Genomic characterization of the germplasm in some South American Solanaceae of economical interest by in situ hybridization, with emphasis in chili peppers

MOSCONE, E. A.

Córdoba (Argentina)

Workshop; Third Research Co-ordination Meeting of FAO/IAEA Co-ordinated Research Project; 2007

Agencia Internacional de Energía Atómica de las Naciones Unidas (IAEA-UNO, Vienna, Austria)

Capsicum is an economically important genus of Solanaceae with 31 recognized species natives of the Americas. Five species are exploited on a global scale because of their valuable fruits, the pungent forms as spices and the sweet ones as vegetables. The cytogenetics of Capsicum has received early attention for breeding purpose with reference to the domesticated taxa and their possible wild ancestors. In this sense, a broad gene mapping and genome characterization program in the genus by molecular cytogenetics will be useful for hybridization and biotechnological approaches. On the bases of the cytogenetic information achieved in the genus by the chief researcher and collaborators (cf. Moscone et al., 1993, 1995, 1996, 2003a), in the present project it is attempted to characterize cytologically species, varieties and cultivars. In particular, it is intended to physically map specific DNA sequences, including genes contributing to crop quality, by fluorescent in situ hybridization (FISH). Furthermore, a program of induced mutagenesis by ionizing radiation to get chromosome rearrangements in one of the cultivated species is carried out. It is expected to achieve reciprocal translocations identified by FISH chromosome landmarks, to which crop quality genes could be assigned in a further step. Marker rearranged chromosomes could be valuable for studies on chromosome pairing and genetic linkage in peppers. In the first four years of this project, a total of 30 samples belonging to 14 species and 5 varieties of peppers were examined (Table 1). The karyotypes of 12 Capsicum taxa, i.e., C. annuum varieties annuum and glabriusculum, C. baccatum varieties baccatum and pendulum, C. praetermissum, C. eximium, C. cardenasii, C. tovarii, C. galapagoense, C. flexuosum, C. recurvatum, and C. rhomboideum, were characterized by fluorescent (CMA/DA/DAPI) banding in respect to amount, type and distribution of constitutive heterochromatin. Additionally, DAPI-C banding was applied to four species, i.e., C. eximium, C. flexuosum, C. recurvatum, and C. rhomboideum. The number, position and size of active nucleolus organizing regions (AgNORs) were determined by silver impregnation in 8 taxa, i.e., C. annuum var. annuum, C. baccatum varieties baccatum and pendulum, C. eximium, C. cardenasii, C. flexuosum, C. recurvatum, and C. rhomboideum. On the other hand, single and double labelling FISH experiments using an Arabidopsis-like telomeric sequence and ribosomal RNA gene (5S and 45S rDNA) probes, respectively, were performed on the somatic chromosomes of 17 accessions from 13 taxa, i.e., the 6 cultivated entities – C. frutescens, C. chinense, C. annuum var. annuum, C. baccatum varieties pendulum and umbilicatum, and C. pubescens – and 7 wild taxa – C. chacoense, C. annuum var. glabriusculum (only for the telomeric sequence), C. galapagoense (only for the telomeric sequence), C. baccatum var. baccatum, C. eximium, C. cardenasii, and C. flexuosum. The molecular characterization of two repeated sequences (pCp-200/17 and pCp-200/33), which are components of the constitutive heterochromatin in C. pubescens was achieved after cloning and testing in FISH experiments several restricition fragments obtained from genomic DNA. One of this sequences, pCp-200/17, was tested in other species by PCR (C. rhomboideum and Lycianthes rantonnei)and FISH experiments (C. annuum var. annuum and C. baccatum var. umbilicatum). In addition, the molecular characterization of the 5S rRNA genes, including the intergenic spacer, was obtained in 15 Capsicum taxa, i.e., C. annuum varieties annuum and glabriusculum, C. baccatum varieties baccatum, pendulum and umbilicatum, C. chacoense, C. chinense, C. eximium, C. flexuosum, C. frutescens, C. praetermissum, C. pubescens, C. recurvatum, C. rhomboideum, and C. tovarii. Sequence comparison of the intergenic spacer from those species allowed to build a phylogenetic tree for peppers. Moreover, 5S, 18S, and 25S rDNA probes specific for Capsicum has been created by PCR from genomic DNA of C. pubescens to be used in FISH experiments. Concerning the induced mutagenesis program, seeds of C. baccatum var. pendulum cultivar “cayenne” were treated with different doses of X-rays (100, 200, and 300 Gy on dried seeds and 20 Gy on presoaked seeds for 16 hs) and the dose effects at the cytological level and plant damage in M1 generation has been estimated. Chromosomal aberrations were examined in the first mitotic cycle by Feulgen staining and physiological effects recorded on seedlings grown in greenhouse. Furthermore, estimations of DNA damage in embryo and root cells by TUNEL test after seed irradiation with the different X-ray doses have been performed. Finally, meiotic and somatic chromosome of the M1, M2, and M3 generations of the different treatments were searched by conventional staining to detect chromosome rearrangements. After a reciprocal translocation of interest has been found in a plant from the M2 generation of the 300 Gy treated line, an extended somatic chromosome analysis was carried out in M2 and M3 generations by conventional staining and banding techniques. Then, it was possible to develop a 300 Gy line with an homozygous translocation valuable as a marker chromosome, i.e., a chromosome pair with NORs in both arms. FISH using ribosomal RNA gene probes together with fluorescent (CMA/DA/DAPI and DAPI-C)- and AgNOR-banding brought out useful markers for: 1) characterizing Capsicum germplasm, 2) identifying chromosomes and establishing chromosome homeologies between species, and 3) mapping further DNA sequences by FISH, including genes contributing to crop quality in peppers. It has been contributed to the knowledge of the genome organization and evolution of Capsicum. Particularly, the lack of intercalary loci of telomeric repeats suggests absence of chromosome rearrangements such as fusions or inversions involving the telomeric region in the recent evolution of the analysed species. Valuable data which cast light on the origin of domesticated chili peppers, i.e., C. annuum var. annuum, the pepper species most widely cultivated, and the cultigen C. pubescens, the appearance of x = 13 in the genus, and the relationships in the C. baccatum complex were obtained. The finding of two repeated sequences, which could be part of retroelements or transposons, as constituent of the heterochromatin in peppers, one of them (pCp-200/17) even present in the related genus Lycianthes, encourages to deepen the exploration. On the other hand, the characterization of the coding region of the 5S rRNA gene in different pepper species allowed to determine the consensus secondary structure of this ribosomal RNA for Capsicum. Furthermore, the sequence variation found in the intergenic 5S spacer was very valuable to establish relationships between taxa and build a strong phylogenetic tree for peppers. The obtainment of specific rDNA probes in this period will allow to achieve more accurate results in the following lapse concerning ribosomal gene localization by FISH. Estimations of X-ray dose effects in cv. “cayenne” M1 generation show that chromosome aberrations and plant damage are correlated with the irradiation dose as expected and are increased by soaking of seeds prior to radiation treatment. On the other hand, estimations of X-ray dose effects by TUNEL test show that the level of DNA damage in the cv. “cayenne” M1 generation increased with the irradiation dose. The results obtained could be useful for programs to achieve a collection of induced reciprocal translocation lines in the selected cultivar. Some chromosome rearrangements were detected in M1 and M2 generation, particularly in the 200 Gy- and 300 Gy-treated lines. A main achievement is a translocated plant line, which bears a conspicuous marker rearranged chromosome with nucleolar organizing regions in both arms, which has been established. The X-ray induced structural rearrangement found could be valuable for mapping genes, including those responsible of crop quality. Part of the results achieved in the course of this project has been included in 6 communications presented at the “4th Argentine-Chilean Meeting on Genetics”, Huerta Grande, Argentina, 21-24.09.2003 (Cecchini et al., 2003; Moscone et al., 2003b), the “33rd Argentine Meeting on Genetics”, Malargüe, Argentina, 26-29.09.2004 (Scaldaferro et al., 2004), the “34th Argentine Meeting on Genetics”, Trelew, Argentina, 11-14.09.2005 (Scaldaferro et al., 2005a), the “1st Bulgarian Symposium on Horticulture”, Plovdiv, Bulgaria, 31.08-01.09.2005 (Scaldaferro et al., 2005b), and  the “PPA/Solanaceae 2006: Genomics Meets Biodiversity (VI International Solanaceae Conference)”, Madison, USA, 23-27.07.2006 (Moscone et al., 2006). Data from these presentations were included in two manuscripts, one of them (Scaldaferro et al., 2006) published in Plant Science (National Centre for Agrarian Sciences, Bulgaria), and the remaining one under review in Acta Horticulturae (International Society for Horticultural Science, Belgium). It should be mentioned that in the ongoing project, efforts have been made to set up collaborations and establish an international Capsicum network with the following leader researchers: Dr. Daniel Ducasse (Institute of Phytopathology and Plant Physiology, Cordoba, Argentina), Dr. Julio Daviña (University of Misiones, Posadas, Argentina), Dr. Claudio Galmarini (National Institute of Agrarian Technology, Mendoza, Argentina), Drs. Friedrich Ehrendorfer and Rose Samuel (Institute of Botany, University of Vienna, Austria), Dr. Luciano de bem Bianchetti (Embrapa – Genetic Resources and Biotechnology, CENARGEN, Brazil), Dr. Nasya Tomlekova (Maritsa Vegetable Crops Research Institute, Plovdiv, Bulgaria), Dr. Ilan Paran (Agricultural Research Organization, The Volcani Center, Bet Dagan, Israel), Drs. Byoung-Cheorl Kang and Byung-Dong Kim (Seoul National University, Seoul, Korea), Dr. Jolanta Juchimiuk (University of Silesia, Katowice, Poland), Dr. Paul Bosland (Director of The Chile Pepper Institute, New Mexico State University, Las Cruces, USA), Dr. Lynn Bohs (University of Utah, Salt Lake City, USA), Dr. Molly Jahn (Cornell University, Ithaca, USA), Dr. Robert Jarret (Plant Genetic Resources Unit, United State Department of Agriculture, Griffin, USA), Dr. Michael Nee (The New York Botanical Garden, Bronx, USA), and Dr. Joshua Tewksbury (University of Washington, Seattle, USA). Finally, an important achievement is that our pepper germplasm bank has been enlarged during the project by collecting 85 seed samples from 16 species and 5 varieties (8 accessions still not identified) to reach at present 280 seed accessions belonging to 21 species and 6 varieties, with 40 samples being still unidentified.     REFERENCES   Cecchini, N.M.; Scaldaferro, M.A.; Barboza, G.E.; Moscone, E.A. 2003. Estudio de cromosomas mitóticos en dos ajíes silvestres de flores púrpuras (Capsicum – Solanaceae) mediante bandeo de fluorescencia. – Actas 4th Jornadas Argentino-Chilenas de Genética, Huerta Grande. J. Bas. Appl. Genet. 15 (Suppl. 2): 91. Moscone, E.A.; Lambrou, M.; Hunziker, A.T.; Ehrendorfer, F. 1993. Giemsa C-banded karyotypes in Capsicum (Solanaceae). Plant Syst. Evol. 186: 213-229. Moscone, E.A.; Loidl, J.; Ehrendorfer, F.; Hunziker, A.T. 1995. Analysis of active nucleolus organizing regions in Capsicum (Solanaceae) by silver staining. Amer. J. Bot. 82: 276-287. Moscone, E.A.; Lambrou, M.; Ehrendorfer, F. 1996. Fluorescent chromosome banding in the cultivated species of Capsicum (Solanaceae). Plant Syst. Evol. 202: 37-63. Moscone, E.A.; Baranyi, M.; Ebert, I.; Greilhuber, J.; Ehrendorfer, F.; Hunziker, A.T. 2003a. Analysis of nuclear DNA content in Capsicum (Solanaceae) by flow cytometry and Feulgen densitometry. Ann. Bot. 92: 21-29. Moscone, E.A.; Seijo, J.G.; Cecchini, N.M.; Ducasse, D.A.; Scaldaferro, M.A.; Acosta, M.C. 2003b. Mapeo de secuencias génicas (rDNA 45S y 5S) y teloméricas en los cromosomas somáticos de algunas especies de Capsicum (Solanaceae) mediante hibridación in situ fluorescente. – Actas 4th Jornadas Argentino-Chilenas de Genética, Huerta Grande. J. Bas. Appl. Genet. 15 (Suppl. 2): 91. Moscone, E. A.; Scaldaferro, M. A.; Grabiele, M.; Cecchini, N. M.; Sánchez García, Y.; Daviña, J. R.; Ducasse, D. A.; Barboza, G. E.; Ehrendofer, F. 2006. 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