C 1999 The Japan Mendel Society Cytologia 64: 117-127, 1999 Karyotype Analysis of Twelve Species belonging to Genus Vigna Gianfranco Venoral, Sebastiano Blangifortil and Roberto Cremonini2 1 Stazione Sperimentale di Granicoltura per la Sicilia, Via Rossini 1, 95041 Caltagirone (CT), Italy 2 Dipartimento Scienze Botaniche, Via Luca Chini 5, 56126 Pisa, Italy Accepted October 26, 1998 Summary Cowpea,Vigna unguiculata (L.) Walp., is an importantcrop for manydeveloping coun- tries. Its potential cannotbe fully achieveddue to scarseresistance to pathogens.Source of resistance are present in wild gene pool, therefore, the introductionof genes for valuable traits from wild species to Vignaunguiculata faced an obstacle due to crossabilitybarriers betweenspecies. The study of the chromosomalmorphology could be usefulin a modernplant breedingapproach of cow- pea. Recentlythe use of image analysissystem to the karyotypingof plant specieswith few chromo- somes has allowed the productionof detailed karyotypesemployed for interspecificcomparisons. Twelvespecies belonging to four differentsubgenera and to six sectionsaccording to Marechalet al. (1978) were analysed by image analysissystem. The karyomorphologicalparameters were utilised for computingkaryosimmetry according to Stebbins(1971). Key words Vignaspecies, Image analysis,Karyotype, Taxonomy. The cowpea, V unguiculata (L.) Walp., is an important leguminous in many developing coun- tries where it represents the principal source of proteins. This species suffers attacks of seed para- sites, that drastically reduces the potential feed of this crop (Rachie and Rawall 1976). The aim of this research is the study of the chromosomal morphology that serves to describe the phyletic relationships (Stebbins 1971, Lackey 1980) and overcoming the hybridisation barriers for the insertion of useful genes from wild species. The chromosomes of the genus Vigna are very small (Parida et al. 1990), and the reports about karyotyping are few (Venora et al. 1995, and refer- ences therein). The phyletic relationships of the species in the genus Vigna were studied not only by morphokaryological analysis but also by isoenzymatic patterns (Jaaska and Jaaska 1988, 1990), nu- clear DNA determination (Parida et al. 1990, Lakhanpaul and Babu 1991) and molecular tech- niques as in situ hybridisation, cloning nucleotid sequence analysis and retrotransposable elements analysis (Fatokun et al. 1993, Galasso et al. 1995, 1997). Recently the use of image analysis in karyotyping of plant species with difficult chromosomes (small and/or not very distinguishable, Fukui 1986, 1998, Fukui and Iijima 1991, lijima et al. 1991, Yanagisawa et al. 1991), has allowed the realisation of detailed karyotypes also in Vigna (Venora and Saccardo 1993, Venora and Padulosi 1997). Using this methodology the complements of twelve species belonging to four different subgenera and six sections (Catiang, Reticulatae, Vigna, Plec- totropis, Leptospron, Sigmoidotropis) were inspected and described, in accordance with the classifi- cation of Marechal et al. (1978). 1 E-mail: [email protected] 2 E-mail: [email protected] 118 Gianfranco Venora, Sebastiano Blangiforti and Roberto Cremonini Cytologia 64 Table 1. Chromosome morphometric data of the species studied 1999 Karyotype Analysis of Genus Vigna 119 Table 1. (continued) Values followed by the same letter are not significantly different, according to the Cluster analysis of Scott and Knott (capital leters P=0.01, small letters P=0.05). 1 Dataffom Venora and Saccardo 120 Gianfranco Venora, Sebastiano Blangiforti and Roberto Cremonini Cytologia 64 1999 Karyotype Analysis of Genus Vigna 121 Materials and methods This study was performed on twelve species of the genus Vigna provided by IITA (Internation- al Institute of Tropical Agriculture, Ibadan, Nigeria) which conserves the world collection of Vigna germplasm. To obtain mitotic chromosomes the traditional approach was adopted: pre-treatment of root apices with 1,4-dichlorobenzene (2 h), and staining with Schiff's reagent after hydrolysis with 5 N HCl for 55 min at room temperature. At least ten metaphase plates for each species were analysed. The microscopic investigation was conducted by using a Zeiss Axioplan 2 microscopy connected with an image analysis system KS400 Zeiss, with dedicated software to karyotyping Ikaros 3.40 (MetaSystems 1996), that enables more reliable results than the traditional handmade karyotype (Venora et al. 1991). The total chro- mosome length, short arms, long arms and satellites were measured with the computer system. All the data for each plate are therefore inserted in a dedicated software "Karyo 95" to the accomplish- ment of the better matching of chromosomes couples, on the base of the data of each chromosome (Pavone et al. 1995). The arm ratio was calculated by adding the length of the satellite to the length of the attached arm. The nomenclature adopted for chromosome classification was that of Levan et al. (1964). Karyological data have been used to produce a classification according to Stebbins's cat- egories, which highlight the degree of karyologic symmetry (Stebbins 1971). The same information has been used for calculating TF% of Huziwara (1962) and the indices REC (Resemblance between chromosomes) and Syi (Symmetric indices) of Greilhuber and Speta (1976), which allow the com- parison between species and evaluation of karyological evolution. All the data referring to V un- guiculata, V oblongifolia, V luteola and V vexillata are from the report of Venora and Saccardo (1993). The results have been statistically tested by applying the cluster analysis method of Scott and Knott (1974). Results In Table 1, the karyotipic data of each of the twelve species are reported, resulted with 2n=22. Two species V ambacensis and V radiata exhibited two satellited pairs, the longest of the comple- ment. All the other species have only one satellited pair, the first, with the exception of V kirkii in Table 2. Heploid complement length, Karyotype formulas, indices and Stebbins categories in Vigna samples 1 Data from Venora and Saccardo 122 Gianfranco Venora, Sebastiano Blangiforti and Roberto Cremonini Cytologia 64 Chromosome 1 Chromosome 2 Chromosome 3 Chromosome 4 Chromosome 5 Chromosome 6 Fig. 2a. Graphical representation of arm ratio (Fs) and length of chromosomes 1-6 of the 12 species . Inside each box, no significantly difference at P=0 .01 (•¬ arm ratio, •¬ length mm). 1999 Karyotype Analysis of Genus Vigna 123 Chromosome 7 Chromosome 8 Chromosome 9 Chromosome 10 Chromosome 11 Fig. 2b. Graphical representation of arm ratio (1/s) and length of chromosomes 7-11 of the 12 species. Inside each box, no significantly differences at P=0.01 (•¬ arm ratio, •¬ length mm). 124 Gianfranco Venora, Sebastiano Blangiforti and Roberto Cremonini Cytologia 64 which the satellited pair is the third. The Fig. 1 illustrates the idiograms of the analysed species . The length of the complement, the karyotype formulas, the Rec , Syi, TF% indices and the classification of Stebbins (1971) are reported in Table 2 . The length of the complement is included between the species V adenatha (19.98 um) and V oblongifolia (12 .12 ƒÊm) which are the species with the longer and shorter complements, respectively . The most widespread karyotipic formula are msc+8m+2sm, common to four species, msc+ 10m is for two species , the other six species show karyotipic formulas specific to each one. The classification of Stebbins (1971) places the twelve species in four different categories. Figs. 2a and 2b show a graphic representation of the arm ratio and length of each chromosome in the twelve analysed species. This graphical representation enabled the identification of typical chromosomes (marker chromosomes) for each species (Table 3). V oblongifolia is the species with five marker chromosomes, while V ambacensis, V radiata and V vexillata have only one . The chro- mosomes that mostly discriminate between the studied species are the 4th , 7th and 8th pairs, that each distinguish four species, while the most homogeneous pairs in the examined species are the 5th and 9th pairs, which enable to identify one species. The symmetry indices have shown that the most asymmetry species is V unguiculata , while V adenantha resulted the most symmetrical. Optimal differentiation between chromosomes of the complement was found in V ambacensis. The spatial representation of the indices highlights four distinct groups. The most evolved karyotype is that of the cultivated species (V unguiculata) , and the most primitive that of V adenantha (Fig. 3a). Infact V unguiculata plot in the lower part of spa- tial representation group 1, on the contrary V adenantha lies in upper part , in the less evolved group. The classification of Marechal et al . (1978) related to the twelve analysed species, based on plant and pollen morphology, highlights six distinct groups (Marechal sections), it has been re- drawn on the spatial representation of the karyologic indices (Fig . 3b). Discussion The chromosomal number 2n=22 for all the species, coincides with previous cytological re- port on other species of the same genus (Venora et al. 1995), with the exception of V ambacensis that has been reported with 2n=20 (Dusseau and Magnaut 1941, Frahm-Leliveld 1965, Galasso et al. 1996, Parida et al. 1990), even Verdcourt (1970) reports 2n=20 both for V ambacensis and for other three species, between which V reticulata and V wittei that always resulted in 2n=22 under Table3. Typicalchromosome