SEIJO Jose Guillermo
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Analysis of a valuable chromosome rearrangement induced by ionizing radiations in a cultivated chili pepper line (Capsicum baccatum var. pendulum – Solanaceae).
Simposio; Symposium on Induced Mutations in Plants (ISIMP).; 2008
Analysis of a valuable chromosome rearrangement induced by ionizing radiations in a cultivated chili pepper line (Capsicum baccatum var. pendulum – Solanaceae)*   M. A. Scaldaferroa, M. Grabielea, J. G. Seijob, H. Debatc, D. A. Ducassec , A. R. Prinad, E. A. Mosconea   a Multidisciplinary Institute of Plant Biology (IMBIV), C.C. 495, 5000-Cordoba, Argentina.   b Botanical Institute from North East (IBONE), C.C. 209, 3400-Corrientes, Argentina.   c Institute of Phytopathology and Plant Physiology (IFFIVE-INTA), Camino a 60 cuadras Km 5½, 5119-Cordoba, Argentina.   d Institute of Genetics “E. A. Favret”, INTA, C.C. 25, 1712-Castelar, Buenos Aires, Argentina.   E-mail address of main author:   * This work was supported by the International Atomic Energy Agency, RBF 12226.   Capsicum (chili peppers) is an economically important genus including five crop species consumed by man as spice and food. Most of the contributions about induced mutagenesis refer to gene mutations, and studies about changes at chromosome level are scarce dealing with polyploid induction [1-3]. Therefore, our research group is involved in a program on induced mutagenesis by ionizing radiations (X-rays) in order to achieve chromosome rearrangements in Capsicum. Ionizing radiations are widely recognized as an effective way to induce chromosome structural changes, particularly reciprocal translocations [4]. In this contribution an interesting structural chromosome rearrangement obtained in C. baccatum var. pendulum (Willd.) Eshbaugh cv. “cayenne” is described.   The analyzed cultivar, from Salta province (Argentina), has 2n=2x=24 and a normal karyotype with 11 m + 1 st pairs, 4 pairs (nos. 1, 3, 10, 12) carrying nucleolar organizing regions (NORs) and associated satellites in the short arm. Dry seeds were treated with different acute doses of X-rays according to the dose range reported for peppers in previous mutation experiments [5], i.e., 100 Gy, 200 Gy, and 300 Gy, and, in addition, 20 Gy after soaking. M1 to M4 generations were developed in greenhouse under controlled conditions of illumination, temperature and humidity.                       In M2 seedlings coming from the only surviving M1 plant after the 300 Gy treatment a rearranged chromosome carrying NORs in both arms was found. In order to analyze this structural change in deep different cytogenetic approaches were applied, i.e., 1) conventional staining technique using Feulgen’s method to observe chromosome number, size and shape; 2) silver impregnation to detect active NORs [6]; 3) fluorescent chromosome banding to reveal type and position of constitutive heterochromatic regions (triple staining technique with the fluorochromes chromomycin A3, distamycin A and 4-6-diamidino-2-phenylindole (CMA/DA/DAPI)] [7]; and 4) fluorescent in situ hybridization (FISH) with the 18S-25S ribosomal DNA repeated sequences using three different probes (R2 from Arabidopsis thaliana, pCa25S-29 from C. annuum var. annuum, and pCf18S-17 from C. frutescens) [8].   A reciprocal translocation between two NOR-bearing chromosomes in the M1 plant has occurred, which gave a viable progeny carrying the chromosomal interchange, so far, without any visible deviating phenotype after four generations, nor in the heterozygous neither in the homozygous for the interchange. The screening of 36 M2 seedlings showed that 44% of them were heterozygous for the interchange, 8% homozygous, and the remaining individuals normal homozygous. Since not any chromosome instability was observed (Comentario: esta inestabilidad se refiere a cortes y roturas de los cromosomas, o a comportamiento anormal de los cromosomas en meiosis, si fuera lo Segundo quizá  habría que poner abnormal meiotic behaviour), it is supposed the occurrence of a small reciprocal interchange between a member of chromosome pair no. 1 and another member of pair no. 3, both carrying active NORs in shorts arms and associated CMA+/DAPI- heterochromatin, according to the normal karyotype of the cultivar. The results of this structural rearrangement were two chromosomes with little change in size, one of them easily recognized by the presence of NORs and CMA+/DAPI- associated heterochromatin in both arms; whilst the other chromosome of difficult identification bears a small heterochromatic CMA+/DAPI- band in the short arm. As the translocation here reported produced a conspicuous rearranged marker chromosome, the obtained plant line is considered very valuable for studies on chromosome pairing and genetic linkage in the genus, including the mapping of genes responsible of crop quality.   Comentario: No se hace mención a los resultados de FISH, quizá habría que poner algo para justificar su empleo, sobre todo que se usan tres sondas para la misma región que se detectó inicialmente con plata.   REFERENCES   [1]  Katiyar, R.B. 1977. Radiocytogenetical studies in Capsicum: induced desynapsis. Caryologia 30:347-350. [2]  Indira, C.; Abraham, S. 1977. Morphological and cytological studies on a radiation induced polyploid in Capsicum annuum Linn. Cytologia 42:371-375. [3]  Kumar O.A.; Raja Rao, K.G. 2003. Cytomorphological studies in gamma-ray induced autotriploids of Capsicum annuum L. Cytologia 68:45-50. [4]  Gaul, H. 1977. Mutagen effects in the first generation after seed treatment: cytological effects. In: FAO/IAEA (eds.). Manual on mutation breeding, pp.91-96. IAEA, Vienna. [5]  Daskalov, S. 1986. Mutation breeding in pepper. In: FAO/IAEA (eds.). Mutation Breeding Review 4:1-26. IAEA, Vienna. [6]  Kodama, Y.; Yoshida, M.C.; Sasaki, M. 1980. An improved silver staining technique for nucleolus organizer regions by using nylon cloth. Jap. J. Hum. Genet. 25:229-233. [7]  Schweizer, D.; Ambros, P.F. 1994. Chromosome banding: stain combinations for specific regions. In: J.R. Gosden (ed.), Chromosome analysis protocols, pp.97-112. Humana Press, Totowa. [8]  Moscone, E.A.; Matzke, M.A.; Matzke, A.J.M. 1996. The use of combined FISH/GISH in conjunction with DAPI counterstaining to identify chromosomes containing transgene inserts in amphidiploid tobacco. Chromosoma 105:231-236.