Fine mapping of major QTL qshgd1 for spontaneous haploid genome doubling in maize (Zea mays L.).

KEY MESSAGE: A large-effect QTL was fine mapped, which revealed 79 gene models, with 10 promising candidate genes, along with a novel inversion. In commercial maize breeding, doubled haploid (DH) technology is arguably the most efficient resource for rapidly developing novel, completely homozygous lines. However, the DH strategy, using in vivo haploid induction, currently requires the use of mutagenic agents which can be not only hazardous, but laborious. This study focuses on an alternative approach to develop DH lines-spontaneous haploid genome duplication (SHGD) via naturally restored haploid male fertility (HMF). Inbred lines A427 and Wf9, the former with high HMF and the latter with low HMF, were selected to fine-map a large-effect QTL associated with SHGD-qshgd1. SHGD alleles were derived from A427, with novel haploid recombinant groups having varying levels of the A427 chromosomal region recovered. The chromosomal region of interest is composed of 45 megabases (Mb) of genetic information on chromosome 5. Significant differences between haploid recombinant groups for HMF were identified, signaling the possibility of mapping the QTL more closely. Due to suppression of recombination from the proximity of the centromere, and a newly discovered inversion region, the associated QTL was only confined to a 25 Mb region, within which only a single recombinant was observed among ca. 9,000 BC1 individuals. Nevertheless, 79 gene models were identified within this 25 Mb region. Additionally, 10 promising candidate genes, based on RNA-seq data, are described for future evaluation, while the narrowed down genome region is accessible for straightforward introgression into elite germplasm by BC methods.

Genetic dissection of Brassica napus seed vigor after aging.

KEY MESSAGE: Stable and novel QTLs that affect seed vigor under different storage durations were discovered, and BnaOLE4, located in the interval of cqSW-C2-3, increased seed vigor after aging. Seed vigor is an important trait in crop breeding; however, the underlying molecular regulatory mechanisms governing this trait in rapeseed remain largely unknown. In the present study, vigor-related traits were analyzed in seeds from a doubled haploid (DH) rapeseed (Brassica napus) population grown in 2 different environments using seeds stored for 7, 5, and 3 years under natural storage conditions. A total of 229 quantitative trait loci (QTLs) were identified and were found to explain 3.78%-17.22% of the phenotypic variance for seed vigor-related traits after aging. We further demonstrated that seed vigor-related traits were positively correlated with oil content (OC) but negatively correlated with unsaturated fatty acids (FAs). Some pleiotropic QTLs that collectively regulate OC, FAs, and seed vigor, such as uq.A8, uq.A3-2, uq.A9-2, and uq.C3-1, were identified. The transcriptomic results from extreme pools of DH lines with distinct seed vigor phenotypes during accelerated aging revealed that various biological pathways and metabolic processes (such as glutathione metabolism and reactive oxygen species) were involved in seed vigor. Through integration of QTL analysis and RNA-Seq, a regulatory network for the control of seed vigor was constructed. Importantly, a candidate (BnaOLE4) from cqSW-C2-3 was selected for functional analysis, and transgenic lines overexpressing BnaOLE4 showed increased seed vigor after artificial aging. Collectively, these results provide novel information on QTL and potential candidate genes for molecular breeding for improved seed storability.

[Genetic dissection of rice resistance to bacterial blight].

Bacterial blight, a major disease in rice, poses a serious impact on rice production. In this study, a doubled haploid (DH) population derived from a cross between the introduced japonica cultivar 'Maybelle' and the indica landrace 'Baiyeqiu' was used to investigate the pathogenicity of four pathogen races causing bacterial blight. The results showed that the pathogenicity of all the pathogen races exhibited continuous, transgressive distribution in the DH population. Moreover, strong correlations existed between every two pathogen races, with the correlation coefficients ranging from 0.3 to 0.6. A total of 12 quantitative trait loci (QTLs) distributed on chromosomes 1, 2, 3, 5, 6, 7, 9, and 12 were detected for rice bacterial blight, explaining 4.95% to 16.05% of the phenotype. Among these QTLs, a major QTL located in the interval RM6024-RM163 on chromosome 5 was detected in three pathogen races. In addition, the pyramiding of the positive alleles can apparently improve the rice resistance to bacterial blight. This study is of great significance for broadening the genetic resources with resistance to bacterial blight in China.

A cyclical switch of gametogenic pathways in hybrids depends on the ploidy level.

The cellular and molecular mechanisms governing sexual reproduction are conserved across eukaryotes. Nevertheless, hybridization can disrupt these mechanisms, leading to asexual reproduction, often accompanied by polyploidy. In this study, we investigate how ploidy level and ratio of parental genomes in hybrids affect their reproductive mode. We analyze the gametogenesis of sexual species and their diploid and triploid hybrids from the freshwater fish family Cobitidae, using newly developed cytogenetic markers. We find that diploid hybrid females possess oogonia and oocytes with original (diploid) and duplicated (tetraploid) ploidy. Diploid oocytes cannot progress beyond pachytene due to aberrant pairing. However, tetraploid oocytes, which emerge after premeiotic genome endoreplication, exhibit normal pairing and result in diploid gametes. Triploid hybrid females possess diploid, triploid, and haploid oogonia and oocytes. Triploid and haploid oocytes cannot progress beyond pachytene checkpoint due to aberrant chromosome pairing, while diploid oocytes have normal pairing in meiosis, resulting in haploid gametes. Diploid oocytes emerge after premeiotic elimination of a single-copied genome. Triploid hybrid males are sterile due to aberrant pairing and the failure of chromosomal segregation during meiotic divisions. Thus, changes in ploidy and genome dosage may lead to cyclical alteration of gametogenic pathways in hybrids.

Comparative characteristics of oat doubled haploids and oat × maize addition lines: Anatomical features of the leaves, chlorophyll a fluorescence and yield parameters.

As a result of oat (Avena sativa L.) × maize (Zea mays L.) crossing, maize chromosomes may not be completely eliminated at the early stages of embryogenesis, leading to the oat × maize addition (OMA) lines development. Introgression of maize chromosomes into oat genome can cause morphological and physiological modifications. The aim of the research was to evaluate the leaves' anatomy, chlorophyll a fluorescence, and yield parameter of oat doubled haploid (DH) and OMA lines obtained by oat × maize crossing. The present study examined two DH and two disomic OMA lines and revealed that they differ significantly in the majority of studied traits, apart from: the number of cells of the outer bundle sheath; light energy absorption; excitation energy trapped in PSII reaction centers; and energy dissipated from PSII. The OMA II line was characterized by larger size of single cells in the outer bundle sheath and greater number of seeds per plant among tested lines.

Development and application of haploid embryonic stem cells.

Haploid cells are a kind of cells with only one set of chromosomes. Compared with traditional diploid cells, haploid cells have unique advantages in gene screening and drug-targeted therapy, due to their phenotype being equal to the genotype. Embryonic stem cells are a kind of cells with strong differentiation potential that can differentiate into various types of cells under specific conditions in vitro. Therefore, haploid embryonic stem cells have the characteristics of both haploid cells and embryonic stem cells, which makes them have significant advantages in many aspects, such as reproductive developmental mechanism research, genetic screening, and drug-targeted therapy. Consequently, establishing haploid embryonic stem cell lines is of great significance. This paper reviews the progress of haploid embryonic stem cell research and briefly discusses the applications of haploid embryonic stem cells.

Discovery of New Genomic Configuration of Mating-Type Loci in the Largest Lineage of Lichen-Forming Fungi.

The genetic architecture of mating-type loci in lichen-forming fungi has been characterized in very few taxa. Despite the limited data, and in contrast to all other major fungal lineages, arrangements that have both mating-type alleles in a single haploid genome have been hypothesized to be absent from the largest lineage of lichen-forming fungi, the Lecanoromycetes. We report the discovery of both mating-type alleles from the haploid genomes of three species within this group. Our results demonstrate that Lecanoromycetes are not an outlier among Ascomycetes.

A non-canonical BZR/BES transcription factor regulates the development of haploid reproductive organs in Marchantia polymorpha.

Gametogenesis, which is essential to the sexual reproductive system, has drastically changed during plant evolution. Bryophytes, lycophytes and ferns develop reproductive organs called gametangia-antheridia and archegonia for sperm and egg production, respectively. However, the molecular mechanism of early gametangium development remains unclear. Here we identified a 'non-canonical' type of BZR/BES transcription factor, MpBZR3, as a regulator of gametangium development in a model bryophyte, Marchantia polymorpha. Interestingly, overexpression of MpBZR3 induced ectopic gametangia. Genetic analysis revealed that MpBZR3 promotes the early phase of antheridium development in male plants. By contrast, MpBZR3 is required for the late phase of archegonium development in female plants. We demonstrate that MpBZR3 is necessary for the successful development of both antheridia and archegonia but functions in a different manner between the two sexes. Together, the functional specialization of this 'non-canonical' type of BZR/BES member may have contributed to the evolution of reproductive systems.

Limits to selection on standing variation in an asexual population.

We consider how a population of N haploid individuals responds to directional selection on standing variation, with no new variation from recombination or mutation. Individuals have trait values z1,…,zN, which are drawn from a distribution ψ; the fitness of individual i is proportional to [Formula: see text] . For illustration, we consider the Laplace and Gaussian distributions, which are parametrised only by the variance V0, and show that for large N, there is a scaling limit which depends on a single parameter NV0. When selection is weak relative to drift (NV0≪1), the variance decreases exponentially at rate 1/N, and the expected ultimate gain in log fitness (scaled by V0), is just NV0, which is the same as Robertson's (1960) prediction for a sexual population. In contrast, when selection is strong relative to drift (NV0≫1), the ultimate gain can be found by approximating the establishment of alleles by a branching process in which each allele competes independently with the population mean and the fittest allele to establish is certain to fix. Then, if the probability of survival to time t∼1/V0 of an allele with value z is P(z), with mean P¯, the winning allele is the fittest of NP¯ survivors drawn from a distribution ψP/P¯. The expected ultimate change is ∼2log(1.15NV0) for a Gaussian distribution, and ∼-12log0.36NV0-log-log0.36NV0 for a Laplace distribution. This approach also predicts the variability of the process, and its dynamics; we show that in the strong selection regime, the expected genetic variance decreases as ∼t-3 at large times. We discuss how these results may be related to selection on standing variation that is spread along a linear chromosome.

Genome-Wide Identification and Analysis of the EIN3/EIL Transcription Factor Gene Family in Doubled Haploid (DH) Poplar.

Ethylene (ET) is an important phytohormone that regulates plant growth, development and stress responses. The ethylene-insensitive3/ethylene-insensitive3-like (EIN3/EIL) transcription factor family, as a key regulator of the ET signal transduction pathway, plays an important role in regulating the expression of ET-responsive genes. Although studies of EIN3/EIL family members have been completed in many species, their role in doubled haploid (DH) poplar derived from another culture of diploid Populus simonii × P. nigra (donor tree, DT) remains ambiguous. In this study, a total of seven EIN3/EIL gene family members in the DH poplar genome were identified. Basic physical and chemical property analyses of these genes were performed, and these proteins were predicted to be localized to the nucleus. According to the phylogenetic relationship, EIN3/EIL genes were divided into two groups, and the genes in the same group had a similar gene structure and conserved motifs. The expression patterns of EIN3/EIL genes in the apical buds of different DH poplar plants were analyzed based on transcriptome data. At the same time, the expression patterns of PsnEIL1, PsnEIN3, PsnEIL4 and PsnEIL5 genes in different tissues of different DH plants were detected via RT-qPCR, including the apical buds, young leaves, functional leaves, xylem, cambium and roots. The findings presented above indicate notable variations in the expression levels of PsnEIL genes across various tissues of distinct DH plants. Finally, the PsnEIL1 gene was overexpressed in DT, and the transgenic plants showed a dwarf phenotype, indicating that the PsnEIL1 gene was involved in regulating the growth and development of poplar. In this study, the EIN3/EIL gene family of DH poplar was analyzed and functionally characterized, which provides a theoretical basis for the future exploration of the EIN3/EIL gene function.

Longitudinal profiling of human androgenotes through single-cell analysis unveils paternal gene expression dynamics in early embryo development.

STUDY QUESTION: How do transcriptomics vary in haploid human androgenote embryos at single cell level in the first four cell cycles of embryo development? SUMMARY ANSWER: Gene expression peaks at the fourth cell cycle, however some androcytes exhibit unique transcriptional behaviors. WHAT IS KNOWN ALREADY: The developmental potential of an embryo is determined by the competence of the oocyte and the sperm. However, studies of the contribution of the paternal genome using pure haploid androgenotes are very scarce. STUDY DESIGN, SIZE, DURATION: This study was performed analyzing the single-cell transcriptomic sequencing of 38 androcytes obtained from 10 androgenote bioconstructs previously produced in vitro (de Castro et al., 2023). These results were analyzed through different bioinformatics software such as g: Profiler, GSEA, Cytoscape, and Reactome. PARTICIPANTS/MATERIALS, SETTING, METHODS: Single cell sequencing was used to obtain the transcriptomic profiles of the different androcytes. The results obtained were compared between the different cycles studied using the DESeq2 program and functional enrichment pathways using g: Profiler, Cytoscape, and Reactome. MAIN RESULTS AND THE ROLE OF CHANCE: A wave of paternally driven transcriptomic activation was found during the third-cell cycle, with 1128 upregulated and 225 downregulated genes and the fourth-cell cycle, with 1373 upregulated and 286 downregulated genes, compared to first-cell cycle androcytes. Differentially expressed routes related to cell differentiation, DNA-binding transcription, RNA biosynthesis and RNA polymerase II transcription regulatory complex, and cell death were found in the third and fourth with respect to the first-cell cycle. Conversely, in the fourth cell cycle, 153 downregulated and 332 upregulated genes were found compared with third cell cycle, associated with differentially expressed processes related to E-box binding and zinc finger protein 652 (ZNF652) transcription factor. Further, significant overexpression of LEUTX, PRAMEF1, DUXA, RFPL4A, TRIM43, and ZNF675 found in androgenotes, compared to biparental embryos, highlights the paternal contributions to zygote genome activation. LARGE SCALE DATA: All raw sequencing data are available through the Gene Expression Omnibus (GEO) under accessions number: GSE216501. LIMITATIONS, REASONS FOR CAUTION: Extrapolation of biological events from uniparental constructs to biparental embryos should be done with caution. Maternal and paternal genomes do not act independently of each other in a natural condition. The absence of one genome may affect gene transcription of the other. In this sense, the haploid condition of the bioconstructs could mask the transcriptomic patterns of the single cells. WIDER IMPLICATIONS OF THE FINDINGS: The results obtained demonstrated the level of involvement of the human paternal haploid genome in the early stages of embryo development as well as its evolution at the transcriptomic level, laying the groundwork for the use of these bioconstructs as reliable models to dispel doubts about the genetic role played by the paternal genome in the early cycles of embryo development. STUDY FUNDING/COMPETING INTEREST(S): This study was funded by Instituto de Salud Carlos III (ISCIII) through the project 'PI22/00924', co-funded by European Regional Development Fund (ERDF); 'A way to make Europe'. F.D. was supported by the Spanish Ministry of Economy and Competitiveness through the Miguel Servet program (CPII018/00002). M.J.E. was supported by Instituto de Salud Carlos III (PI19/00577 [M.J.E.]) and FI20/00086. P.dC. was supported by a predoctoral grant for training in research into health (PFIS PI19/00577) from the Instituto de Salud Carlos III. All authors declare having no conflict of interest with regard to this trial.

Acute Lymphoblastic Leukemia With Near-haploid Karyotype and Philadelphia Chromosome.

BACKGROUND/AIM: In precursor B-cell lineage acute lymphoblastic leukemia (BCP-ALL), leukemic cells harbor genetic abnormalities that play an important role in the diagnosis, prognosis, and treatment. A subgroup of BCP-ALL is characterized by the presence of a Philadelphia (Ph) chromosome and a chimeric BCR::ABL1 gene, whereas in another subgroup, leukemic cells exhibit near-haploidy with chromosome number 24-30. This study presents the third documented case of BCP-ALL in which a near haploid clone concurrently displayed a Ph chromosome/BCR::ABL1. CASE REPORT: Bone marrow cells obtained at diagnosis from a 25-year-old man with BCP-ALL were genetically investigated using G-banding, fluorescence in situ hybridization, and array comparative genomic hybridization. Leukemic cells had an abnormal karyotype 28,X,-Y,+6,+10,+18,+21,+ der(22) t(9;22)(q34;q11)[13]/28,idem, del(10)(q24),der(12) t(1;12) (q21;p13)[2]/46,XY[3], retained heterozygosity of the disomic chromosomes 6, 10, 18, and 21, had breakpoints in introns 1 of ABL1 and BCR, and carried a BCR::ABL1 chimera encoding the 190 kDa BCR::ABL1 protein. CONCLUSION: The coexistence of the BCR::ABL1 chimera and near-haploidy in the same cytogenetic clone suggested a possible synergistic role in leukemogenesis, with the former activating signaling pathways and the latter disrupting gene dosage balance.

Constructing telomere-to-telomere diploid genome by polishing haploid nanopore-based assembly.

Draft genomes generated from Oxford Nanopore Technologies (ONT) long reads are known to have a higher error rate. Although existing genome polishers can enhance their quality, the error rate (including mismatches, indels and switching errors between paternal and maternal haplotypes) can be significant. Here, we develop two polishers, hypo-short and hypo-hybrid to address this issue. Hypo-short utilizes Illumina short reads to polish an ONT-based draft assembly, resulting in a high-quality assembly with low error rates and switching errors. Expanding on this, hypo-hybrid incorporates ONT long reads to further refine the assembly into a diploid representation. Leveraging on hypo-hybrid, we have created a diploid genome assembly pipeline called hypo-assembler. Hypo-assembler automates the generation of highly accurate, contiguous and nearly complete diploid assemblies using ONT long reads, Illumina short reads and optionally Hi-C reads. Notably, our solution even allows for the production of telomere-to-telomere diploid genomes with additional manual steps. As a proof of concept, we successfully assembled a fully phased telomere-to-telomere diploid genome of HG00733, achieving a quality value exceeding 50.

Induction of somatic cell haploidy by premature cell division.

Canonical mitotic and meiotic cell divisions commence with replicated chromosomes consisting of two sister chromatids. Here, we developed and explored a model of premature cell division, where nonreplicated, G0/G1-stage somatic cell nuclei are transplanted to the metaphase cytoplasm of mouse oocytes. Subsequent cell division generates daughter cells with reduced ploidy. Unexpectedly, genome sequencing analysis revealed proper segregation of homologous chromosomes, resulting in complete haploid genomes. We observed a high occurrence of somatic genome haploidization in nuclei from inbred genetic backgrounds but not in hybrids, emphasizing the importance of sequence homology between homologs. These findings suggest that premature cell division relies on mechanisms similar to meiosis I, where genome haploidization is facilitated by homologous chromosome interactions, recognition, and pairing. Unlike meiosis, no evidence of recombination between somatic cell homologs was detected. Our study offers an alternative in vitro gametogenesis approach by directly reprogramming diploid somatic cells into haploid oocytes.

Interactions between U and V sex chromosomes during the life cycle of Ectocarpus.

In many animals and flowering plants, sex determination occurs in the diploid phase of the life cycle with XX/XY or ZW/ZZ sex chromosomes. However, in early diverging plants and most macroalgae, sex is determined by female (U) or male (V) sex chromosomes in a haploid phase called the gametophyte. Once the U and V chromosomes unite at fertilization to produce a diploid sporophyte, sex determination no longer occurs, raising key questions about the fate of the U and V sex chromosomes in the sporophyte phase. Here, we investigate genetic and molecular interactions of the UV sex chromosomes in both the haploid and diploid phases of the brown alga Ectocarpus. We reveal extensive developmental regulation of sex chromosome genes across its life cycle and implicate the TALE-HD transcription factor OUROBOROS in suppressing sex determination in the diploid phase. Small RNAs may also play a role in the repression of a female sex-linked gene, and transition to the diploid sporophyte coincides with major reconfiguration of histone H3K79me2, suggesting a more intricate role for this histone mark in Ectocarpus development than previously appreciated.

Cyto-swapping in maize by haploid induction with a cenh3 mutant.

Maize mutants of the centromeric histone H3 (CENP-A/CENH3) gene can form haploids that inherit only chromosomes of the pollinating parent but the cytoplasm from the female parent. We developed CENH3 haploid inducers carrying a dominant anthocyanin colour marker for efficient haploid identification and harbouring cytoplasmic male sterile cytoplasm, a type of cytoplasm that results in male sterility useful for efficient hybrid seed production. The resulting cytoplasmic male sterility cyto-swapping method provides a faster and cheaper way to convert commercial lines to cytoplasmic male sterile compared to conventional trait introgression.

Stable demographic ratios of haploid gametophyte to diploid sporophyte abundance in macroalgal populations.

Macroalgal populations often consist of free-living haploid (gametophyte) and diploid (sporophyte) stages. Various ecological studies have been conducted to examine the demographic diversity of haploid-diploid populations with regard to the dominant stage. Here, I relaxed the assumption of classical research that the life history parameters of haploids and diploids are identical and developed a generalized haploid-diploid model that explicitly accounts for population density dependence and asexual reproduction. Analysis of this model yielded an exact solution for the abundance ratio of haploids to diploids in a population in which the ratio is determined by the balance of four demographic forces: sexual reproduction by haploids, sexual reproduction by diploids, asexual reproduction by haploids, and asexual reproduction by diploids. Furthermore, the persistence of a haploid-diploid population and its total biomass are shown to be determined by the basic reproductive number (R0), which is shown to be a function of these four demographic forces. When R0 is greater than one, the haploid-diploid population stably persists, and the ploidy ratio obtained by the analytical solution is realized.

Prediction of matrilineal specific patatin-like protein governing in-vivo maternal haploid induction in maize using support vector machine and di-peptide composition.

The mutant matrilineal (mtl) gene encoding patatin-like phospholipase activity is involved in in-vivo maternal haploid induction in maize. Doubling of chromosomes in haploids by colchicine treatment leads to complete fixation of inbreds in just one generation compared to 6-7 generations of selfing. Thus, knowledge of patatin-like proteins in other crops assumes great significance for in-vivo haploid induction. So far, no online tool is available that can classify unknown proteins into patatin-like proteins. Here, we aimed to optimize a machine learning-based algorithm to predict the patatin-like phospholipase activity of unknown proteins. Four different kernels [radial basis function (RBF), sigmoid, polynomial, and linear] were used for building support vector machine (SVM) classifiers using six different sequence-based compositional features (AAC, DPC, GDPC, CTDC, CTDT, and GAAC). A total of 1170 protein sequences including both patatin-like (585 sequences) from various monocots, dicots, and microbes; and non-patatin-like proteins (585 sequences) from different subspecies of Zea mays were analyzed. RBF and polynomial kernels were quite promising in the prediction of patatin-like proteins. Among six sequence-based compositional features, di-peptide composition attained > 90% prediction accuracies using RBF and polynomial kernels. Using mutual information, most explaining dipeptides that contributed the highest to the prediction process were identified. The knowledge generated in this study can be utilized in other crops prior to the initiation of any experiment. The developed SVM model opened a new paradigm for scientists working in in-vivo haploid induction in commercial crops. This is the first report of machine learning of the identification of proteins with patatin-like activity.

Evolution of parental care in haploid-diploid plants.

In bryophytes that alternate between haploid gametophytes and diploid sporophytes through sexual reproduction, sporophytes are often attached to and nurtured on the female gametophyte. A similar phenomenon is seen in Florideophyceae (a group of red algae). These systems in which a gametophyte (mother) invests nutrients in sporophytes (offspring) are ideal for studying the evolution of 'parental care' in non-animal organisms. Here, we propose a model of a haploid-diploid life cycle and examine the evolution of maternal investment in sporophytes focusing on two effects: the degree of paternal or maternal control of investment and the number of sporophytes. We find that when the female dominantly controls the investment, the evolutionarily stable level of investment is that which maximizes the expected reproductive success of the female gametophyte. The genomic conflict between maternal and paternal alleles complicates the evolutionary outcome; however, a greater male allelic effect and a higher number of sporophytes favour a higher energy investment, which may lead to evolutionary branching or run-away escalation of the investment level. This suggests that the selfishness of the paternal gene is the evolutionary driver of parental care and that complex structures such as fusion cells in red algae may have evolved to suppress it.