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Abstract
The present investigations are conducted to find out the phylogenetic relationships between five Vicia species by biochemical and molecular cytogenetical studies including SDS-PAGE for seed storage protein electrophoretic patterns (water-soluble and water non soluble fractions), karyotype study of chromosomes, chromosome banding (C-banding) patterns via DAPI stain and fluorescent in situ hybridization (FISH) technique. This thesis has been carried out at plant genetic and cytology laboratory - genetic and cytology department - genetic engineering and biotechnology division - National Research center - Egypt, during the period from 2005 to 2009. The seeds of Vicia species (Vicia macrocarpa (Moris) Betol., Vicia sativa (L.) ssp. Sativa convar var. sativa, Vicia narbonensis (L.) var. narbonensis and Vicia ervilia (L.) Willd) are obtained from the germplasm collection of the Institut for Plant Genetics and Crop Plant Research (IPK), Gatersleben, Germany. Except Vicia faba var. sakha 3 is obtained from Agricultural Research Center, Giza, Egypt.
The results could be summarized as follows:
I. Biochemical studies
1– Seed storage proteins profiles (SDS-PAGE)
a- Water soluble protein
The five Vicia species showed a total number of 39 different bands, of which 27 bands were detected in Vicia narbonensis and 21 bands in Vicia ervilia, 24 bands in Vicia macrocarpa as well as in Vicia sativa and 25 bands in Vicia faba. Based on UPGMA analysis using Jaccard′s coefficient revealed the percentage of similarity between studied species. It was found that Vicia narbonensis is highly related to Vicia ervilia (77.8%) then Vicia macrocarpa to Vicia narbonensis (64.5%) then Vicia macrocarpa to Vicia sativa (60%) while Vicia faba is less related to all studied species.
b- Water non soluble protein
The five Vicia species showed a total number of 40 different bands, of which 27 bands were detected in Vicia narbonensis and 23 bands in Vicia ervilia whereas 24 bands in Vicia macrocarpa and 18 bands in Vicia sativa while 26 bands were detected in Vicia faba. Based on UPGMA analysis using Jaccard′s coefficient the results revealed a percentage of similarity between studied species, it indicating that Vicia narbonensis is highly related to Vicia macrocarpa (70%) then Vicia macrocarpa to Vicia sativa (61.5%) then Vicia narbonensis related to Vicia ervilia (61.3%) while Vicia faba is less related to all studied species.
II. Molecular cytogenetics studies
1- Karyotype analysis:-
The somatic chromosome number of the two species V. narbonensis and V. ervilia is 2n = 14, while in the other three species V. sativa, V .macrocarpa and V. faba, it is 2n = 12. All the examined species have one secondary constriction except V. sativa has two secondary constriction.
Vicia macrocarpa
Chromosomes no. 1, 5 and 6 are metacentric whereas chromosomes no. 2 and 3 are submetacentric, and chromosome no. 4 is subtelocentric. Chromosome no. 6 has the secondary constriction region on its relatively short arm.
Vicia sativa
Chromosomes no. 2, 3 and 5 are metacentric while chromosomes no. 1, 4 and 6 are subtelocentric. This species is characterized by the presence of two secondary constriction regions on the long arm of chromosomes no. 1 and relatively long arm of chromosome no. 3.
Vicia narbonensis
Chromosome no.7 is metacentric whereas chromosomes no. 1, 2, 3, 5 and 6 are submetacentric, and chromosome no. 4 is subtelocentric. Chromosome no. 6 in this species has the secondary constriction region on its long arm.
Vicia ervilia
Chromosomes no. 1, 2 and 6 are metacentric while chromosome no., 3 is submetacentric and chromosomes no. 4, 5 and 7 are subtelocentric. Chromosome no. 7 has the secondary constriction region on its long arm.
Vicia faba
Chromosome no. 1 is metacentric while other chromosomes are subtelocentric. The longest chromosome of this species (chromosome no. 1) has the secondary constriction region on its relatively short arm.
2- DAPI chromosome banding patterns and rDNA loci revealed by flourescence in situ hybridization:-
Double color FISH with digoxigenin labelled 25S rDNA and biotin labelled 5S rDNA. Probes revealed one locus of 25S rDNA associated with nucleolar organizer region (NOR) for all the examined species except V. sativa, which has two sites. All loci were associated with the secondary constriction region.
Vicia macrocarpa
The relatively short arm of chromosome no. 6 has one locus for 25S rDNA gene, while the short arm of chromosome no. 3 has one locus for 5S rDNA gene. Chromosomes no. 1 has one interstitial band on relatively short arm and two interstitial bands on relatively long arm also chromosome no. 2 has one interstitial band on short arm and two interstitial bands on long arm. Chromosome no. 3 has three interstitial bands on long arm and chromosome no.4 has one interstitial band on long arm. The centromeric band is found only in chromosome no. 6.
Vicia sativa
There are two loci for 25S rDNA gene on the long arm of chromosomes no. 1 and relatively long arm of chromosome no. 3. In addition chromosome no. 3 has one locus for 5S rDNA gene on its relatively short arm. The long arms of chromosomes no. 1 and relatively long arm of chromosomes no. 3 have two interstitial bands while relatively long arm of chromosomes no. 2 and long arm of chromosomes no. 4 have one interstitial band. The short arms of chromosomes no. 1 and relatively short arms of chromosomes no. 2 have one interstitial band. The centromeric band is only found in chromosome no. 6.
Vicia narbonensis
The long arm of chromosome no. 6 has one locus for 25S rDNA, while the short arm of chromosome no. 1 has one 5S rDNA gene locus. The short arm of chromosome no. 1 has one interstitial band while short arms of other chromosomes have only telomeric bands. The long arm of chromosomes no. 1, 4, 5 and 6 and relatively long arm of chromosomes no. 7 have telomeric bands and chromosomes no. 2, 3, 4 and 6 have one interstitial band on long arms. The centromeric band found only on chromosome no. 6 and 7.
Vicia ervilia
This species has two loci for 5S rDNA gene, one of them on the relatively short arm of chromosome no. 2 and the other on the long arm of chromosome no. 5, the long arm of chromosome no. 7 has one locus for 25S rDNA. The relatively short arm of chromosomes no. 1 and 2 and short arm of chromosomes no. 3, 4 and 5 have telomeric band also the telomeric band is present on relatively long arm of chromosome no. 6. The chromosome no. 1 has one interstitial band on relatively short arm but the relatively long arm has two interstitial bands. The relatively long arm of chromosome no. 2 and long arm of chromosome no. 5 and 7 have one interstitial band. This species is characterized by the presence of a centromeric band on all chromosomes.
Vicia faba
The relatively short arm of chromosome no. 1 has one locus for 25S rDNA gene and two loci for 5S rDNA gene. The relatively long arm of chromosome no. 1 has one interstitial band while the relatively short arm has two interstitial bands. The chromosomes no. 2, 3, 5 and 6 have one interstitial band on long arms. The short arm of chromosome no. 4 has one interstitial band.