The dynamics of genome size and GC contents evolution in genus Nicotiana / A dinâmica do tamanho do genoma e a evolução dos conteúdos de GC no gênero Nicotiana

Abstract Hybridization and Polyploidization are most common of the phenomenon observed in plants, especially in the genus Nicotiana leading to the duplication of genome. Although genomic changes associated with these events has been studied at various levels but the genome size and GC content variation is less understood because of absence of sufficient genomic data. In this study the flow cytometry technique was used to uncover the genome size and GC contents of 46 Nicotiana species and we compared the genomic changes associated with the hybridization events along evolutionary time scale. The genome size among Nicotiana species varied between 3.28 pg and 11.88 pg whereas GC contents varied between 37.22% and 51.25%. The tetraploid species in genus Nicotiana including section Polydiclae, Repandae, Nicotiana, Rustica and Sauveolentes revealed both up and downsizing in their genome sizes when compared to the sum of genomes of their ancestral species. The genome sizes of three homoploid hybrids were found near their ancestral species. Loss of large genome sequence was observed in the evolutionary more aged species (>10 Myr) as compared to the recently evolved one's (<0.2 Myr). The GC contents were found homogenous with a mean difference of 2.46% among the Nicotiana species. It is concluded that genome size change appeared in either direction whereas the GC contents were found more homogenous in genus Nicotiana.

Resumo A hibridização e a poliploidização são os fenômenos mais comuns observados em plantas, principalmente no gênero Nicotiana, levando à duplicação do genoma. Embora as mudanças genômicas associadas a esses eventos tenham sido estudadas em vários níveis, o tamanho do genoma e a variação do conteúdo de GC são menos compreendidos devido à ausência de dados genômicos suficientes. Neste estudo, a técnica de citometria de fluxo foi usada para descobrir o tamanho do genoma e o conteúdo de GC de 46 espécies de Nicotiana, e comparamos as mudanças genômicas associadas aos eventos de hibridização ao longo da escala de tempo evolutiva. O tamanho do genoma entre as espécies de Nicotiana variou entre 3,28 pg e 11,88 pg, enquanto os conteúdos de GC variaram entre 37,22% e 51,25%. As espécies tetraploides do gênero Nicotiana, incluindo as seções Polydiclae, Repandae, Nicotiana, Rustica e Sauveolentes, revelaram aumento e redução do tamanho do genoma quando comparados à soma dos genomas de suas espécies ancestrais. Os tamanhos do genoma de três híbridos homoploides foram encontrados perto de suas espécies ancestrais. A perda da grande sequência do genoma foi observada nas espécies evolutivas mais velhas (> 10 Myr) em comparação com as que evoluíram recentemente (< 0,2 Myr). Os teores de GC foram homogêneos com diferença média de 2,46% entre as espécies de Nicotiana. Conclui-se que a mudança no tamanho do genoma apareceu em ambas as direções, enquanto os conteúdos de GC foram encontrados mais homogêneos no gênero Nicotiana.

The dynamics of genome size and GC contents evolution in genus Nicotiana / A dinâmica do tamanho do genoma e a evolução dos conteúdos de GC no gênero Nicotiana

Hybridization and Polyploidization are most common of the phenomenon observed in plants, especially in the genus Nicotiana leading to the duplication of genome. Although genomic changes associated with these events has been studied at various levels but the genome size and GC content variation is less understood because of absence of sufficient genomic data. In this study the flow cytometry technique was used to uncover the genome size and GC contents of 46 Nicotiana species and we compared the genomic changes associated with the hybridization events along evolutionary time scale. The genome size among Nicotiana species varied between 3.28 pg and 11.88 pg whereas GC contents varied between 37.22% and 51.25%. The tetraploid species in genus Nicotiana including section Polydiclae, Repandae, Nicotiana, Rustica and Sauveolentes revealed both up and downsizing in their genome sizes when compared to the sum of genomes of their ancestral species. The genome sizes of three homoploid hybrids were found near their ancestral species. Loss of large genome sequence was observed in the evolutionary more aged species (>10 Myr) as compared to the recently evolved one’s (<0.2 Myr). The GC contents were found homogenous with a mean difference of 2.46% among the Nicotiana species. It is concluded that genome size change appeared in either direction whereas the GC contents were found more homogenous in genus Nicotiana.

A hibridização e a poliploidização são os fenômenos mais comuns observados em plantas, principalmente no gênero Nicotiana, levando à duplicação do genoma. Embora as mudanças genômicas associadas a esses eventos tenham sido estudadas em vários níveis, o tamanho do genoma e a variação do conteúdo de GC são menos compreendidos devido à ausência de dados genômicos suficientes. Neste estudo, a técnica de citometria de fluxo foi usada para descobrir o tamanho do genoma e o conteúdo de GC de 46 espécies de Nicotiana, e comparamos as mudanças genômicas associadas aos eventos de hibridização ao longo da escala de tempo evolutiva. O tamanho do genoma entre as espécies de Nicotiana variou entre 3,28 pg e 11,88 pg, enquanto os conteúdos de GC variaramentre 37,22% e 51,25%. As espécies tetraploides do gênero Nicotiana, incluindo as seções Polydiclae, Repandae, Nicotiana, Rustica e Sauveolentes, revelaram aumento e redução do tamanho do genoma quando comparados à soma dos genomas de suas espécies ancestrais. Os tamanhos do genoma de três híbridos homoploides foram encontrados perto de suas espécies ancestrais. A perda da grande sequência do genoma foi observada nas espécies evolutivas mais velhas (> 10 Myr) em comparação com as que evoluíram recentemente (< 0,2 Myr). Os teores de GC foram homogêneos com diferença média de 2,46% entre as espécies de Nicotiana. Conclui-se que a mudança no tamanho do genoma apareceu em ambas as direções, enquanto os conteúdos de GC foram encontrados mais homogêneos no gênero Nicotiana.

The dynamics of genome size and GC contents evolution in genus Nicotiana

Abstract Hybridization and Polyploidization are most common of the phenomenon observed in plants, especially in the genus Nicotiana leading to the duplication of genome. Although genomic changes associated with these events has been studied at various levels but the genome size and GC content variation is less understood because of absence of sufficient genomic data. In this study the flow cytometry technique was used to uncover the genome size and GC contents of 46 Nicotiana species and we compared the genomic changes associated with the hybridization events along evolutionary time scale. The genome size among Nicotiana species varied between 3.28 pg and 11.88 pg whereas GC contents varied between 37.22% and 51.25%. The tetraploid species in genus Nicotiana including section Polydiclae, Repandae, Nicotiana, Rustica and Sauveolentes revealed both up and downsizing in their genome sizes when compared to the sum of genomes of their ancestral species. The genome sizes of three homoploid hybrids were found near their ancestral species. Loss of large genome sequence was observed in the evolutionary more aged species (>10 Myr) as compared to the recently evolved ones ( 0.2 Myr). The GC contents were found homogenous with a mean difference of 2.46% among the Nicotiana species. It is concluded that genome size change appeared in either direction whereas the GC contents were found more homogenous in genus Nicotiana.

Resumo A hibridização e a poliploidização são os fenômenos mais comuns observados em plantas, principalmente no gênero Nicotiana, levando à duplicação do genoma. Embora as mudanças genômicas associadas a esses eventos tenham sido estudadas em vários níveis, o tamanho do genoma e a variação do conteúdo de GC são menos compreendidos devido à ausência de dados genômicos suficientes. Neste estudo, a técnica de citometria de fluxo foi usada para descobrir o tamanho do genoma e o conteúdo de GC de 46 espécies de Nicotiana, e comparamos as mudanças genômicas associadas aos eventos de hibridização ao longo da escala de tempo evolutiva. O tamanho do genoma entre as espécies de Nicotiana variou entre 3,28 pg e 11,88 pg, enquanto os conteúdos de GC variaram entre 37,22% e 51,25%. As espécies tetraploides do gênero Nicotiana, incluindo as seções Polydiclae, Repandae, Nicotiana, Rustica e Sauveolentes, revelaram aumento e redução do tamanho do genoma quando comparados à soma dos genomas de suas espécies ancestrais. Os tamanhos do genoma de três híbridos homoploides foram encontrados perto de suas espécies ancestrais. A perda da grande sequência do genoma foi observada nas espécies evolutivas mais velhas (> 10 Myr) em comparação com as que evoluíram recentemente ( 0,2 Myr). Os teores de GC foram homogêneos com diferença média de 2,46% entre as espécies de Nicotiana. Conclui-se que a mudança no tamanho do genoma apareceu em ambas as direções, enquanto os conteúdos de GC foram encontrados mais homogêneos no gênero Nicotiana.

Genome sizes of four important medicinal species in Kadsura by flow cytometry / 中草药·英文版

Objective: Dianjixueteng is a geoherb in Yunnan Province, the source plant of which is Kadsura interior. However, the formation of this geoherb is not clear in genetic mechanism, in which genome size is the first step that should be known on the genomic level. In this study we aimed to estimate the genome sizes of source plants of K. interior and three related herbs K. heteroclita, K. longipedunculata, and K. coccinea by flow cytometry (FCM) and make a comparison. Methods: The genome sizes of K. interior, K. heteroclita, K. longipedunculata and K. coccinea, i.e., the source plants of Dianjixueteng and its relative medicinal materials, were estimated by FCM. The nuclei of K. interior were isolated using modified LB01 buffer, for the rest species, by the Galbraith's buffer. Results: The genome sizes of K. interior, K. heteroclita, K. longipedunculata, and K. coccinea were 7.36, 7.12, 7.01, and 5.15 pg/1C, respectively. Genome size of K. interior had no significant variation with those of K. heteroclita and K. longipedunculata (P = 0.296), which was significantly larger than that of K. coccinea. Conclusion: Genome size can not distinguish K. interior from K. heteroclita and K. longipedunculata, but could distinguish them from K. coccinea, which lays the foundation for future studies on genetic mechanism of the geoherb formation.

First report on DNA content of three species of Amorphophallus

The Amorphophallus genus is a perennial herb which belongs to the family Araceae. There are more than 170 species in this genus, which is widely distributed in tropical and subtropical areas. As a kind of food and medicine Amorphophallus has been used for more than 2000 years in China. Because of the high content of konjac glucomannan (KGM) and dietary fiber, it has attracted more attention worldwide. In this article, the DNA contents of A. konjac, A. albus and A. bulbifer in China, A. albus, A. paeoniifolius and A. muelleri in Indonesia were estimated by using flow cytometry. In the samples of China, the DNA contents were 12.95 ± 0.73 pg/2C in A. konjac, 10.51 ± 0.05 pg/2C in A. albus and 17.61 pg/2C in A. bulbifer, and for Indonesia, 14.16 ± 0.48 pg/2C in A. albus (flowering), 8.49 ± 0.2 pg/2C in A. paeoniifolius and 17.84 ± 1.46 pg/2C in A. muelleri were used. Interspecific variation was found significantly (P \0.01), suggesting that DNA content might be a parameter that can be used to differentiate the species. Intraspecific variation has also been found significantly (P \ 0.01), whether in the same region or between two regions. As far as we know, this is the first report on genome size estimation of the A. konjac, A. albus and A. muelleri using flow cytometry. Understanding the genome size of Amorphophallus species will help to sequence the genome and analyse the genetic diversity, evolutionary relationship and geographical variation pattern of Amorphophallus species.

Genome survey study of Alpinia katsumadai based on Illumina high throughput sequencing / 中草药

Objective: To analyze the genome survey of medicinal and edible plant Alpinia katsumadai and complete its genome genetic information. Methods: This study was based on high throughput sequencing platform Illumina, and K-mer analysis was applied to estimate the genome size and heterozygosity rate of A. katsumadai. Meanwhile, simple sequence repeat (SSR) loci that were suitable as markers were identified by MISA software. Results: The estimated genome size of A. katsumadai was 1.60 Gb, with a 0.44% heterozygosity rate and 72.72% repeats; In the genome sequence, 364 395 simple sequence repeats (SSRs) were detected by SSR molecular marker analysis, among which mono-nucleotide, di-nucleotide and tri-nucleotide repetitive motifs ranked the higher percentages of 64.25%, 24.05% and 10.31%, summed up to 98.61%; From the 350 bp library obtained by sequencing, 10 000 single-end reads were randomly selected and blasted with NT bank, the results showed that its genetically close species Alpinia zerumbet and Elettaria cardamomum were blasted with the reads of 12.89% and 12.36% in NT bank. Conclusion: The genome size, heterozygosity rate and SSR molecular marker analysis' genome survey study on A. katsumadai indicated that the genome of A. katsumadai species was a complex, highly repetitive and large genome, which provided genetic information support for the resource protection, genetic diversity analysis and variety breeding of A. katsumadai.

Genome Size Evolution Mediated by Gypsy Retrotransposons in Brassicaceae / 基因组蛋白质组与生物信息学报·英文版

The dynamic activity of transposable elements (TEs) contributes to the vast diversity of genome size and architecture among plants. Here, we examined the genomic distribution and transposition activity of long terminal repeat retrotransposons (LTR-RTs) in Arabidopsis thaliana (Ath) and three of its relatives, Arabidopsis lyrata (Aly), Eutrema salsugineum (Esa), and Schrenkiella parvula (Spa), in Brassicaceae. Our analyses revealed the distinct evolutionary dynamics of Gypsyretrotransposons, which reflects the different patterns of genome size changes of the four species over the past million years. The rate of Gypsy transposition in Aly is approximately five times more rapid than that of Ath and Esa, suggesting an expanding Aly genome. Gypsy insertions in Esa are strictly confined to pericentromeric heterochromatin and associated with dramatic centromere expansion. In contrast, Gypsy insertions in Spa have been largely suppressed over the last million years, likely as a result of a combination of an inherent molecular mechanism of preferential DNA removal and purifying selection at Gypsy elements. Additionally, species-specific clades of Gypsy elements shaped the distinct genome architectures of Aly and Esa.

Flow cytometry and K-mer analysis estimates of genome size of Sophora alopecuroides / 中草药

Objective: In this study, flow cytometry and K-mer analysis were used to estimate genome size and hybridity percentage of Sophora alopecuroides, so as to provide reference for its genome sequencing. Methods: Glycine max served as an internal reference, the multiple relationship between DNA content in Glycine max and S. alopecuroides cells was detected by flow cytometry, and the genome size of S. alopecuroides was calculated. Genome sequencing of S. alopecuroides was carried out using NGS technology, genome size and hybridity percentage of S. alopec uroides were estimated by K-mer analysis. Results: The genome size of S. alopecuroides was about 1 749 Mb measured by flow cytometry. K-mer analysis showed that the genome size of S. alopecuroides was about 1 648 Mb, the hybridity percentage was 1.12%, and the percentage of repetitive sequence was high. Conclusion: The genome size of S. alopecuroides was about 1.7 Gb with high hybridity percentage. The technology of combining Illumina and PacBio is recommended for future genome sequencing.

Estimation of genome sizes of Astragalus membranaceus based on flow cytometry and K-mer analysis / 中草药

Objective: To determinate the genome size and complexity of Astragalus membranaceus by using flow cytometry (FCM) and K-mer analysis, which can lay the foundation for the screening of functional genes of A. membranaceus. Methods Lycopersicon esculentum was served as an internal reference in this study. The mixed sample of A. membranaceus cell nucleus and L. esculentum cell nucleus was stained using propidium iodide (PI). The PI fluorescence intensities of the sample were measured by FCM. The genome size of A. membranaceus was calculated by comparing the multiple relationship between the peak of DNA content in the cells of A. membranaceus and L. esculentum. The genome of A. membranaceus was sequenced by using high-throughput sequencing technologies. The genome size of A. membranaceus was calculated by K-mer analysis. The hybridity percentage, repetitive sequence, and GC of A. membranaceus were estimated by bioinformatics analysis. Results The genome size of A. membranaceus was about 1 426 Mb. For K-mer analysis, more than 95 Gb high quality data from the genome was generated. The average genome size and sequencing coverage depth of A. membranaceus was about 1 456 Mb and 39 times respectively. The genome of A. membranaceus had obvious hybridity peak by K-mer method, and the hybridity percentage as high as 2.1%. Conclusion The genome size of A. membranaceus was about 1.45 Gb and the heterozygosity is high. These data would provide a reference for the genomic research in A. membranaceus.

Genome survey and characteristic analysis of SSR in Callicarpa nudiflora / 中国中药杂志

Callicarpa nudiflora,which is a big brand of Li nationality medicine with Hainan characteristics,has the effects of dissolving stasis,hemostasis,anti-inflammatory and antibacterial. At present,there is a lack of information about the reference genome of C. nudiflora. The study of the genome size,heterozygosity rate and characteristics of SSR of C. nudiflora,can provide an effective basis for the formulation of the whole genome de novo sequencing strategy and development of SSR molecular markers of C. nudiflora. To realize this purpose,high throughput sequencing platform Illumina Hiseq was used to sequence the genome structure of C. nudiflora and K-mer analysis was applied to estimate genome size,repeat sequences and heterozygosity rate. Simple-sequence repeat( SSR) loci that are suitable as markers were identified by MISA software. The results showed the estimated genome size of C. nudiflora was 822. 43 Mb,with a 0. 85% heterozygosity rate and 71. 67% repeats,and the GC content of genome was about 49. 20%. Therefore,C. nudiflora belongs to a complex genome with high heterozygosity and repetition. SSR molecular genetic markers were analyzed in the genome sequence,and a total of 206 049 SSRs were identified,among which mono-nucleotide,di-nucleotide and tri-nucleotide repetitive motifs summed up to 198 993,accounting for 96. 57% of the total SSRs. Among the 2-6 nucleotide repeats,AT/AT,AAT/ATT,AGCC/CTGG,AAAAT/ATTTT and AGATAT/ATATCT have the largest number,respectively. This report represents the first genome-wide characterization of C. nudiflora,and provides a reference for the construction of the library for the fine sequencing of the genome,and a molecular basis for the development of SSR molecular markers as well as for the protection and utilization of gene resources.

Estimation of Genome Size of Ginseng Based on Herbgenomics by Flow Cytometric Analysis And High-throughput Sequence / 世界科学技术-中医药现代化

Ginseng is the dried root and rhizome of Panax ginseng.The lack of genomic data has restricted the development of ginseng industry and basic research.The genome size of P.ginseng was estimated to be 3.42 Gb by using the genome data of Oryza sativa ssp.Nipponbare and Glycine max (L.) Merrill as the reference and the flow cytometric analysis.Meanwhile,shotgun libraries with the insert size of 250 bp and 500 bp were constructed,and sequenced for double terminal PE 150 by using Illumina Hiseq X Ten platform.Totally,183.82 Gb high quality data was obtained after filtering the raw data.The genome size of P.ginseng was 3.35 Gb and the sequencing depth was 54.87 X by K-mer analysis.In this study,flow cytometry and K-mer analysis were used to identify the genome size of ginseng,which provided basic data for the further whole genome sequencing and herbgenomics studies.

Estimation of Nuclear DNA Content in Tannin-rich Medicinal Plant Cornus officinalis by Flow Cytometry / 中草药·英文版

Objective: The amount of nuclear DNA (C-value) is a key biodiversity character that provides strong unifying elements in revealing the phylogenetic regularity and relationship between genome size and functional traits for plant resource. The estimation of C-values could primarily extend our knowledge on the genetic background and genome diversity for medicinal plants, and thereby the variation of pharmacological constituents and phylogenetic mechanism of medicinal plant taxa will be revealed. However, a large number of medicinal plants (e.g. Cornus officinalis) typically contain a series of secondary metabolites, especially tannic acid, which would significantly affect the estimation of DNA content by flow cytometry (FCM). Methodological discussions and improvement need to be made to solve this problem. Methods: Two isolation buffers LB01 and Otto 1 were selected to prepare nuclear suspension with additional treatments of pre-soaking and centrifugation combination of gradient centrifugal force and duration. The best isolation and estimation methods were determined by FCM measurement in C. officinalis. Results: The dry leaves were pre-soaked in Otto I buffer for 15 min and the Otto I nuclear suspension was centrifugated at 1.0103 g for 2 min. The results showed that debris and nuclei were better separated and the scatterplots of good quality were obtained with low coefficient of variation (CV). Contrarily, the nuclear DNA content of C. officinalis could not be accurately estimated for nuclei extracted by LB01 buffer. Finally, 2C-value and genome size of C. officinalis were first estimated as 5.92 pg and 2893 Mbp, respectively. Conclusion: The new methods proposed here are able to accurately estimate DNA content of C. officinalis, which provides valuable references for the estimation of genome size in other tannin-rich medicinal plants. © 2014 Tianjin Press of Chinese Herbal Medicines.

Transposable Elements and Genome Size Variations in Plants

Although the number of protein-coding genes is not highly variable between plant taxa, the DNA content in their genomes is highly variable, by as much as 2,056-fold from a 1C amount of 0.0648 pg to 132.5 pg. The mean 1C-value in plants is 2.4 pg, and genome size expansion/contraction is lineage-specific in plant taxonomy. Transposable element fractions in plant genomes are also variable, as low as ~3% in small genomes and as high as ~85% in large genomes, indicating that genome size is a linear function of transposable element content. Of the 2 classes of transposable elements, the dynamics of class 1 long terminal repeat (LTR) retrotransposons is a major contributor to the 1C value differences among plants. The activity of LTR retrotransposons is under the control of epigenetic suppressing mechanisms. Also, genome-purging mechanisms have been adopted to counter-balance the genome size amplification. With a wealth of information on whole-genome sequences in plant genomes, it was revealed that several genome-purging mechanisms have been employed, depending on plant taxa. Two genera, Lilium and Fritillaria, are known to have large genomes in angiosperms. There were twice times of concerted genome size evolutions in the family Liliaceae during the divergence of the current genera in Liliaceae. In addition to the LTR retrotransposons, non-LTR retrotransposons and satellite DNAs contributed to the huge genomes in the two genera by possible failure of genome counter-balancing mechanisms.

Detection of diploid males in a natural colony of the cleptobiotic bee Lestrimelitta sp (Hymenoptera, Apidae)

When working at quantifying the genome size of stingless bees, it was observed that males of Lestrimelitta sp possessed the same amount of nuclear DNA as the females. Thus, we used flow cytometry (FCM) and cytogenetic analysis to confirm the ploidy of these individuals. The males analyzed proved to be diploid, since, through cytometric analysis, it was demonstrated that the mean genome size of both males and females was the same (C = 0.463 pg), and, furthermore, cytogenetic analysis demonstrated that both had 2n = 28 chromosomes.

Karyotype and nuclear DNA content of hexa-, octo-, and duodecaploid lines of Bromus subgen. Ceratochloa

The subgenus Ceratochloa of the genus Bromus includes a number of closely related allopolyploid forms or species that present a difficult taxonomic problem. The present work combines data concerning chromosome length, heterochromatin distribution and nuclear genome size of different 6x, 8x and 12x accessions in this subgenus. Special attention is paid to the karyotype structure and genomic constitution of duodecaploid plants recently found in South America. Hexaploid lineages possess six almost indistinguishable genomes and a nuclear DNA content between 12.72 pg and 15.10 pg (mean 1Cx value = 2.32 pg), whereas octoploid lineages contain the same six genomes (AABBCC) plus two that are characterized by longer chromosomes and a greater DNA content (1Cx = 4.47 pg). Two duodecaploid accessions found in South America resemble each other and apparently differ from the North American duodecaploid B. arizonicus as regards chromosome size and nuclear DNA content (40.00 and 40.50 pg vs. 27.59 pg). These observations suggest that the South American duodecaploids represent a separate evolutionary lineage of the B. subgenus Ceratochloa, unrecognized heretofore.

Incongruent evolution of chromosomal size in rice

To investigate genome size evolution, it is usually informative to compare closely related species that vary dramatically in genome size. A whole genome duplication (polyploidy) that occurred in rice (Oryza sativa) about 70 million years ago has been well documented based on current genome sequencing. The presence of three distinct duplicate blocks from the polyploidy, of which one duplicated segment in a block is intact (no sequencing gap) and less than half the length of its syntenic duplicate segment, provided an excellent opportunity for elucidating the causes of their size variation during the post-polyploid time. The results indicated that incongruent patterns (shrunken, balanced and inflated) of chromosomal size evolution occurred in the three duplicate blocks, spanning over 30 Mb among chromosomes 2, 3, 6, 7, and 10, with an average of 20.3% for each. DNA sequences of chromosomes 2 and 3 appeared to had become as short as about half of their initial sequence lengths, chromosomes 6 and 7 had remained basically balanced, and chromosome 10 had become dramatically enlarged (approximately 70%). The size difference between duplicate segments of rice was mainly caused by variations in non-repetitive DNA loss. Amplification of long terminal repeat retrotransposons also played an important role. Moreover, a relationship seems to exist between the chromosomal size differences and the nonhomologous combination in corresponding regions in the rice genome. These findings help shed light on the evolutionary mechanism of genomic sequence variation after polyploidy and genome size evolution.