The field experiment was conducted in the field laboratory of the Department of Plant Pathology, Bangladesh Agricultural University, Mymensingh during the cropping season Rabi (October 2011 to April, 2012) (Ahmed et al. 1998). The in vitro experiments were conducted at the Plant Disease Clinic, IPM Laboratory and net house, Department of Plant Pathology, Bangladesh Agricultural University, Mymensingh and Biotechnology Laboratory; Plant Breeding Division, Bangladesh Institute of Nuclear Agriculture, Mymensingh.
Source of seed, seedling raising and transplantation: Two lines of different morphological characters from each of F3 to F7 generation were selected. The selected line characteristics were: F3 – Laffa purple globose and Dohazari green long, F4 – Dohazari green long and Laffa purple globose, F5 – Dohazari green long and Laffa green globose, F6 – Dohazari green long and Laffa green globose, F7 – Dohazari green long and Laffa purple globose. The materials were obtained from IPM Lab, Department of Plant Pathology, Bangladesh Agricultural University, Mymensingh, Bangladesh.
Seedlings were raised in the net house in plastic trays with proper care and management. Trays were prepared by mixing soil, sand, and cow dung. Twenty-eight days old healthy seedlings were transplanted in the main experimental field. The experiment was designed in a Randomized Complete Block Design (RCBD) with three replications in the Field. In each subplot, four seedlings were planted maintaining 60 cm distance between plants and 60 cm between lines. Fertilizers and manures were applied in the experimental field as per guideline of BARC (Anon. 2005). Weeding, irrigation and other intercultural operations were done whenever necessary.
Inoculation of eggplant by Phomopsis vexans: The plants in the field were inoculated at the flowering stage and again at the fruiting stage. Seventy milliliter spore suspension (5 x 106 spore/ml) prepared with all Phomopsis vexans isolates available at IPM Lab, was sprayed on each plant. The spraying was done at afternoon. Inoculated plants were kept moist and covered with transparent polythene sheet for 24 hours for ensuring better infection. One plot of four plants from each advance line was kept uninoculated (control). Culturing P. vexans isolates and preparation of spore suspension were done following the procedure of Islam (2006).
Assessment of Phomopsis fruit rot: After inoculation, symptoms on fruits were observed at seven-day interval up to 21 days. Data on fruit infection (%) were recorded and the disease severity was calculated according to the standard rating scale (1-5) (Islam et al. 1990).
Harvesting and data recording: The characters responsible for yield of eggplant were studied and the data were collected at fruiting stage. The mature fruits were harvested at the edible stage at an interval of seven days. Four fruits per entry were allowed to ripe and seeds were collected from them for growing plants in the next year.
Statistical analysis: All the characters were analyzed according to design used to find out the statistical significance following Steel and Torrie (1960) and differences between the genotypes were performed by Duncan's Multiple Range Test (DMRT).
Molecular characterization of F3 to F7 population of eggplant through RAPD analysis: Molecular characterization of the ten lines was done through random amplified polymorphic DNA (RAPD) technique. Fresh leaf samples collected from the 20 days old seedlings were used as a source of genomic DNA. Modified CTAB mini-prep method was followed to extract DNA from leaf samples (Kabir, 2007).
Approximately, 2g of leaf tissues were cut into small pieces and taken into a 1.5 ml Eppendorf tube. For digestion, 670 μl extraction buffer (50 mM Tris-HCl, 25mm Ethyline di-amine tetra acetic acid, 300 mM NaCl) 40μl SDS ( Sodium Dodecyl Sulphate) were taken into a tube and grind with the help of pre cooled mortar pestle. The grinded samples were vortexed for 20 seconds for proper mixing and incubated at 65ºC for 10 minutes in hot water bath.100 μl NaCl and100 μl CTAB were added in the samples and then again incubated at the same temperature (65ºC) for 10 minutes for proper digestion. After that the suspension was transferred to another tube and 900 μl chloroform (chloroform: isomylalcohol: 24:1, v/v) was added and mixed well by a shaker. To allow precipitation of the cell debris, the content was centrifuged for 15 minutes at 12000 rpm with a micro centrifuge. After that the supernatant was transferred into new Eppendorf tubes and then 600 μl ice cold isopropanol was added and shaken slowly. The supernatant was decanted and air dried for at least 1 hour. Pellets were washed with 70% ethanol (200 μl), spinned for 10 minutes at 12000 rpm and then air-dried for ½ hours. Then the liquid was completely removed without disturbing the pellet of DNA and air dried. Finally the pellets were resuspended in 30.0 μl TE buffer (10 mM Tris-HCl, 1mM EDTA) and stored at -20ºC.
The quality of the DNA was verified by electrophoreses on a 0.8% agarose gel in TBE (Tris-boric acid-EDTA) buffer. The concentration of DNA samples was determined using a UV Spectrophotometer at 260 nm.
PCR amplification and Electrophoresis: RAPD amplification reactions were maintained following Williams et al. (1990) with some modifications. Fifteen arbitrary decamer primers (Bengalore Genei, India) were screened against DNA from parental cultivars. Three primers producing good scoreable and reproducible bands were selected for subsequent RAPD analysis of eggplant cultivars and phenotypes.
PCR reactions were performed on each DNA sample in a 10 μl reaction mixture containing 1x PCR buffer (10 mM Tris HCl pH 8.5, 50mM KCl and 15 mM MgCl2), 10 mM each dNTPs, 5 pmols primer, 2 U of Taq DNA polymerase (Bengalore Genei, India), 100 ng of genomic DNA and rest amount of sterile deionized water. DNA amplification was carried out in a DNA thermocycler (Biometra, Germany) as the following thermal profile: initial denaturation for 3 min at 94°C followed by 41 cycles of 1 min denaturation at 94°C, 1 min annealing at 35°C and extension at 72°C for 2 min. A final extension step at 72°C for 7 min was allowed for complete extension of all amplified fragments (Kabir, 2007). The PCR products were kept at 4°C until electrophoresis. Reaction mixtures were mixed with 2.0 μl 6X loading dye. Amplified fragments were separated on a 1.5% agarose (Bengalore Genei, India) gel in 0.5 X TBE buffer along with 20 bp DNA weight marker (Bengalore Genei, India) for 2 hours at 100V. Gel was stained with Ethidium bromide solution (0.1 μg ml-1) for 15 min. Finally fragments were visualized under UV-transilluminator and photographed by Gel Documentation System (Biometra, Germany).
Scoring and Data analysis: Since RAPD markers are dominant; we assumed that each band represented the phenotype at a single allelic locus (Williams et al. 1990). The amplified bands were visually scored as present (1) and absent (0) separately for each individual and each primer. The scores obtained were pooled to create -a single data matrix. This was used to estimate polymorphic loci, Nei’s (1972) gene diversity, population differentiation (Gst), gene flow (Nm), genetic distance (D) and to construct a UPGMA (Unweighted Pair Group Method of Arithmetic Means) dendrogram among populations using a computer program, POPGENE (Version 1 .31) (Yeh et al. 1999).The same program was also used to perform test of homogeneity in different loci between population pairs.