Quantitative testing of the methodology for genome size estimation in plants using flow cytometry: a case study of the Primulina genus.
ABSTRACT: Flow cytometry (FCM) is a commonly used method for estimating genome size in many organisms. The use of FCM in plants is influenced by endogenous fluorescence inhibitors and may cause an inaccurate estimation of genome size; thus, falsifying the relationship between genome size and phenotypic traits/ecological performance. Quantitative optimization of FCM methodology minimizes such errors, yet there are few studies detailing this methodology. We selected the genus Primulina, one of the most representative and diverse genera of the Old World Gesneriaceae, to evaluate the methodology effect on determining genome size. Our results showed that buffer choice significantly affected genome size estimation in six out of the eight species examined and altered the 2C-value (DNA content) by as much as 21.4%. The staining duration and propidium iodide (PI) concentration slightly affected the 2C-value. Our experiments showed better histogram quality when the samples were stained for 40 min at a PI concentration of 100 ?g ml(-1). The quality of the estimates was not improved by 1-day incubation in the dark at 4°C or by centrifugation. Thus, our study determined an optimum protocol for genome size measurement in Primulina: LB01 buffer supplemented with 100 ?g ml(-1) PI and stained for 40 min. This protocol also demonstrated a high universality in other Gesneriaceae genera. We report the genome size of nine Gesneriaceae species for the first time. The results showed substantial genome size variation both within and among the species, with the 2C-value ranging between 1.62 and 2.71 pg. Our study highlights the necessity of optimizing the FCM methodology prior to obtaining reliable genome size estimates in a given taxon.
Project description:Genome size is of fundamental biological importance with significance in predicting structural and functional attributes of organisms. Although abundant evidence has shown that the genome size can be largely explained by differential proliferation and removal of non-coding DNA of the genome, the evolutionary and ecological basis of genome size variation remains poorly understood. Nitrogen (N) and phosphorus (P) are essential elements of DNA and protein building blocks, yet often subject to environmental limitation in natural ecosystems. Using phylogenetic comparative methods, we test this hypothesis by determining whether leaf N and P availability affects genome sizes in 99 species of Primulina (Gesneriaceae), a group of soil specialists adapted to limestone karst environment in south China. We find that genome sizes in Primulina are strongly positively correlated with plant N content, but the correlation with plant P content is not significant when phylogeny history was taken into account. This study shows for the first time that N limitation might have been a plausible driver of genome size variation in a group of plants. We propose that competition for nitrogen nutrient between DNA synthesis and cellular functions is a possible mechanism for genome size evolution in Primulina under N-limitation.
Project description:The limestone regions of Yunnan-Guangxi-Guizhou in southern and southwestern China are regarded as some of biodiversity's hotspots for China's Gesneriaceae where numerous rare new species of Primulina have been, or are being, described over the past two decades. Primulina flexusa, a new lithophytic species of Gesneriaceae from a limestone hill in a Karst area, from Guizhou, China, is described here with color photographs. It is similar to P. curvituba, but can be easily distinguished by a combination of characteristics, especially in the shape and length of its capsule. We found only one population with approximately 100 mature individuals at the type locality. This new species is provisionally assessed as Critically Endangered [CR C1] by using IUCN criteria.
Project description:Primulina jiuyishanica K. Liu, D.C. Meng & Z.B. Xin, a new species of Gesneriaceae from Hunan, China, is described and illustrated. The new species is morphologically similar to Primulina fimbrisepala (Hand.-Mazz.) Yin Z. Wang, but differs in its elliptic to broadly elliptic leaf blade with broadly cuneate base, peduncle densely pubescent with sparse glandular hairs, corolla throat with no purple spots inside, the yellow patch in the throat densely glandular-pubescent and pistil densely glandular-pubescent. Photographs and descriptions of the new species are provided below.
Project description:BACKGROUND:Primulina cardaminifolia Yan Liu & W.B. Xu (Gesneriaceae), a distinct new species with imparipinnate leaves, is described and illustrated from a limestone valley in Guangxi Zhuangzu Autonomous Region, China. To assure its generic placement and phylogenetic affinity, phylogenetic analyses were performed using DNA sequences of nuclear ITS and chloroplast trnL-F intron spacer region. Additionally, somatic chromosome number was counted and pollen stainability was tested. RESULTS:Phylogenetic analyses support its placement in Primulina; however, two phylogenetically distinct ITS sequence types were detected, suggesting a probable hybrid origin. Its pollen stainability is 100% and its chromosome number, 2n = 36, is congruent with all known counts of diploid species of the genus. CONCLUSION:All available data support the recognition of the new species Primulina cardaminifolia and suggest that it could have derived from homoploid hybrid speciation. Color plates, line drawings and a distribution map are provided to aid in identification.
Project description:BACKGROUND:The Gesneriaceae genus Chiritopsis, confined almost exclusively to cave or cave-like microhabitats of limestone karsts of southern China, was described to distinguish it from Chirita by much smaller flowers and generally miniature plant sizes in the former genus. However, molecular phylogenetic analyses showed that Chiritopsis is polyphyletic and its species delimitation has been problematic. To understand how many times Chiritopsis-like species have evolved from within the recircumscribed Primulina and to further clarify their species identification, we sampled all but two recently described species of Chiritopsis-like Primulina and reconstructed their phylogenetic relationship based on DNA sequences of nuclear ITS and chloroplast trnL-F and trnH-psbA. RESULTS:With 182 accessions of 165 taxa of Primulina sampled, our analyses placed the 40 accessions of 25 taxa of Chiritopsis-like Primulina in 17 unrelated positions, indicating at least 17 independent origins of the traits associated with caves or cave-like microhabitats. Of the 17 clades containing Chiritopsis-like Primulina, Clade 1 is composed of P. bipinnatifida, P. cangwuensis, P. jianghuaensis, P. lingchuanensis, and P. zhoui, as well as additional samples that show variable and overlapping morphology in leaf shapes. Clade 10 includes P. cordifolia, P. huangii, and P. repanda, while Primulina repanda var. guilinensis is not placed within Clade 10. Primulina glandulosa var. yangshuoensis is not placed in the same clade of P. glandulosa. CONCLUSIONS:Based on our data, P. cangwuensis, P. jianghuaensis, and P. lingchuanensis are proposed to synonymize under P. bipinnatifida, with P. zhoui treated as a variety of P. bipinnatifida. Primulina repanda var. guilinensis is transferred as P. subulata var. guilinensis comb. nov. and Primulina pseudoglandulosa nom. nov. is proposed for P. glandulosa var. yangshuoensis. One new species is named P. chingipengii to honor the late Dr. Ching-I Peng (1950-2018).
Project description:Five new species of Primulina (Gesneriaceae) are described and illustrated here, namely P.purpureokylin F. Wen, Yi Huang & W. Chuen Chou, P.persica F. Wen, Yi Huang & W. Chuen Chou, P.cerina F. Wen, Yi Huang & W. Chuen Chou, P.niveolanosa F. Wen, S. Li & W. Chuen Chou and P.leiyyi F. Wen, Z.B. Xin & W. Chuen Chou. The characteristic traits of these species, together with photographs, detailed descriptions, notes on etymology, distribution, and habitat, as well as comparisons with morphologically similar species, are provided.
Project description:Primulina Hance is an emerging model for studying evolutionary divergence, adaptation and speciation of the karst flora. However, phylogenetic relationships within the genus have not been resolved due to low variation detected in the cpDNA regions. Chloroplast genomes can provide important information for phylogenetic and population genetic studies. Recent advances in next-generation sequencing (NGS) techniques greatly facilitate sequencing whole chloroplast genomes for multiple individuals. Consequently, novel strategies for development of highly polymorphic loci for population genetic and phylogenetic studies based on NGS data are needed.For development of high polymorphic loci for population genetic and phylogenetic studies, two novel strategies are proposed here. The first protocol develops lineage-specific highly variable markers from the true high variation regions (Con_Seas) across whole cp genomes, instead of traditional noncoding regions. The pipeline has been integrated into a single perl script, and named "Con_Sea_Identification_and_PIC_Calculation". The second method assembles chloroplast fragments (poTs) and sub-super-marker (CpContigs) through our "SACRing" pipeline. This approach can fundamentally alter the strategies used in phylogenetic and population genetic studies based on cp markers, facilitating a transition from traditional Sanger sequencing to RAD-Seq. Both of these scripts are available at https://github.com/scbgfengchao/ .Three complete Primulina chloroplast genomes were assembled from genome survey data, and then two novel strategies were developed to yield highly polymorphic markers. For experimental evaluation of the first protocol, a set of Primulina species were used for PCR amplification. The results showed that these newly developed markers are more variable than traditional ones, and seem to be a better choice for phylogenetic and population studies in Primulin a. The second method was also successfully applied in population genetic studies of 21 individuals from three natural populations of Primulina.These two novel strategies may provide a pathway for similar research in other non-model species. The newly developed high polymorphic loci in this study will promote further the phylogenetic and population genetic studies in Primulina and other genera of the family Gesneriaceae.
Project description:Premise:We developed single-copy nuclear DNA markers for a perennial herb, Oreocharis auricula (Gesneriaceae), to help infer the evolutionary history of the genus Oreocharis in subtropical China. Methods and Results:We screened 834 putative single-copy nuclear genes from transcriptomes of 11 species of Primulina using the Illumina HiSeq 2000 platform. Based on the screening results, we developed 16 primer sets for accessions of O. auricula representing three wild populations. The number of alleles per locus ranged from three to 34. The levels of expected and observed heterozygosity varied from 0.000 to 0.372 and 0.000 to 0.650, respectively. The markers were successfully cross-amplified in the related species O. magnidens, O. speciosa, O. maximowiczii, and O. henryana. Conclusions:The newly developed markers will facilitate further studies on genetic diversity and phylogeographic structure throughout the distributional range of O. auricula. Additionally, these markers may be useful for other related species in Gesneriaceae.
Project description:Importance:Strategies to procure high-quality core-needle biopsy (CNB) specimens are critical for making basic tissue diagnoses and for ancillary testing. Objectives:To investigate acquisition of fluorescence confocal microscopy (FCM) images of interventional radiology (IR)-guided CNB in real time in the radiology suite and to compare the accuracy of FCM diagnoses with those of hematoxylin-eosin (H&E)-stained CNB sections. Design, Setting, and Participants:In this diagnostic study, FCM imaging of IR-guided CNBs was performed in the radiology suite at a major cancer center for patients with an imaging abnormality from August 1, 2016, to April 30, 2019. The time taken to acquire FCM images and the quality of FCM images based on percentage of interpretable tissue with optimal resolution was recorded. The FCM images were read by 2 pathologists and categorized as nondiagnostic, benign/atypical, or suspicious/malignant; these diagnoses were compared with those made using H&E-stained tissue sections. Cases with discrepant diagnosis were reassessed by the pathologists together for a consensus diagnosis. Data were analyzed from June 3 to July 19, 2019. Interventions:Each IR-guided CNB was stained with 0.6mM acridine orange, subjected to FCM imaging, and then processed to generate H&E-stained sections. Main Outcomes and Measures:Mean time taken for acquisition of FCM images, quality of FCM images based on interpretable percentage of the image, and accuracy of diagnostic categorization based on FCM images compared with H&E-stained sections. Results:A total of 105 patients (57 male [54.3%]; mean [SD] age, 63  years) underwent IR-guided CNBs in a mean (SD) of 7 (2) minutes each. The FCM images showed at least 20% of the tissue with optimal quality in 101 CNB specimens (96.2%). The FCM images were accurately interpreted by the 2 pathologists in 100 of 105 cases (95.2%) (2 false-positive and 3 false-negative) and 90 of 105 cases (85.7%) (6 false-positive and 9 false-negative). A reassessment of 14 discordant diagnoses resulted in consensus diagnoses that were accurate in 101 of 105 cases (96.2%) (1 false-positive and 3 false-negative). Conclusions and Relevance:The ease of acquisition of FCM images of acceptable quality and the high accuracy of the diagnoses suggest that FCM may be useful for rapid evaluation of IR-guided CNBs. This approach warrants further investigation.
Project description:One new species and one new variety of Gesneriaceae in Vietnam are described, viz. Paraboea sinensis var. glabrissimavar. nov. and Primulina xuansonensis sp. nov. These two new taxa grow in limestone regions in North Vietnam. The former new variety differs from Paraboea sinensis (Oliv.) Burtt var. sinensis in its young leaf abaxially, stem and peduncle sparsely and thin pannose, acute top of leaves, pistil glandular-pubescent or pubescent. The latter new species differs in its bracts 1.6-2.5 × 1.3-1.5 cm, corolla 3-3.5 cm long, corolla tube slightly curved near the base and inflated on the adaxial surface near the corolla lobes, and corolla abaxial lip lobes rounded. The two new taxa grow at Xuan Son National Natural Reserve, North Vietnam and remain undisturbed with low risk of extinction.