Genome Size Determination Research Articles

Variability in genome size of Trigonella foenum-graecum, Trigonella corniculata and Trigonella caerulea as estimated by flow cytometry indicates complex evolutionary history of fenugreek.

The determination of genome size is a fundamental step which provides a basis to initiate studies aimed at deciphering the genetic similarity of a species and to carry out other genomics based investigations. Fenugreek (Trigonella spp.) is an important spice crop which has numerous health promoting phytochemicals. Many species within this genus are known for their various health benefits owing to the presence of a wide diversity of important phytochemicals like diosgenin, trigonelline, fenugreekine, galactomannan, 4-hydroxy isoleucine, etc. It is a multipurpose crop being cultivated for food, animal feed and industrial purposes. Despite its importance, research on the genomics aspect of fenugreek remains scant. In the absence of sufficient genomic information, crop improvement in fenugreek is severely lagging. Estimation of genome size of a species is the preliminary step for initiation of any genomic studies and therefore in the present study we have estimated the genome size for fenugreek. Here, we have determined the genome sizes of three different Trigonella spp. namely T. foenum-graecum, T. corniculata and T. caerulea through flow cytometry (FC). The 2C DNA content values were found to be 6.05 pg (T. foenum-graecum), 1.83 pg (T. corniculata) and 1.96 pg (T. caerulea). The genome size of T. foenum-graecum is approximately three times the genome size of T. corniculata and T. caerulea. This variation in genome size of more than three-fold indicates the level of genetic divergence among the three species, though within the same genus. The differences observed in the genome sizes of the three species provide conclusive evidence of their genetic divergence. Additionally, the information about the genome size would provide an impetus to the structural and functional genomics-based research in this crop.

Genome sequencing and analysis of black flounder (Paralichthys orbignyanus) reveals new insights into Pleuronectiformes genomic size and structure

Black flounder (Paralichthys orbignyanus, Pleuronectiformes) is a commercially significant marine fish with promising aquaculture potential in Argentina. Despite extensive studies on Black flounder aquaculture, its limited genetic information available hampers the crucial role genetics plays in the development of this activity. In this study, we first employed Illumina sequencing technology to sequence the entire genome of Black flounder. Utilizing two independent libraries—one from a female and another from a male—with 150 bp paired-end reads, a mean insert length of 350 bp, and over 35 X-fold coverage, we achieved assemblies resulting in a genome size of ~ 538 Mbp. Analysis of the assemblies revealed that more than 98% of the core genes were present, with more than 78% of them having more than 50% coverage. This indicates a somehow complete and accurate genome at the coding sequence level. This genome contains 25,231 protein-coding genes, 445 tRNAs, 3 rRNAs, and more than 1,500 non-coding RNAs of other types. Black flounder, along with pufferfishes, seahorses, pipefishes, and anabantid fish, displays a smaller genome compared to most other teleost groups. In vertebrates, the number of transposable elements (TEs) is often correlated with genome size. However, it remains unclear whether the sizes of introns and exons also play a role in determining genome size. Hence, to elucidate the potential factors contributing to this reduced genome size, we conducted a comparative genomic analysis between Black flounder and other teleost orders to determine if the small genomic size could be explained by repetitive elements or gene features, including the whole genome genes and introns sizes. We show that the smaller genome size of flounders can be attributed to several factors, including changes in the number of repetitive elements, and decreased gene size, particularly due to lower amount of very large and small introns. Thus, these components appear to be involved in the genome reduction in Black flounder. Despite these insights, the full implications and potential benefits of genome reduction in Black flounder for reproduction and aquaculture remain incompletely understood, necessitating further research.

Phylogenomic analysis of Wolbachia genomes from the Darwin Tree of Life biodiversity genomics project.

The Darwin Tree of Life (DToL) project aims to sequence all described terrestrial and aquatic eukaryotic species found in Britain and Ireland. Reference genome sequences are generated from single individuals for each target species. In addition to the target genome, sequenced samples often contain genetic material from microbiomes, endosymbionts, parasites, and other cobionts. Wolbachia endosymbiotic bacteria are found in a diversity of terrestrial arthropods and nematodes, with supergroups A and B the most common in insects. We identified and assembled 110 complete Wolbachia genomes from 93 host species spanning 92 families by filtering data from 368 insect species generated by the DToL project. From 15 infected species, we assembled more than one Wolbachia genome, including cases where individuals carried simultaneous supergroup A and B infections. Different insect orders had distinct patterns of infection, with Lepidopteran hosts mostly infected with supergroup B, while infections in Diptera and Hymenoptera were dominated by A-type Wolbachia. Other than these large-scale order-level associations, host and Wolbachia phylogenies revealed no (or very limited) cophylogeny. This points to the occurrence of frequent host switching events, including between insect orders, in the evolutionary history of the Wolbachia pandemic. While supergroup A and B genomes had distinct GC% and GC skew, and B genomes had a larger core gene set and tended to be longer, it was the abundance of copies of bacteriophage WO who was a strong determinant of Wolbachia genome size. Mining raw genome data generated for reference genome assemblies is a robust way of identifying and analysing cobiont genomes and giving greater ecological context for their hosts.

100 Years of Chromosome Research in Rye, Secale L.

Although microscopy and genetics were still in their infancy, there are cytological results produced a hundred years ago that are still relevant today. Since the 1920s, rye has been a subject of chromosome research. It started by plotting its mitotic and meiotic chromosomes to determine genome size. After controversial evidence, it became clear that the base number is n = 7. However, structural differences exist between species within the genus Secale. Some rye populations even carry accessory chromosomes evolutionary derived from the A genome. The development of tetraploid strains significantly promoted chromosome analysis. Various techniques have tried to stabilize the disturbed chromosome pairing of the induced tetraploids. Although slight improvements could be achieved, they did not lead to a breakthrough. However, the various aneuploid derivatives of the polyploids found major advances in the genetic analysis of rye. Trisomics, telo-trisomics, and reciprocal translocation have served as important tools for gene mapping. Since the 1970s, various chromosome banding techniques have stimulated scientific progress. The seven haploid chromosomes could be diagnosed unequivocally, not only in S. cereale but also in related species. These findings led to a clear homoeologous assignment to the genomes of related grass species such as wheat, barley, rice, etc. Current applications of in situ fluorescence staining methods, such as GISH and FISH, allow even more precise results, depending on the specificity of the DNA samples. Advanced preparation techniques are supplemented by the variety of innovations in the field of molecular genome analysis. They replace complex cytological examinations. In this way, introgressions can be safely detected by DNA markers and be much more detailed. In addition, CRISPR/CAS-mediated chromosome engineering will become an important method of the future.

Chromosome and ploidy analysis of winter hardy Hibiscus species by FISH and flow cytometry

Determination of nuclear DNA content, genome size, and ploidy level and, information on cytogenetic characteristics are all prerequisite of modern plant breeding. However, identification of individual chromosomes of Hibiscus species is extremely difficult due to high number, small size and similar shape of mitotic chromosomes. The goal of the study was to ascertain the chromosome number, karyomorphology, distribution of 5S and 18S rDNA signals, chromosome length, and centromere positions as well as the ploidy level, genome sizes, 2C - DNA content of winter-hardy Hibiscus (H. syriacus ‘Saejamyung’, H. sinosyriacus ‘Seobong’, H. moscheutos ‘Luna Red’ and, H. paramutabilis). 5S rDNA and 18S rDNA signals were detected by fluorescence in situ hybridization (FISH). According to the FISH results, there are two 5S rDNA signals (green) in H. syriacus, H. sinosyriacus, and H. moscheutos, and four 5S rDNA signals in H. paramutabilis. The range in length of somatic chromosomes in H. syriacus, H. sinosyriacus, H. moscheutos, and H. paramutabilis is 2.66–7.06, 3.18–7.31, 2.91–5.23, and 4.75–7.60, respectively. The 2C - DNA content of H. syriacus, H. sinosyriacus, and H. paramutabilis are very similar, the amount was 4.06, 4.11, and 4.18 pg, respectively whereas, H. moscheutos has nearly half and that amount was 2.06 pg. These findings will contribute to the detailed cytogenetic assessment of Hibiscus and thus benefit plant breeding in this genus.

Genome size distributions in bacteria and archaea are strongly linked to evolutionary history at broad phylogenetic scales.

The evolutionary forces that determine genome size in bacteria and archaea have been the subject of intense debate over the last few decades. Although the preferential loss of genes observed in prokaryotes is explained through the deletional bias, factors promoting and preventing the fixation of such gene losses often remain unclear. Importantly, statistical analyses on this topic typically do not consider the potential bias introduced by the shared ancestry of many lineages, which is critical when using species as data points because of the potential dependence on residuals. In this study, we investigated the genome size distributions across a broad diversity of bacteria and archaea to evaluate if this trait is phylogenetically conserved at broad phylogenetic scales. After model fit, Pagel’s lambda indicated a strong phylogenetic signal in genome size data, suggesting that the diversification of this trait is influenced by shared evolutionary histories. We used a phylogenetic generalized least-squares analysis (PGLS) to test whether phylogeny influences the predictability of genome size from dN/dS ratios and 16S copy number, two variables that have been previously linked to genome size. These results confirm that failure to account for evolutionary history can lead to biased interpretations of genome size predictors. Overall, our results indicate that although bacteria and archaea can rapidly gain and lose genetic material through gene transfers and deletions, respectively, phylogenetic signal for genome size distributions can still be recovered at broad phylogenetic scales that should be taken into account when inferring the drivers of genome size evolution.

Genome size, ploidy levels, and development of novel SSR primer to evaluate genetic diversity of Corylopsis Siebold & Zucc. germplasm collections

Corylopsis Seibold & Zucc. is a genus of flowering shrubs with few cultivars and little information on genome size, ploidy levels, and genetic relationships among taxa. The United States National Arboretum and collaborating gardens in North America maintain a diverse germplasm collection of Corylopsis including cultivated and wild-collected taxa. The objectives of this study were to determine genome size, ploidy, and genetic diversity in Corylopsis. Flow cytometry revealed a ploidy series of diploid, tetraploid, pentaploid, and hexaploid taxa in Corylopsis with 2C genome sizes ranging from 1.9 pg to 5.7 pg. Representative taxa were selected for root tip cytology, revealing diploid (C. platypetala Rehder & E.H. Wilson), tetraploid (C. gotoana Makino), pentaploid (unnamed hybrid selection), and hexaploid (C. willmottiae Rehder & E.H. Wilson) taxa. Seventeen genomic simple sequence repeat markers were developed and used to determine the genetic diversity of Corylopsis. These markers had an average of 7.4 alleles per locus with a range of three to 17 alleles. A dendrogram was generated by unweighted pair group method with arithmetic mean (UPGMA) cluster analysis using the Jaccard similarity coefficient to visualize genetic relationships and ploidy series within taxa. Information gained from this study will further breeding efforts and aid management of germplasm collections of Corylopsis.

Genome Size, Cytotype Diversity and Reproductive Mode Variation of Cotoneaster integerrimus (Rosaceae) from the Balkans.

Cotoneaster integerrimus represents a multiploid and facultative apomictic system of widely distributed mountain populations. We used flow cytometry to determine genome size, ploidy level, and reproduction mode variation of the Balkan populations, supplemented by analysis of nuclear microsatellites in order to address: (i) geographic distribution and variation of cytotypes among the populations; (ii) variation of reproduction mode and the frequency of sexuality; (iii) pathways of endosperm formation among the sampled polyploids and their endosperm balance requirements; (iv) genotypic diversity and geographic distribution of clonal lineages of polyploids. The prevalence of apomictic tetraploid cytotype followed by sexual diploids and extremely rare triploids was demonstrated. This prevalence of tetraploids affected the populations’ structure composed from clonal genotypes with varying proportions. The co-occurrence of diploids and tetraploids generated higher cytotype, reproductive mode, and genotypic diversity, but mixed-ploidy sites were extremely rare. The endosperm imbalance facilitates the development and the occurrence of intermediate triploids in mixed-ploidy populations, but also different tetraploid lineages elsewhere with unbalanced endosperm. All these results showed that the South European populations of C. integerrimus have higher levels of cytotype and reproductive diversity compared to the Central European ones. Therefore, the South European populations can be considered as a potential reservoir of regional and global diversity for this species.

Determination of Genetic Distance, Genome Size and Chromosome Numbers to Support Breeding in Ornamental Lavandula Species

Knowledge of phylogenetic relatedness and cytogenetic characteristics can facilitate breeding programs and interspecific hybridization in ornamentals. In this study genetic relationships, genome sizes and chromosome numbers were determined in a collection of 82 lavender genotypes, including 15 hybrids. Amplified Fragment Length Polymorphism (AFLP) marker analysis revealed 5 clusters, corresponding to the sections Lavandula, Stoechas, Dentatae, Pterostoechas and Subnudae. Genome sizes varied between 0.76 ± 0.02 pg 2C−1 and 4.80 ± 0.06 pg 2C−1 and chromosome numbers ranged from 2n = 22 to 2n = 100. Genome sizes of hybrids were intermediate to the progenitors genome sizes. All analyzed hybrid genotypes were characterized by even chromosome numbers, with 2n = 48 or 50. Using Principal Coordinate Analysis (PCoA) separate hybrid clusters were identified between the progenitor species clusters. Polyploidy and/or aneuploidy was observed in two hybrids genotypes: L. × intermedia ‘Heavenly Angel’ (2n = 100) and L. × heterophylla ‘Big Boy James’ (2n = 66). Results obtained in this study can be used to predict cross compatibility among different species, thus enabling targeted breeding towards the creation of new improved Lavandula cultivars.

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

Genome size variation and karyotype diversity in eight taxa of Sorbus sensu stricto (Rosaceae) from China.

Eight taxa of Sorbus Linnaeus, 1753 sensu stricto (Rosaceae) from China have been studied karyologically through chromosome counting, chromosomal measurement and karyotype symmetry. Genome size was also estimated by flow cytometry. Six taxa, S. amabilis Cheng ex T.T.Yu et K.C.Kuan, 1963, S. hupehensis var. paucijuga (D.K. Zang et P.C. Huang, 1992) L.T. Lu, 2000, S. koehneana C.K. Schneider, 1906, S. pohuashanensis (Hance, 1875) Hedlund, 1901, S. scalaris Koehne, 1913 and S. wilsoniana C.K. Schneider, 1906 are diploids with 2n = 34, whereas two taxa, S. filipes Handel-Mazzetti,1933 and S. ovalis McAllister, 2005 are tetraploid with 2n = 68. In general, the chromosome size is mainly small, and karyotypes are symmetrical with predominance of metacentric chromosomes. Genome size variation of diploids and tetraploids is 1.401 pg –1.676 pg and 2.674 pg –2.684 pg, respectively. Chromosome numbers of S. amabilis and S. hupehensis var. paucijuga, and karyotype and genome size of eight taxa studied are reported for the first time. This study emphasised the reliability of flow cytometry in genome size determination to infer ploidy levels in Chinese native Sorbus species.

Genome size variation and karyotype diversity in eight taxa of Sorbus sensu stricto (Rosaceae) from China

Eight taxa of Sorbus Linnaeus, 1753 sensu stricto (Rosaceae) from China have been studied karyologically through chromosome counting, chromosomal measurement and karyotype symmetry. Genome size was also estimated by flow cytometry. Six taxa, S. amabilis Cheng ex T.T.Yu et K.C.Kuan, 1963, S. hupehensis var. paucijuga (D.K. Zang et P.C. Huang, 1992) L.T. Lu, 2000, S. koehneana C.K. Schneider, 1906, S. pohuashanensis (Hance, 1875) Hedlund, 1901, S. scalaris Koehne, 1913 and S. wilsoniana C.K. Schneider, 1906 are diploids with 2n = 34, whereas two taxa, S. filipes Handel-Mazzetti,1933 and S. ovalis McAllister, 2005 are tetraploid with 2n = 68. In general, the chromosome size is mainly small, and karyotypes are symmetrical with predominance of metacentric chromosomes. Genome size variation of diploids and tetraploids is 1.401 pg –1.676 pg and 2.674 pg –2.684 pg, respectively. Chromosome numbers of S. amabilis and S. hupehensis var. paucijuga, and karyotype and genome size of eight taxa studied are reported for the first time. This study emphasised the reliability of flow cytometry in genome size determination to infer ploidy levels in Chinese native Sorbus species.

First report on genome size and ploidy determination of five indigenous coffee species using flow cytometry and stomatal analysis

The nuclear 2C DNA content and ploidy level determination of five indigenous coffee species from India was carried out using flow cytometry and by assessing the stomatal characteristics. The nuclear DNA content (2C/pg) analyzed varied from 1.29 pg in Coffea wightiana Wall. ex Wight & Arn. to 1.48 pg in Coffea jenkinsii Hook.f. Based on the genome size, all five species were divided into two groups. The first group comprises Coffea travancorensis Wight & Arn and C. wightiana with lower genome size (2C DNA 1.29–1.30 pg), and the second group consists of Coffea bengalensis Roxb.ex Schult, Coffea khasiana (Korth.) Hook.f and C. jenkinsii with larger genome size (2C DNA 1.45–1.48 pg). The species included in the first group were inhabited in dry environmental conditions in contrast to the species included in the second group which was predominantly from mesic habitat. Among the stomatal characteristics, no significant variation in stomatal density was observed among the species, although stomatal guard cell length and stomatal chloroplast number recorded significant variation among some species. Based on flow cytometry and stomatal characteristics, all five endangered coffee species analyzed in the study were identified as diploids. The study revealed that stomatal guard cell length and stomatal chloroplast number could be fast, inexpensive, and reliable methods for determining the ploidy status of indigenous coffee species.

Do pentaploid hybrids mediate gene flow between tetraploid Senecio disjunctus and hexaploid S. carniolicus s. str. (S. carniolicus aggregate, Asteraceae)?

Polyploids with odd-ploidy levels may play an important evolutionary role as they enable backcrosses with their parental cytotypes. We chose the ploidy-variable Senecio carniolicus species group to address the overarching question if pentaploid individuals, which occur at high frequencies in immediate contact zones of parental tetraploids and hexaploids, mediate effective interploidy gene flow. We employed a set of approaches including relative genome size (RGS) determination and viability tests of pollen, RGS of the embryo, viability tests and germination experiments of seeds, and RGS and survival experiments of seedlings to tackle the following specific questions. Is pollen of pentaploid individuals viable suggesting regular meiosis in spite of the odd-ploidy level? Are the five sets of chromosomes split into 2.5 compliments, or rather into two and three compliments? Do crossings between pentaploid individuals produce viable seeds and seedlings, suggesting that pentaploids are an independent evolutionary unit? We found that pollen stainability is not strongly reduced in pentaploids and that the five chromosome complements are most often split into 2.5 complements. Seeds originating from homoploid crossings of pentaploids show a very low viability whereas backcrosses with tetraploids or hexaploids were more successful. No significant difference in seedling survival was observed among the different crossings but seedlings emerging from 5x5x crosses were significantly smaller than both, seedlings from backcrosses and from homoploid parental crosses. Altogether, our results indicate that pentaploids may play an important evolutionary role as mediators of gene flow but that they rather do not form an independent evolutionary unit.

Selected perennial plants do provide convenient standards for the determination of genome sizes with flow cytometry

Flow cytometry delivers a key value to detect differences between species and hybrids and to determine ploidy. In addition, DNA weight per nucleus appears to be related to various properties, such as the size of the cell and the nucleus, the duration of mitosis and meiosis and the generation time. The variation in DNA weight is also useful to analyze biodiversity, genome evolution and relationships between related taxa and recognizing ploidy-chimeras. Moreover, it is essential to know the size of a genome before one starts to determine the base sequences of DNA of a plant. To be able to measure the amount of DNA per nucleus, a standard that is genetically stable with constant genome size, easy to use and commonly available is recommended for flow cytometry. In this study, standards from selected perennial plants instead of annuals are recommended as these are more stable than individual seedlings and don’t need greenhouse facilities. Four different methods are used here to determine genome sizes or 2C-value (the amount of DNA per nucleus) for the standard values of perennial plants: 1. Eight selected standards are measured separately against four primary standards 2. Starting with the lowest two standards, each result is used cascade-wise as a standard for the next plant. 3/4. Combining six to nine standard plants in a single chopping event: 3. Calculating all standards against a single standard and 4. Calculate all plants cascade-wise. The last combination 3/4 was also used to compare the results from buffers of Bharathan, Galbraith, Otto and Doležel. The aim of this study was to present selected perennial plants to be useful in flow cytometry. Selected perennial plants have several advantages like permanent availability, surviving for weeks without watering and the absence of variation as is possible in seedlings of annuals. The obtained 2C-values of perennial plants, ranging from 2.7 to 70.5 pg, are presented as a valuable tool in the determination of genome sizes. Agave americana’Aureomarginata’ is presented as the preferred reference standard.

Gigantic Genomes Provide Empirical Tests of Transposable Element Dynamics Models

Transposable elements (TEs) are a major determinant of eukaryotic genome size. The collective properties of a genomic TE community reveal the history of TE/host evolutionary dynamics and impact present-day host structure and function, from genome to organism levels. In rare cases, TE community/genome size has greatly expanded in animals, associated with increased cell size and changes to anatomy and physiology. Here, we characterize the TE landscape of the genome and transcriptome in an amphibian with a giant genome — the caecilianIchthyophis bannanicus, which we show has a genome size of 12.2 Gb. Amphibians are an important model system because the clade includes independent cases of genomic gigantism. The I. bannanicus genome differs compositionally from other giant amphibian genomes, but shares a low rate of ectopic recombination-mediated deletion. We examine TE activity using expression and divergence plots; TEs account for 15% of somatic transcription, and most superfamilies appear active. We quantify TE diversity in the caecilian, as well as other vertebrates with a range of genome sizes, using diversity indices commonly applied in community ecology. We synthesize previous models that integrate TE abundance, diversity, and activity, and test whether the caecilian meets model predictions for genomes with high TE abundance. We propose thorough, consistent characterization of TEs to strengthen future comparative analyses. Such analyses will ultimately be required to reveal whether the divergent TE assemblages found across convergent gigantic genomes reflect fundamental shared features of TE/host genome evolutionary dynamics.

Evaluation of Genome Size, Chromosome Number and Karyotype in <i>Zygophyllum xanthoxylon</i>

Zygophyllum xanthoxylon, a super-xerophytic shrub in drylands, is widely used for afforestation in the arid and barren mountains of central Asia. Understanding the karyotype and genome size could provide basic information for genome sequencing of species. To date, few data on the DNA content and chromosomal characterization of Z. xanthoxylon have been reported. Here, we present both the karyotype analysis and genome size determination of Z. xanthoxylon based on the traditional pressing and flow cytometry methods. Chromosome counting showed that Z. xanthoxylon is diploid with a chromosome number of 22. Karyotype analysis revealed that the length of chromosomes ranges from 0.88 ± 0.08 μm to 2.36 ± 0.19 μm, the chromosomes are metacentric or submetacentric, and the karyotype formula is 2n = 2x = 22 = 18m + 4sm. Flow cytometry analysis estimated that the nuclear genome size of Z. xanthoxylon is 460 ± 7.05 Mbp. Interestingly, our results indicated the seedlings of Z. xanthoxylon exhibit endopolyploidy, which may confer better ecological adaptation. Collectively, the present study will provide an important cytological basis for the study of the origin, evolution and utilization of Z. xanthoxylon.

Development and quality of pollen in Lachenalia cultivars with determination of genome size and chromosome number

The genus Lachenalia J. Jacq. ex Js. Murray (Asparagaceae) comprises 136 species endemic to South Africa and Namibia. These bulbous geophytes are considered to have excellent horticultural potential due to their morphological and genetic diversity, and thus have been chosen for the initiation of a breeding program to develop new cultivars that suitable for commercial use. From breeding perspective, studies on pollen viability and morphology are of great importance, since pollen quality represents an essential factor that influences crossbreeding processes. Therefore, to comprehensively characterize the pollen of selected Lachenalia cultivars, we conducted a multi-level analysis in which we investigated the processes of microsporogenesis and pollen grain development, as well as evaluated pollen morphology and viability. We also performed somatic chromosome counts and flow cytometry, thereby providing, for the first time, valuable cytogenetic data on the chromosome numbers and nuclear DNA contents of these cultivars. Morphologically, all the pollen grains were classified as medium-sized, subspheroidal in shape, and monosulcate with a single elongated aperture. Their exine ornamentation was categorized as reticulate with a heterobrochate reticulum. The pollen viability analysis revealed one of the cultivars to be nearly completely sterile, however, we were able to explain this phenomenon and found that this cultivar was affected by meiotic abnormalities associated with its triploid nature. Based on the obtained results, three of the studied cultivars may be considered as suitable pollinizers. From the perspective of applied research, the findings of our research offer important information for selecting appropriate donors for cross-pollination in the development of new Lachenalia cultivars.

Nuclear Genome Size Determination Of Christia Vespertilionis Via Flow Cytometry

Abstract Christia vespertilionis (butterfly wing plant) is an ornamental plant originated from South East Asia with reported usage in traditional medicine practice and potential as an anticancer and antitumor. This research aims to estimate the genome size of C. vespertilionis via flow cytometry (FCM) method. The research was conducted with the optimisation of nuclear suspension preparation followed by the genome size estimation. Two chopping techniques [manual chopping (MC) and BDTM Medimachine (MM)] and two lysis buffers (Otto and LBO1) were tested. Otto buffer with manual chopping was found to be the most suitable method, generated fine DNA peak with minimum debris background, and coefficient of variation (CV) value less than 3%. Five replicates of the FCM analysis were made for the genome size determination. The estimated genome size of C. vespertilionis was found to be 3.22 pg by using Glycine max cv. Polanka (2C=2.5pg) as an external reference standard. Further comparison with other Christia species was not possible due to the lack of data on genome size. The genome size data of C. vespertilionis can be useful for future morphology and genetics studies of Christia species.

Improved reliability in production of maize inbred lines by the combination of the R1-navajo marker with flow cytometry or microsatellite genotyping

Doubled haploid (DH) technology is an essential component in producing inbred lines for a competitive maize (Zea mays L.) breeding program. The R1-navajo (R1-nj) gene provides phenotypic marker that insures only variable reliability for seed selection of haploid embryos. Therefore, in the present study we outline a complex protocol for early stage genome size determination that integrates the phenotypic screening with the flow cytometry of nuclei from root tips and with the use of DNA isolated from seedlings for molecular marker-based genotyping. In a representative experiment with three genotypes, only 59% of the color marker pre-selected seeds were confirmed to be haploid by cytometric analysis of nuclei isolated from root tips. As a novel tool we have identified the UMC1152 SSR marker being polymorphic between the haploid inducer line (K405) and the K4390 hybrid as parents to screen seedlings pre-selected with the R1-navajo marker. Using this molecular marker, alleles characteristic for the inducer K405 line could not be detected in 83% of seedlings previously selected as haploid candidate. Seedlings identified as haploids were exposed to 0.06% colchicine solution for rediploidization. This procedure resulted in doubled haploids with 3% frequency relative to the initial population as it was quantified by the number of mature maize plants with fertile tassel. The described complex approach can support safer identification of haploids at early seedling stage in a hybrid population derived from crossing with a haploid inducer line.