Mitochondrial DNA variation and substructure among the tribal populations of Andhra Pradesh, India.

We analyzed mtDNA HVR-I variation among six tribal populations-Andh, Pardan, Gond, Naikpod, Kolam and Chenchu--from Andhra Pradesh. These tribes belong to the Dravidian and Indo-European linguistic group. Except for Chenchu, the rest of the tribal samples were collected from two or more than two locations. The analysis of molecular variance (AMOVA) of the sequences yields a significant F(ST) value (0.045), suggesting a fair degree of genetic differentiation among these tribes. When the tribal samples collected from different locations were considered as subpopulations in AMOVA, it is found that the variation among the subunits within the tribal groups is smaller than among the tribes. However, when Chenchu is removed from the analysis, the magnitude of within and between groups diversity becomes similar. In the multidimensional scaling plot based on F(ST) distances the Chenchu is found to be the extreme outlier. Exclusion of Chenchu from AMOVA analysis and multidimensional scaling plot does not result in any specific pattern of population clustering. Mismatch distribution suggest that Chenchu might have undergone a bottleneck effect and does not show evidence of past demographic expansion as shown by the other five tribal groups. A comparison of AP tribes with some other caste and tribal populations of India suggests common maternal genetic heritage.

Optimization of RQRT-pCR protocols to measure beta-1,3-glucanase mRNA levels in infected tissues of rubber tree (Hevea brasiliensis).

RQRT-PCR technique was evaluated for its validity as an alternative to Northern blotting for quantification of plant gene expression in diseased tissues of Hevea. Reliable RT-PCR results could be obtained by co-amplification of housekeeping actin gene as the internal control along with the gene of interest. The product of interest was quantified relative to that of the internal control by measuring net intensity of bands. Expression levels of defense-related beta-1,3-glucanase gene was studied in the pathogen infected tissues of rubber. The beta-1,3-glucanase gene was found to be induced in infected leaf tissues and reached a peak at 48 h after inoculation. The beta-1,3-glucanase gene expression during pathogen infection was determined through Northern blot hybridization also, using 18S RNA as the internal control. RQRT-PCR and Northern hybridization showed almost similar results, thereby validating the use of this technique to study the gene expression in rubber.

Induction and differential expression of beta-1,3-glucanase mRNAs in tolerant and susceptible Hevea clones in response to infection by Phytophthora meadii.

Most cultivated rubber tree (Hevea brasiliensis Willd. ex A. Juss.) clones in India are susceptible to abnormal leaf fall disease (ALF), which is caused by various Phytophthora species and results in yield losses of up to 40%. Because the conventional breeding programs for this perennial tree crop are complex and time consuming, we attempted to find a molecular solution to increase the tolerance of rubber trees to ALF. The expression patterns of the gene coding for the pathogenesis-related beta-1,3-glucanase (beta-glu) enzyme in a tolerant (RRII 105) and a highly susceptible (RRIM 600) clone of rubber tree were examined, following infection with ALF-causing Phytophthora meadii McRae. Infected leaf samples were collected at different times after inoculation, and RNA was extracted and subjected to Northern blot hybridization and reverse transcriptase polymerase chain reaction (RT-PCR). On hybridization with a 1.25 kb beta-glu probe, Northern blots showed a marked increase in beta-glu transcript levels in both clones 48 h after inoculation. However, compared with the susceptible RRIM 600 clone, the tolerant RRII 105 clone had a higher rate of increase and a more prolonged induction, with beta-glu transcript levels remaining high for 4 days after inoculation. In RRIM 600, the mRNA levels decreased significantly 48 h after inoculation. On re-hybridization with an 18S rRNA probe, uniform signals were detected in all the lanes, indicating that an equal amount of total RNA was present in all samples. Similar results were obtained in relative quantitative RT-PCR experiments with the housekeeping actin gene as an internal control. Thus, although induction of the beta-glu gene occurred in both tolerant and susceptible clones, the predominant difference between clones was in the intensity and duration of the response. The tolerance of clone RRII 105 may be associated with the prolonged expression of the gene following infection. The antifungal activity of these hydrolase enzymes makes them rational candidates for overexpression by genetic transformation to produce disease resistant crops.