Cytological and molecular characterization of three gametoclones of Citrus clementina

Abstract Background Three gametoclonal plants of Citrus clementina Hort. ex Tan., cv. Nules, designated ESP, FRA, and ITA (derived from three labs in Spain, France, and Italy, respectively), were selected for cytological and molecular characterization in order to elucidate genomic rearrangements provoked by haploidization. The study included comparisons of their ploidy, homozygosity, genome integrity, and gene dosage, using chromosome counting, flow cytometry, SSR marker genotyping, and array-Comparative Genomic Hybridization (arrayCGH). Results Chromosome counting and flow cytometry revealed that ESP and FRA were haploid, but ITA was tri-haploid. Homozygous patterns, represented by a single peak (allele), were observed among the three plants at almost all SSR loci distributed across the entire diploid donor genome. Those few loci with extra peaks visualized as output from automated sequencing runs, generally low or ambiguous, might result from amplicons of paralogous members at the locus, non-specific sites, or unexpected recombinant alleles. No new alleles were found, suggesting the genomes remained stable and intact during gametogenesis and regeneration. The integrity of the haploid genome also was supported by array-CGH studies, in which genomic profiles were comparable to the diploid control. Conclusions The presence of few gene hybridization abnormalities, corroborated by gene dosage measurements, were hypothetically due to the segregation of hemizygous alleles and minor genomic rearrangements occurring during the haploidization procedure. In conclusion, these plants that are valuable genetic and breeding materials contain completely homozygous and essentially intact genomes

Patient concerns about accessing sexual and reproductive health services outside of primary care: A survey in rural and urban settings in the Pacific Northwest.

OBJECTIVES: While primary care providers are a major source of sexual and reproductive health (SRH) services in the United States, particularly in rural areas, not all primary care settings offer a full range of SRH services. We aimed to understand primary care patient concerns regarding accessing SRH services, including abortion care, outside of their primary care clinic and if those concerns differed by urban or rural setting. STUDY DESIGN: An anonymous survey was distributed over a 2-week period between December 2019 to March 2020 to all adult patients in four primary care clinics in Idaho, Washington, and Wyoming. The survey assessed patient concerns regarding accessing SRH services outside of their primary care clinic and their willingness to travel to access SRH services. RESULTS: The overall response rate was 69% (745/1086). Over 85% of respondents identified at least one concern to seeking SRH services outside of a primary care setting, with cost, insurance coverage, length of wait time, and lack of an established relationship being the most frequently reported concerns. A majority of respondents were willing to travel a maximum of 1 hour for most SRH services. Respondents from rural-serving clinics were significantly more likely to be willing to travel longer amounts of time for medication abortion, aspiration abortion, and intrauterine device placement. CONCLUSION: Our findings highlight that a majority of both urban and rural primary care patients have concerns regarding accessing SRH services outside of their primary care clinic and are unwilling to travel more than 1 hour to access most SRH services. IMPLICATIONS: A majority of primary care patients have concerns regarding accessing SRH services outside of primary care settings. Health care policy changes should aim to strengthen the SRH services available in primary care settings to alleviate the burdens primary care patients face in accessing SRH services outside of their primary care clinic, particularly for rural populations

RANK links senescence to stemness in the mammary epithelia, delaying tumor onset but increasing tumor aggressiveness.

Rank signaling enhances stemness in mouse and human mammary epithelial cells (MECs) and mediates mammary tumor initiation. Mammary tumors initiated by oncogenes or carcinogen exposure display high levels of Rank and Rank pathway inhibitors have emerged as a new strategy for breast cancer prevention and treatment. Here, we show that ectopic Rank expression in the mammary epithelia unexpectedly delays tumor onset and reduces tumor incidence in the oncogene-driven Neu and PyMT models. Mechanistically, we have found that ectopic expression of Rank or exposure to Rankl induces senescence, even in the absence of other oncogenic mutations. Rank leads to DNA damage and senescence through p16/p19. Moreover, RANK-induced senescence is essential for Rank-driven stemness, and although initially translates into delayed tumor growth, eventually promotes tumor progression and metastasis. We uncover a dual role for Rank in the mammary epithelia: Rank induces senescence and stemness, delaying tumor initiation but increasing tumor aggressiveness

Cytological and molecular characterization of three gametoclones of Citrus clementina

Three gametoclonal plants of Citrus clementina Hort. ex Tan., cv. Nules, designated ESP, FRA, and ITA (derived from three labs in Spain, France, and Italy, respectively), were selected for cytological and molecular characterization in order to elucidate genomic rearrangements provoked by haploidization. The study included comparisons of their ploidy, homozygosity, genome integrity, and gene dosage, using chromosome counting, flow cytometry, SSR marker genotyping, and array-Comparative Genomic Hybridization (arrayCGH). Results Chromosome counting and flow cytometry revealed that ESP and FRA were haploid, but ITA was tri-haploid. Homozygous patterns, represented by a single peak (allele), were observed among the three plants at almost all SSR loci distributed across the entire diploid donor genome. Those few loci with extra peaks visualized as output from automated sequencing runs, generally low or ambiguous, might result from amplicons of paralogous members at the locus, non-specific sites, or unexpected recombinant alleles. No new alleles were found, suggesting the genomes remained stable and intact during gametogenesis and regeneration. The integrity of the haploid genome also was supported by array-CGH studies, in which genomic profiles were comparable to the diploid control. Conclusions The presence of few gene hybridization abnormalities, corroborated by gene dosage measurements, were hypothetically due to the segregation of hemizygous alleles and minor genomic rearrangements occurring during the haploidization procedure. In conclusion, these plants that are valuable genetic and breeding materials contain completely homozygous and essentially intact genomes

Ploidy manipulation and citrus breeding, genetics and genomics

Polyploidy appears to have played a limited role in citrus germplasm evolution. However, today, ploidy manipulation is an important component of citrus breeding strategies. For varieties, the main objective is to develop triploid seedless varieties. For rootstock, the aim is to cumulate interesting traits in tetraploid hybrids and to improve adaptation to biotic and abiotic stresses. In this chapter we make a review of the recent knowledge acquired on the natural mechanisms of citrus polyploidization, and teraploid meiosis. Chromosome doubling of nucellar cells is frequent in apomictic citrus and results in tetraploid seedling production. Unreduced gametes are also frequently produced, mainly by second division restitution for ovules. First division restitution was described for pollen as well as alternative mechanisms for both ovules and pollen. Tetraploid plants display tetrasomic to disomic segregations in relation with their genome structure (autoteraploid versus allotetraploid) and the divergence of the parental species. The implications of the origin of diploid gametes, on the genetic diversity of polyploid progenies, are discussed. The biotechnological tools (haplo-methods, chromosome doubling by chemichal treatments, somatic hybridization and cytogenetic/molecular tools for polyploid genome studies) to optimize ploidy manipulation are presented. The interest of haploids and polyploid genotypes for basic genetic and genomic studies is discussed. The following research area are reviewed: haploids and doubled haploid for genome sequencing and haplotyping, centromere mapping from unreduced gametes, marker-trait association study in polyploids, phenome and gene expression in polyploids with a special focus on polyploidy and adaptation. Finally, we give an overview of the recent advances of concrete polyploid citrus breeding programs in China, Florida and the Mediterranean Basin