controlling long panicle) sequence of H8 was different from many other neo-tetraploid rice and most of the diploid and autotetraploid lines. The qRT-PCR results showed that LP was up-regulated in the hybrid compared to its parents at very young stage of panicle development. Conclusions These results suggested that H8 could overcome the intersubspecific autotetraploid hybrid rice sterility caused by embryo sac and pollen sterility loci. Notably, long panicles of H8 showed dominance phenomenon and played an important role in yield heterosis, which is a complex molecular mechanism. The neo-tetraploid rice is a useful germplasm to attain high yield of polyploid rice.

In this study, the effects of methyl jasmonate (MeJA) on the phytomass and triterpenoid production of diploid and tetraploid Centella asiatica hairy roots were investigated. Hairy root cultures were obtained from diploid and induced tetraploid plants of C. asiatica infected by Agrobacterium rhizogenes strain ATCC 43057. MeJA triggered triterpenoid production in both ploidy hairy roots, whereas triterpenoids were not produced in the untreated hairy roots. Among the treatments, the 50 µM MeJA treatment yielded the maximum triterpenoid production in diploid hairy roots of 27.25 ± 0.27 µg/mg Dry weight (DW) total triterpenoid at day 21. For the tetraploid hairy root cultures, the 28th-day hairy root culture produced a maximum amount of triterpenoids of 16.29 ± 6.32 µg/mg DW in response to the 50 µM MeJA treatment, whereas the 100 µM MeJA treatment produced a similar triterpenoid amount (16.31 ± 9.24 µg/mg DW) at day 14. Moreover, in response to 50 µM MeJA, we obtained different ratios of aglycone to glycoside, i.e., 1:7 and 1:2, between the diploid and tetraploid hairy root cultures. Asiaticoside was the dominant phytochemical, followed by asiatic acid and madecassic acid. This study provides valuable information for producing triterpenoids for C. asiatica commercial products and preparations by using hairy root cultures.

Background Tetraploid Thinopyrum elongatum (2n = 4x = 28) is a promising source of useful genes, including those related to adaptability and resistance to diverse biotic (Fusarium head blight, rust, powdery mildew, and yellow dwarf virus) and abiotic (cold, drought, and salt) stresses. However, gene transfer rates are low for this species and relatively few species-specific molecular markers are available. Results The wheat-tetraploid Th. elongatum line K17–841-1 derived from a cross between a hexaploid Trititrigia and Sichuan wheat cultivars was characterized based on sequential genomic and fluorescence in situ hybridizations and simple sequence repeat markers. We revealed that K17–841-1 is a 1E (1D) chromosomal substitution line that is highly resistant to stripe rust pathogen strains prevalent in China. By comparing the sequences generated during genotyping-by-sequencing (GBS), we obtained 597 specific fragments on the 1E chromosome of tetraploid Th. elongatum. A total of 235 primers were designed and 165 new Th. elongatum-specific markers were developed, with an efficiency of up to 70%. Marker validation analyses indicated that 25 specific markers can discriminate between the tetraploid Th. elongatum chromosomes and the chromosomes of other wheat-related species. An evaluation of the utility of these markers in a F2 breeding population suggested these markers are linked to the stripe rust resistance gene on chromosome 1E. Furthermore, 28 markers are unique to diploid Th. elongatum, tetraploid Th. elongatum, or decaploid Thinopyrum ponticum, which carry the E genome. Finally, 48 and 74 markers revealed polymorphisms between Thinopyrum E-genome- containing species and Thinopyrum bessarabicum (Eb) and Pseudoroegneria libanotica (St), respectively. Conclusions This new substitution line provide appropriate bridge–breeding–materials for alien gene introgression to improve wheat stripe rust resistance. The markers developed using GBS technology in this study may be useful for the high-throughput and accurate detection of tetraploid Th. elongatum DNA in diverse materials. They may also be relevant for investigating the genetic differences and phylogenetic relationships among E, Eb, St, and other closely-related genomes and for further characterizing these complex species.

Key message Twelve potato accessions were selected to represent two principal views on potato taxonomy. The genomes were sequenced and analyzed for structural variation (copy number variation) against three published potato genomes. Abstract The common potato (Solanum tuberosum L.) is an important staple crop with a highly heterozygous and complex tetraploid genome. The other taxa of cultivated potato contain varying ploidy levels (2X–5X), and structural variations are common in the genomes of these species, likely contributing to the diversification or agronomic traits during domestication. Increased understanding of the genomes and genomic variation will aid in the exploration of novel agronomic traits. Thus, sequencing data from twelve potato landraces, representing the four ploidy levels, were used to identify structural genomic variation compared to the two currently available reference genomes, a double monoploid potato genome and a diploid inbred clone of S. chacoense. The results of a copy number variation analysis showed that in the majority of the genomes, while the number of deletions is greater than the number of duplications, the number of duplicated genes is greater than the number of deleted ones. Specific regions in the twelve potato genomes have a high density of CNV events. Further, the auxin-induced SAUR genes (involved in abiotic stress), disease resistance genes and the 2-oxoglutarate/Fe(II)-dependent oxygenase superfamily proteins, among others, had increased copy numbers in these sequenced genomes relative to the references.

Background Meiosis of newly formed allopolyploids frequently encounter perturbations induced by the merging of divergent and hybridizable genomes. However, to date, the meiotic properties of allopolyploids with dysploid parental karyotypes have not been studied in detail. The allotetraploid Cucumis ×hytivus (HHCC, 2n = 38) was obtained from interspecific hybridization between C. sativus (CC, 2n = 14) and C. hystrix (HH, 2n = 24) followed by chromosome doubling. The results of this study thus offer an excellent opportunity to explore the meiotic properties of allopolyploids with dysploid parental karyotypes. Results In this report, we describe the meiotic properties of five chromosomes (C5, C7, H1, H9 and H10) and two genomes in interspecific hybrids and C. ×hytivus (the 4th and 14th inbred family) through oligo-painting and genomic in situ hybridization (GISH). We show that 1) only two translocations carrying C5-oligo signals were detected on the chromosomes C2 and C4 of one 14th individual by the karyotyping of eight 4th and 36 14th plants based on C5- and C7-oligo painting, and possible cytological evidence was observed in meiosis of the 4th generation; 2) individual chromosome have biases for homoeologous pairing and univalent formation in F1 hybrids and allotetraploids; 3) extensive H-chromosome autosyndetic pairings (e.g., H-H, 25.5% PMCs) were observed in interspecific F1 hybrid, whereas no C-chromosome autosyndetic pairings were observed (e.g. C-C); 4) the meiotic properties of two subgenomes have significant biases in allotetraploids: H-subgenome exhibits higher univalent and chromosome lagging frequencies than C-subgenome; and 5) increased meiotic stability in the S14 generation compared with the S4 generation, including synchronous meiosis behavior, reduced incidents of univalent and chromosome lagging. Conclusions These results suggest that the meiotic behavior of two subgenomes has dramatic biases in response to interspecific hybridization and allopolyploidization, and the meiotic behavior harmony of subgenomes is a key subject of meiosis evolution in C. ×hytivus. This study helps to elucidate the meiotic properties and evolution of nascent allopolyploids with the dysploid parental karyotypes.

Tetraploid johnsongrass [Sorghum halepense (L.) Pers.] is a sexually-compatible weedy relative of diploid sorghum [Sorghum bicolor (L.) Moench]. To determine the extent of interspecific hybridization between male sterile grain sorghum and johnsongrass and the ploidy of their progeny, cytoplasmic (CMS), genetic (GMS) and chemically induced male sterile lines of Tx623 and Tx631 were pollinated with johnsongrass pollen. At maturity 1% and 0.07% of the developing seeds of Tx623 and Tx631 respectively were recovered. Ninety-one percent of recovered hybrids were tetraploid and two percent were triploid, the tetraploids resulting from 2n gametes present in the sorghum female parent. Their formation appears to be genotype dependent as more tetraploids were recovered from Tx623 than Tx631. Because a tetraploid sorghum x johnsongrass hybrid has a balanced genome, they are male and female fertile providing opportunities for gene flow between the two species. Given the differences in 2n gamete formation among Tx623 and Tx631, seed parent selection may be one way of reducing the likelihood of gene flow. These studies were conducted in controlled and optimum conditions; the actual outcrossing rate in natural conditions is expected to be much lower. More studies are needed to assess the rates of hybridization, fitness, and fertility of the progeny under field conditions.

Root-knot nematode is a very destructive pathogen, to which most peanut cultivars are highly susceptible. Strong resistance is present in the wild diploid peanut relatives. Previously, QTLs controlling nematode resistance were identified on chromosomes A02, A04 and A09 of Arachis stenosperma. Here, to study the inheritance of these resistance alleles within the genetic background of tetraploid peanut, an F2 population was developed from a cross between peanut and an induced allotetraploid that incorporated A. stenosperma, [Arachis batizocoi x A. stenosperma]4×. This population was genotyped using a SNP array and phenotyped for nematode resistance. QTL analysis allowed us to verify the major-effect QTL on chromosome A02 and a secondary QTL on A09, each contributing to a percentage reduction in nematode multiplication up to 98.2%. These were validated in selected F2:3 lines. The genome location of the large-effect QTL on A02 is rich in genes encoding TIR-NBS-LRR protein domains that are involved in plant defenses. We conclude that the strong resistance to RKN, derived from the diploid A. stenosperma, is transferrable and expressed in tetraploid peanut. Currently it is being used in breeding programs for introgressing a new source of nematode resistance and to widen the genetic basis of agronomically adapted peanut lines.

Tetraploidisation is considered a common event in the evolution of chromosomal instability (CIN) in cancer cells. The current model for how tetraploidy drives CIN in mammalian cells is that a doubling of the number of centrioles that accompany the genome doubling event leads to multipolar spindle formation and chromosome segregation errors. By exploiting the unusual scenario of mouse blastomeres, which lack centrioles until the ~64-cell stage, we show that tetraploidy can drive CIN by an entirely distinct mechanism. Tetraploid blastomeres assemble bipolar spindles dictated by microtubule organising centres, and multipolar spindles are rare. Rather, kinetochore-microtubule turnover is altered, leading to microtubule attachment defects and anaphase chromosome segregation errors. The resulting blastomeres become chromosomally unstable and exhibit a dramatic increase in whole chromosome aneuploidies. Our results thus reveal an unexpected mechanism by which tetraploidy drives CIN, in which the acquisition of chromosomally-unstable microtubule dynamics contributes to chromosome segregation errors following tetraploidisation., During cell division, tetraploidy can drive chromosomal instability (CIN) via supernumerary centrosomes, but it is unclear if this is the only route to CIN. Here the authors show that, in early mouse embryos, tetraploidy can drive chromosomal instability by altering microtubule dynamics and attachment.