Draft Genome Sequence of a Diploid and Hybrid Candida Strain, Candida sanyaensis UCD423, Isolated from Compost in Ireland

Candida sanyaensis is a CUG-Ser1 clade yeast that is associated with soil. Assembly of short-read and long-read data shows that C. sanyaensis has a diploid and hybrid genome, with approximately 97% identity between the haplotypes. The haploid genome size is approximately 15.4 Mb.

Karyotype and genome size determination of Jarilla chocola, an additional sister clade of Carica papaya

Jarilla chocola is an herbaceous plant species that belongs to the Jarilla genus and the Caricaceae family. No information on chromosome number or genome size has been reported for J. chocola that confirms the occurrence of dysploidy events and explore the existence of heteromorphic sex chromosomes. Therefore, the total number of chromosomes of this species was determined by karyotyping and counting the number of chromosomes observed, and the genome size of female and male plants was estimated separately by flow cytometry. Results showed that J. chocola has eight pairs of chromosomes (2n = 2x = 16), and its chromosomes are classified as metacentric for five pairs, submetacentric for two pairs and telocentric for one pair. The nuclear DNA content (1C-value) in picograms and diploid genome size was estimated separately from female and male plants using two species as the standards, Phaseolus vulgaris (1C = 0.60 pg) and Carica papaya (1C = 0.325 pg), to look for the possible existence of heteromorphic sex chromosomes. C. papaya proved to be a better standard for the determination of J. chocola DNA content and diploid genome size. No significant difference on the DNA content was observed between female (1C = 1.02 ± 0.003 pg) and male (1C = 1.02 ± 0.008 pg) plants. The estimated genome size of J. chocola per haploid genome in base pairs was calculated from the obtained C-values. Results showed an estimated genome size per haploid genome of 1018.44 ± 3.07 Mb and 1022.08 ± 7.76 Mb for female and male plants, respectively. Due to the observed chromosome number and genome size, only the occurrence of one of two previously reported dysploidy events in Jarilla could be confirmed for J. chocola and no evidence of heteromorphic sex chromosomes was found. These results provide fundamental information of the J. chocola genome and will expedite investigation of sex chromosomes and genome evolution in this species, the Jarilla genus and the Caricaceae family

Usefulness of Adapted Exotic Maize Lines Developed By Doubled Haploid and Single Seed Descent Methods

Adapted exotic maize (Zea mays L.) germplasm, such as BS39, provides a unique opportunity for broadening the genetic base of U.S. Corn Belt germplasm. In vivo doubled haploid (DH) technology has been used to efficiently exploit exotic germplasm. It can help to purge deleterious recessive alleles. The objectives of this study were to determine the usefulness of BS39-derived inbred lines using both SSD and DH methods, to determine the impact of spontaneous as compared to artificial haploid genome doubling on genetic variance among BS39-derived DH lines, and to identify SNP markers associated with agronomic traits among BS39 inbreds monitored at testcross level. We developed two sets of inbred lines directly from BS39 by DH and SSD methods, named BS39_DH and BS39_SSD. Additionally, two sets were derived from a cross between BS39 and A427 (SHGD donor) by DH and SSD methods, named BS39×A427_DH and BS39×A427_SSD, respectively. Grain yield, moisture, plant height, ear height, stalk lodging, and root lodging were measured to estimate genetic parameters. For genome-wide association (GWAS) analysis, inbred lines were genotyped using Genotype-by-Sequencing (GBS) and Diversity Array Technology Sequencing (DArTSeq). Some BS39-derived inbred lines performed better than elite germplasm inbreds and all sets showed significant genetic variance. The presence of spontaneous haploid genome doubling genes did not affect performance of inbred lines. Five SNPs were significant and three of them located within genes related to plant development or abiotic stresses. These results demonstrate the potential of BS39 to add novel alleles to temperate elite germplasm.

Haploid Genome Analysis Reveals a Tandem Cluster of Four HSP20 Genes Involved in the High-Temperature Adaptation of Coriolopsis trogii

Heat stress is one of the most frequently encountered environmental stresses for most mushroom-forming fungi. Currently available fungal genomes are mostly haploid because high heterozygosity hinders diploid genome assembly.

Gametophyte genome activation occurs at pollen mitosis I in maize

Flowering plants alternate between multicellular haploid (gametophyte) and diploid (sporophyte) generations. One consequence of this life cycle is that plants face substantial selection during the haploid phase (1-3). Pollen actively transcribes its haploid genome (4), providing phenotypic diversity even among pollen grains from a single plant. Currently, the timing that pollen precursors first establish this independence is unclear. Starting with an endowment of transcripts from the diploid parent, when do haploid cells generated by meiosis begin to express genes? Here, we follow the shift to haploid expression in maize pollen using allele-specific RNA-sequencing (RNA-Seq) of single pollen precursors. We observe widespread biallelic expression for 11 days after meiosis, indicating that transcripts synthesized by the diploid sporophyte persist long into the haploid phase. Subsequently, there was a rapid and global conversion to monoallelic expression at pollen mitosis I (PMI), driven by active new transcription from the haploid genome. Genes expressed during the haploid phase showed reduced rates of nonsynonymous relative to synonymous substitutions (dn/ds) if they were expressed after PMI, but not before, consistent with purifying selection acting on the haploid gametophyte. This work establishes the timing with which haploid selection may act in pollen and provides a detailed time-course of gene expression during pollen development.

P–800 Generation of artificial oocytes by two distinct mechanisms

Study question Are haploid genome replication and somatic cell haploidization feasible mechanisms for generating parentally genotyped oocytes? Summary answer Artificial oocytes can be generated by haploid genome replication and somatic cell haploidization. The latter is more efficient and capable of generating live offspring. What is known already A low number of mature oocytes is one of the major limitations to treating infertile women who have impaired ovarian reserve. Although it has been proposed that competent oocytes can be created by a phenomenon known as somatic cell haploidization (SCH), its clinical value has yet to be examined due to its poorly understood mechanism. On the other hand, spindle transfer has been clinically applied for mitochondrial replacement therapy. Therefore, we propose to utilize G2-phase haploid pseudo-blastomere (HpB), generated by parthenogenesis, as a nuclear donor to create oocyte replica. Study design, size, duration In the past 7 months, individual G0 phase cumulus cells (CCs) were transferred into 1,066 ooplasts for SCH. HpBs obtained from the activation of 80 oocytes were transferred into 464 ooplasts. Both cohorts were ICSI-inseminated and placed in the time lapse for embryo development. Another 379 unmanipulated oocytes were ICSI-inseminated, serving as control. Pre-implantation development was monitored and compared for both neogametogenesis techniques. Fully expanded blastocysts were transferred to obtain live pups. Participants/materials, setting, methods CCs were isolated from the cumulus oophorus of B6D2F1 mice. HpBs were obtained via oocyte activation, cultured to the 8-cell stage, and subsequently treated by nocodazole to synchronize at the G2-phase. In two experimental groups, CCs or HpBs were individually transferred into the perivitelline space of the ooplasts with inactivated Sendai virus. Reconstructed oocytes presenting with a pseudo-meiotic spindle were fertilized by piezo-actuated ICSI. Blastocysts were transferred into a pseudo-pregnant CD–1 surrogate to obtain pups. Main results and the role of chance A total of 1,769 oocytes underwent enucleation to generate ooplasts, with a survival rate of 97%. Survived ooplasts were allocated to SCH (n = 1,034) and HpB-SCNT (n = 458). To generate HpBs, 80 unmanipulated oocytes were activated; 58 of them progressed to the 8-cell stage and generated 464 HpB for SCNT. For SCH, CCs were selected based on morphology with a diameter <10 micron. Nuclear transfer of CCs and HpB yielded survival rates of 98.6% and 93.2%, respectively. Following SCH and HpB-SCNT, spindle development for SCH and HpB-SCNT was comparable at 63.5% for SCH and 66.7% for HpB-SCNT. The ICSI survival rates for SCH and HpB-SCNT were 58.9% and 64.9%, respectively, but lower than the control at 73.9% (P < 0.001). Fertilization rates for SCH and HpB-SCNT were also comparable at 61.3% and 64.3%, respectively, but lower than the control at 89.6% (P < 0.00001). Full pre-implantation development was achieved for both experimental groups. While the SCH group yielded a development rate of 24.6% (n = 94), the HpB-SCNT group yielded a lower rate at 12.4% (n = 23) (P < 0.001), both lower than the control (71.7%, P < 0.00001); however, the morphokinetics of the embryo development was retained. To date, only 3 live pups were obtained from SCH group. Limitations, reasons for caution While these techniques to manufacture oocytes are very new and highly experimental, our findings show a lower blastulation rate for oocytes generated by HpB. Both techniques require refinement and improvement of reliability and consistency before they can be considered a feasible technique for human reproduction. Wider implications of the findings: The study confirms the potential to create artificial oocytes capable of supporting full pre-implantation development and, in some cases, live pups. If further streamlining of both procedures demonstrates their safety, they may both represent a viable option to generate de novo gametes Trial registration number N/A

Detection of copy number variation of alpha amylase genes in domestic pigs (Sus scrofa domesticus) and wild boars (Sus scrofa)

Copy numbers of alpha amylase genes (AMY), which encode starch-digesting enzymes, are markedly increased in modern humans and domesticated dogs as an adaptive evolutionary mechanism in response to increased consumption of starch-rich foods acquired either by farming or domestication. In this study, we surveyed total AMY gene copy numbers in 150 domestic pigs (50 pigs of Berkshire breed, 50 of Landrace breed, and 50 of Large White breed) and 51 wild boars (30 Sus scrofa leucomystax and 21 S. s. riukiuanus) to identify whether the gene copy number has changed during the domestication of pigs. The relative copy number of AMY genes was measured using a quantitative polymerase chain reaction (qPCR) and it varied from 2.7 to 10.8 per haploid genome among individuals. However, in the four remaining populations, excluding S. s. riukiuanus, the average copy number was approximately six, and no significant differences were observed between the three selected pig breeds and S. s. leucomystax wild boar. Conversely, S. s. riukiuanus had an average of 7.2 copies. The results indicating six AMY copies per haploid genome were consistent with the porcine genome reference sequence (Sscrofa11.1). These results suggest that there has been no significant increase in the AMY gene copy number during the domestication process of pigs.

Haploid mouse germ cell precursors from embryonic stem cells reveal Xist activation from a single X chromosome

SummaryMammalian haploid cells have applications for genetic screening and substituting gametic genomes. Here we characterize a culture system for obtaining haploid primordial germ cell-like cells (PCGLCs) from haploid mouse embryonic stem cells (ESCs). We find that a haploid genome is maintained in PGCLCs with a high frequency indicating a substantially lower rate of diploidization than somatic cells. Characterization of the differentiating haploid ESCs reveals that Xist is activated from the single X chromosome. This observation suggests that X chromosome inactivation is initiated in haploid cells consistent with a model where autosomal blocking factors set a threshold for X-linked activators. The germline segregates from the epiblast and differs from somatic lineages in gene expression and epigenetic mechanisms. The ability of primordial germ cells for repressing Xist might contribute to the maintenance of a haploid genome.