FISH Karyotype Analysis of Four Wild Cucurbitaceae Species Using 5S and 45S rDNA Probes and the Emergence of New Polyploids in Trichosanthes kirilowii Maxim

Waminal, Nomar Espinosa,Kim, Hyun Hee Korean Society of Horticultural Science 2015 원예과학기술지 Vol.33 No.6

Wild relative species of domesticated crops are useful genetic resources for improving agronomic traits. Cytogenetic investigations based on chromosome composition provide insight into basic genetic and genomic characteristics of a species that can be exploited in a breeding program. Here, we used FISH analysis to characterize the ploidy level, chromosome constitution, and genomic distribution o f 5S and 4 5S r ibosomal DNA (rDNA) in four wild Cucurbitaceae species, namely, Citrullus lanatus (Thunb.) Mansf. var. citroides L. H. Bailey (2n = 22), Melothria japonica Maxim. (2n = 22), Sicyos angulatus L. (2n = 24), and Trichosanthes kirilowii Maxim. (2n = 66, 88, 110 cytotypes), collected in different areas of Korea. All species were diploids, except for T. kirilowii, which included hexa-, octa-, and decaploid cytotypes (2n = 6x = 66, 8x = 88, and 10x = 110). All species have small metaphase chromosomes in the range of $2-5{\mu}m$. The 45S rDNA signals were localized distally compared to the 5S rDNA. C. lanatus var. citroides and M. japonica showed one and two loci of 45S and 5S rDNA, respectively, with co-localization of rDNA signals in one M. japonica chromosome. S. angulatus showed two co-localized signals of 5S and 45S rDNA loci. The hexaploid T. kirilowii cytotype showed five signals each for 45S and 5S rDNA, with three being co-localized. This is the first report of hexaploid and decaploid cytotypes in T. kirilowii. These results will be useful in future Cucurbitaceae breeding programs.

A refined Panax ginseng karyotype based on an ultra-high copy 167-bp tandem repeat and ribosomal DNAs

Waminal, Nomar Espinosa,Choi, Hong-Il,Kim, Nam-Hoon,Jang, Woojong,Lee, Junki,Park, Jee Young,Kim, Hyun Hee,Yang, Tae-Jin The Korean Society of Ginseng 2017 Journal of Ginseng Research Vol.41 No.4

Background: Panax ginseng Meyer (Asian ginseng) has a large nuclear genome size of > 3.5 Gbp in haploid genome equivalent of 24 chromosomes. Tandem repeats (TRs) occupy significant portions of the genome in many plants and are often found in specific genomic loci, making them a valuable molecular cytogenetic tool in discriminating chromosomes. In an effort to understand the P. ginseng genome structure, we characterized an ultrahigh copy 167-bp TR (Pg167TR) and explored its chromosomal distribution as well as its utility for chromosome identification. Methods: Polymerase chain reaction amplicons of Pg167TR were labeled, along with 5S and 45S rDNA amplicons, using a direct nick-translation method. Direct fluorescence in situ hybridization (FISH) was used to analyze the chromosomal distribution of Pg167TR. Results: Recently, we reported a method of karyotyping the 24 chromosome pairs of P. ginseng using rDNA and DAPI (4',6-diamidino-2-phenylindole) bands. Here, a unique distribution of Pg167TR in all 24 P. ginseng chromosomes was observed, allowing easy identification of individual homologous chromosomes. Additionally, direct labeling of 5S and 45S rDNA probes allowed the identification of two additional 5S rDNA loci not previously reported, enabling the refinement of the P. ginseng karyotype. Conclusion: Identification of individual P. ginseng chromosomes was achieved using Pg167TR-FISH. Chromosome identification is important in understanding the P. ginseng genome structure, and our method will be useful for future integration of genetic linkage maps and genome scaffold anchoring. Additionally, it is a good tool for comparative studies with related species in efforts to understand the evolution of P. ginseng.

A refined Panax ginseng karyotype based on an ultra-high copy 167-bp tandem repeat and ribosomal DNAs

Nomar Espinosa Waminal,Hong-Il Choi,Nam-Hoon Kim,Woojong Jang,Junki Lee,Jee Young Park,Hyun Hee Kim,Tae-Jin Yang 고려인삼학회 2017 Journal of Ginseng Research Vol.41 No.4

Background: Panax ginseng Meyer (Asian ginseng) has a large nuclear genome size of > 3.5 Gbp in haploid genome equivalent of 24 chromosomes. Tandem repeats (TRs) occupy significant portions of the genome in many plants and are often found in specific genomic loci, making them a valuable molecular cytogenetic tool in discriminating chromosomes. In an effort to understand the P. ginseng genome structure, we characterized an ultrahigh copy 167-bp TR (Pg167TR) and explored its chromosomal distribution as well as its utility for chromosome identification. Methods: Polymerase chain reaction amplicons of Pg167TR were labeled, along with 5S and 45S rDNA amplicons, using a direct nick-translation method. Direct fluorescence in situ hybridization (FISH) was used to analyze the chromosomal distribution of Pg167TR. Results: Recently, we reported a method of karyotyping the 24 chromosome pairs of P. ginseng using rDNA and DAPI (40,6-diamidino-2-phenylindole) bands. Here, a unique distribution of Pg167TR in all 24P. ginseng chromosomes was observed, allowing easy identification of individual homologous chromosomes. Additionally, direct labeling of 5S and 45S rDNA probes allowed the identification of two additional 5S rDNA loci not previously reported, enabling the refinement of the P. ginseng karyotype. Conclusion: Identification of individual P. ginseng chromosomes was achieved using Pg167TR-FISH. Chromosome identification is important in understanding the P. ginseng genome structure, and our method will be useful for future integration of genetic linkage maps and genome scaffold anchoring. Additionally, it is a good tool for comparative studies with related species in efforts to understand the evolution of P. ginseng.

Five-color fl uorescence in situ hybridization system for karyotyping of Panax ginseng

Nomar Espinosa Waminal,Tae-Jin Yang,인준교,HyunHeeKim 한국원예학회 2020 Horticulture, Environment, and Biotechnology Vol.61 No.5

Chromosomal mapping of repetitive elements is invaluable in understanding genome structure and evolution. Repetitive elementsconstitute ~ 80% of the allotetraploid ginseng ( Panax ginseng ) genome. Preparing sporophytic metaphase chromosomesof ginseng is laborious; therefore, it would be advantageous to maximize the information obtained from a single slide. Here,we investigated the suitability of simultaneous fi ve-color fl uorescence in situ hybridization using major ginseng repeats,namely PgDel1 , PgDel2 , PgTel, and Pg167TR. For Pg167TR, we generated two degenerate probe libraries (Pg167TRa andPg167TRb) based on the chromosomal target coverage. We labeled the probes with dark-red, blue, red, orange, and greenfl uorochromes and used excitation/emission fi lter sets specifi c to each fl uorochrome to detect fl uorescence in situ hybridizationsignals. PgDel1 was distributed across all 24 chromosome pairs, except for the secondary constriction region of chromosome16, whereas PgDel2 was distributed over 12 of the 24 pairs. PgTel was localized in the termini of chromosomesand in an intercalary region in chromosome 13. Pg167TRa and Pg167TRb were distributed among 22 chromosome pairswith loci polymorphisms. These results showed the utility of fi ve-color fl uorescence in situ hybridization for chromosomalmapping of fi ve repeats to expedite karyotyping and facilitate genome evolution studies in ginseng and other plant species.

Cytogenetic mapping of Panax ginseng major DNA repeats: Evidence for allotetraploidy and utility for chromosome identification

Nomar Espinosa Waminal,Hong-Il Choi,Hyun Hee Kim,Tae-Jin Yang 한국육종학회 2014 한국육종학회 심포지엄 Vol.2014 No.07

There is a growing number of plant genomes that are being sequenced, but most of these available assemblies do not cover the entire genome mainly due to the highly repetitive sequences found in most plant genomes. Nevertheless, these repeats, although a challenge in assembly algorithms, provide relevant information about a genome’s history that could help explain its structure and complexity. Here, we cytogenetically mapped previously and presently characterized major repeats of Panax ginseng genome, including several LTR retrotransposons (PgDel2, PgDel3, PgTat1, PgTat2, PgTork) and one tandem repeat, PgTR Fluorescence in situ hybridization (FISH) results showed differential accumulation of Ty3/gypsy LTR retrotransposons into different chromosomal regions or subgenomes, suggesting a non-random preferential amplification of retrotransposons in these regions and an allopolyploid origin of P. ginseng. In silico analysis based on 1x whole genome sequence reads suggests that PgTR is the most abundant tandem repeat in ginseng, which was further corroborated by FISH analysis. More importantly, its unique distribution pattern among the 24 ginseng chromosomes, coupled with the non-random distribution of LTR retrotransposons and rDNA arrays, allowed us to discriminate and characterize each individual ginseng chromosome. These different newly characterized cytogenetic markers allowed reorganization of previously reported ginseng karyotype with better resolution, demonstrating the irutility in ginseng chromosome identification. These information give us insight about the genomic structure of P. ginseng, and should be useful for future comparative cytogenetics studies among closely related species to unravel its genomic history. This work was supported by the Next-Generation BioGreen21 Program (No. PJ008202), Rural Development Administration, Republic of Korea.

Karyotypic Analyses of Six Horticultural Species by DAPI Staining and Fluorescence in Situ Hybridization Methods

Nomar Espinosa Waminal,Shin Jae Kang,Jee Hee Lee,Seong Han Sohn,Do Sun Kim,Jang-Ho Hahn,Dong Hern Kim,Yoon Jung Hwang,Hyun Hee Kim 한국원예학회 2014 한국원예학회 학술발표요지 Vol.2014 No.10

Phylogeny of Cucurbitaceae species in Korea based on 5S rDNA non-transcribed spacer

Nomar Espinosa Waminal,김현희,류광복,박보름 한국유전학회 2014 Genes & Genomics Vol.36 No.1

The 5S rDNA coding region and its spacer havebeen successfully utilized in phylogenetic studies of plants. However, it has not been utilized in the phylogeneticanalysis of Cucurbitaceae. Here, we obtained the 5S rDNAsequences of 12 Cucurbitaceae species by direct PCR orcloning. The 5S rDNA sequences ranged from 275 to359 bp, and the coding regions of all species were 120 bplong, except for that of Cucurbita, which was 119 bp. Some genus-specific SNPs were observed in the codingregions of Cucurbita, Lagenaria, Melothria, and Tricosanthes. The GC content of the coding regions was generallyhigher than that of the NTS regions, and thedifference in GC content between the coding and NTSregions varied among species, with Gynostemma pentaphyllumhaving the greatest difference of 20.3. The phylogenetictrees generated using maximum parsimony andmaximum likelihood were congruent and well supportedby the recently published classification of Cucurbitaceae. These results demonstrated the utility of the 5S rDNAsequence in inferring phylogenetic relationships among 12Cucurbitaceae species, and its utility could be extended byusing a greater number of species in future studies.

Repeat Evolution in Brassica rapa (AA), B. oleracea (CC), and B. napus (AACC) Genomes

( Nomar Espinosa Waminal ),( Sampath Perumal ),( Jonghoon Lee ),( Hyun Hee Kim ),( Tae Jin Yang ) 한국육종학회 2016 Plant Breeding and Biotechnology Vol.4 No.2

The genus Brassica is an important resource for major agricultural products such as oils, vegetable and fodder. The Brassiceae tribe-specific whole-genome triplication that occurred ~15.9 million years ago influenced the speciation and morphological diversification that has been exploited in agriculture, making Brassica an excellent model system for studying polyploidization- mediated evolution. Genome sequencing and comparative genome analysis have revealed conserved structures and uncovered the genome evolution of Brassica species. While chromosome shuffling and asymmetric subgenome gene retention are widely reported in Brassica species, limited information is available about the dynamics of repetitive elements (REs), which are central to epigenetic mechanisms and thus play a pivotal role in plant genome adaptation and evolution. The assembled reference genome sequences of B. rapa (AA) and B. oleracea (CC), and their derived allotetraploid, B. napus (AACC), cover 58%, 86%, and 75% of their respective estimated genome sizes. The remaining non-assembled genome portions vary between these three genome sequences, and the major components remain hidden in each genome. Here, we review the dynamics of the major Brassica repeats that have played roles in speciation of the AA, CC, and AACC genomes. We show that 10 major Brassica repeats appear to occupy more than 50% of each respective unassembled genome sequence, yet represent less than 1% of assembled reference genome sequences. We have estimated their genome proportions using whole-genome Illumina reads and cytogenetic analyses in an attempt to understand the role of these repeats in genome evolution.

FISH Karyotype Analyses of Four Cucurbitaceae Species Using 5S and 45S rDNA Probes

Nomar Espinosa Waminal,Bo Reum Park,Hadassah Roa Belandres,Yoon Jung Hwang,Hyun Hee Kim 한국원예학회 2014 한국원예학회 학술발표요지 Vol.2014 No.10

FISH Karyotype Analysis of Four Wild Cucurbitaceae Species Using 5S and 45S rDNA Probes and the Emergence of New Polyploids in Trichosanthes kirilowii Maxim.

Nomar Espinosa Waminal,Hyun Hee Kim 한국원예학회 2015 원예과학기술지 Vol.33 No.6

Wild relative species of domesticated crops are useful genetic resources for improving agronomic traits. Cytogenetic investigations based on chromosome composition provide insight into basic genetic and genomic characteristics of a species that can be exploited in a breeding program. Here, we used FISH analysis to characterize the ploidy level, chromosome constitution, and genomic distribution o f 5S and 4 5S r ibosomal DNA ( rDNA) in f our wild C ucurbitaceae s pecies, namely, Citrullus lanatus (Thunb.) Mansf. var. citroides L. H. Bailey (2n = 22), Melothria japonica Maxim. (2n = 22), Sicyos angulatus L. (2n = 24), and Trichosanthes kirilowii Maxim. (2n = 66, 88, 110 cytotypes), collected in different areas of Korea. All species were diploids, except for T. kirilowii, which included hexa-, octa-, and decaploid cytotypes (2n = 6x = 66, 8x = 88, and 10x = 110). All species have small metaphase chromosomes in the range of 2–5 μm. The 45S rDNA signals were localized distally compared to the 5S rDNA. C. lanatus var. citroides and M. japonica showed one and two loci of 45S and 5S rDNA, respectively, with co-localization of rDNA signals in one M. japonica chromosome. S. angulatus showed two co-localized signals of 5S and 45S rDNA loci. The hexaploid T. kirilowii cytotype showed five signals each for 45S and 5S rDNA, with three being co-localized. This is the first report of hexaploid and decaploid cytotypes in T. kirilowii. These results will be useful in future Cucurbitaceae breeding programs.