FISH karyotype and GISH meiosis analyses of a fertile xBrassicoraphanus and its parental lines

Hadassah Roa Belandres,Nomar Espinosa Waminalp,Yoon Jung Hwang,Soo Sung Lee,Jin Hoe Huh,Hyun Hee Kim 한국육종학회 2014 한국육종학회 심포지엄 Vol.2014 No.07

xBrassicoraphanus, a new synthetic intergeneric hybrid between Brassica rapa L. ssp. pekinensis and Raphanus sativus L., also locally known as ‘Baemoochae’, is an interesting subject for studying polyploidy and genome plasticity in the family Brassicaceae, but very few genomic and cytogenetic information. Here, we analysed the chromosome complements and pairing of the most fertile lines, BB1 and BB5, using dual-color fluorescence in situ hybridization (FISH) and genomic in situ hybridization (GISH) to check their chromosomal segregation stability. The somatic chromosome complement of B. rapa was confirmed to be 2n=20 (2.8~4.8μm), of R.sativus, 2n=18 (2.0~3.3μm), and of xBrassicoraphanus, 2n=38 (2.2~5.0μm). There were eight, eight, and seventeen metacentric pairs and two, one, and two submetacentric pairs in B. rapa, R. sativus, and xBrassicoraphanus, respectively. Additionally, three, two, and five pairs of 5S rDNA and five, three, and eight pairs of 45S rDNA were observed in B. rapa, R. sativus, and xBrassicoraphanus, respectively. This suggests that both B. rapa (AA) and R. sativus (RR) genomes, particularly the rDNA arrays, co-exist in xBrassicoraphanus (AARR) genome. In meiosis I, nineteen bivalents were most frequent, and GISH analysis showed ten bivalents from the A genome. This study would provide a useful information for further genomic study of xBrassicoraphanus and its improvement as a new promising breeding variety.

Cytogenetic Analyses of xBrassicoraphanus Lines through FISH and GISH

Hadassah Roa Belandres,Nomar Espinosa Waminal,Soo Sung Lee,Jin Hoe Huh,Yoon Jung Hwang,Hyun Hee Kim 한국원예학회 2014 한국원예학회 학술발표요지 Vol.2014 No.10

FISH Karyotype and GISH Meiotic Pairing Analyses of a Stable Intergeneric Hybrid xBrassicoraphanus Line BB#5

Belandres, Hadassah Roa,Waminal, Nomar Espinosa,Hwang, Yoon-Jung,Park, Beom-Seok,Lee, Soo-Seong,Huh, Jin Hoe,Kim, Hyun Hee Korean Society of Horticultural Science 2015 원예과학기술지 Vol.33 No.1

xBrassicoraphanus line BB#5, a new synthetic intergeneric hybrid between Brassica rapa L. ssp. pekinensis and Raphanus sativus L. var. rafiphera induced by N-methyl-N-nitroso-urethane mutagenesis in microspore culture, shows high seed fertility and morphological uniformity. Dual-color fluorescence in situ hybridization (FISH) using 5S and 45S rDNA probes and genomic in situ hybridization (GISH) using B. rapa genomic DNA probe were carried out to analyze the chromosome composition and the meiosis pairing pattern compared to its parental lines. The somatic chromosome complement is 2n = 38, which consists of 17 metacentric and two submetacentric chromosomes with lengths of 2.18 to $5.01{\mu}m$. FISH karyotype analysis showed five and eight pairs of 5S and 45S rDNA loci. GISH meiosis pairing analysis showed that 19 complete bivalents were most frequent and accounted for 42% of the 100 pollen mother cells examined. Based on chromosome number, size, morphology, rDNA distribution, and meiosis pairing pattern, both parental genomes of B. rapa and R. sativus appear to exist in xBrassicoraphanus line BB#5, demonstrating its genome integrity. Such stable chromosome constitutions and meiotic pairing patterns in somatic and meiotic cells are very rare in natural and synthetic intergeneric hybrids. Chromosomal studies and genetic and phenotypic changes in allopolyploids a re discussed. The results p resented h erein will b e usef ul f or f urther g enomic s tudy o f xBrassicoraphanus lines and their improvement as promising new breeding varieties.

FISH Karyotype and GISH Meiotic Pairing Analyses of a Stable Intergeneric Hybrid xBrassicoraphanus Line BB#5

Hadassah Roa Belandres,Nomar Espinosa Waminal,Yoon-Jung Hwang,Beom-Seok Park,Soo-Seong Lee,Jin Hoe Huh,Hyun Hee Kim 한국원예학회 2015 원예과학기술지 Vol.33 No.1

xBrassicoraphanus line BB#5, a new synthetic intergeneric hybrid between Brassica rapa L. ssp. pekinensis and Raphanus sativus L. var. rafiphera induced by N-methyl-N-nitroso-urethane mutagenesis in microspore culture, shows high seed fertility and morphological uniformity. Dual-color fluorescence in situ hybridization (FISH) using 5S and 45S rDNA probes and genomic in situ hybridization (GISH) using B. rapa genomic DNA probe were carried out to analyze the chromosome composition and the meiosis pairing pattern compared to its parental lines. The somatic chromosome complement is 2n = 38, which consists of 17 metacentric and two submetacentric chromosomes with lengths of 2.18 to 5.01 ㎛. FISH karyotype analysis showed five and eight pairs of 5S and 45S rDNA loci. GISH meiosis pairing analysis showed that 19 complete bivalents were most frequent and accounted for 42% of the 100 pollen mother cells examined. Based on chromosome number, size, morphology, rDNA distribution, and meiosis pairing pattern, both parental genomes of B. rapa and R. sativus appear to exist in xBrassicoraphanus line BB#5, demonstrating its genome integrity. Such stable chromosome constitutions and meiotic pairing patterns in somatic and meiotic cells are very rare in natural and synthetic intergeneric hybrids. Chromosomal studies and genetic and phenotypic changes in allopolyploids a re discussed. The results p resented h erein will b e usef ul f or f urther g enomic s tudy o f xBrassicoraphanus lines and their improvement as promising new breeding varieties.

Cytogenetic Analyses Revealed Different Genome Rearrangement Footprints in Four ×Brassicoraphanus Lines with Different Fertility Rates

Hadassah Roa Belandres,Hui Chao Zhou,Nomar Espinosa Waminal,이수영,허진회,김현희 한국육종학회 2019 Plant Breeding and Biotechnology Vol.7 No.2

×Brassicoraphanus (AARR, 2n = 38) is a synthetic intergeneric allopolyploid between Brassica rapa L. ssp. pekinensis (AA, 2n = 20) and Raphanus sativus L. var. rafiphera (RR, 2n = 18). Abnormalities in meiosis are main causes for infertility, especially in recent intergeneric allopolyploids. Several ×Brassicoraphanus lines showing varied fertility rates were produced previously, but no cytogenetic data specifying the reasons for infertility have been reported. In this study, we performed cytogenetic analyses in BB4, BB6, BB12, and BB50 lines to evaluate their chromosomal composition and behavior during meiosis. The four lines had relatively small chromosomes, ranging in length from 1.82 to 5.72 mm. BB6, BB12, and BB50 have euploid chromosome number of 2n = 38, whereas BB4 is an aneuploid with 2n ‒ 1 = 37. Fluorescent in situ hybridization karyotype analysis by using 5S/45S rDNA revealed 5/7, 6/7, 5/5 and 5/5 pairs in BB4, BB6, BB12 and BB50, respectively. Genomic in situ hybridization analysis on cells in prophase I revealed varying frequencies of tetravalent pairing and sticky, ring, rod, and laggard chromosomes across the lines, which were more abundant in BB4 and BB6. Unlike BB4 and BB6, both BB12 and BB50 are known to have relatively higher seed fertility and uniform plant morphology. The varied degrees of chromosomal pairing stability during meiosis could explain the different fertility rates among the four ×Brassicoraphanus lines in this study. These data might facilitate breeding programs of ×Brassicoraphanus and further cytogenomic analyses.

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

Karyotypes of Three Exotic Cucurbit Species Based on Triple-Color FISH Analysis

Remnyl Joyce Pellerin,Nomar Espinosa Waminal,Hadassah Roa Belandres,Hyun Hee Kim 한국원예학회 2018 원예과학기술지 Vol.36 No.3

Cytogenetic investigations based on chromosome composition provide insight into basic genetic and genomic characteristics of a species that in turn facilitate species identification and breeding programs. Tandem repeats (TRs) like the 45S rDNA, 5S rDNA and telomeric repeats are ubiquitous in nuclear genomes and are good cytogenetic markers for karyotyping. In this study, we analyzed the karyotypes of three exotic cucurbit species, namely Cucumis melo var. flexuosus (L.) Naudin (2n = 24), Melothria pendula L. (2n = 24) and Trichosanthes anguina L. (2n = 22), based on the cytogenetic distribution of the 45S, 5S and Arabidopsis-type telomeric TRs through triple-color fluorescence in situ hybridization. T. anguina had larger chromosomes (3.2-5.4 μm) compared to C. melo var. flexuosus and M. pendula (1.5-2.2 μm and 1.8-2.5 μm). One and two pairs of 5S and 45S rDNA signals were observed in C. melo var. flexuosus, respectively; while M. pendula and T. anguina had four and three pairs of 45S rDNA, respectively, and two pairs of 5S rDNA. Co-localized signals of 5S and 45S rDNA were observed in M. pendula and T. anguina, but not in C. melo var. flexuosus. Telomeric repeats were observed at chromosome ends of all chromosomes. This information will be useful in future cytogenomic and phylogenetic studies facilitating cucurbit breeding programs.