Pine oleoresin is a complex mixture of volatile and nonvolatile terpenes and is exploited for commercial production in India. Pines have long reproductive cycle vegetative phase extending upto many years. Therefore, there is an urgent need to identify high resin pine yielders at an early stage. Due to the strong influence of genetic factors on resin yield in pine species, marker assisted selection may serve as a potential tool for early identification of the genotype of interest. This study was planned to identify high resin pine yielders at early stage to avoid exploitation of natural germplasm of chir pine. Protein sequences of different terpene synthases were downloaded from the NCBI database and were multiply aligned to identify conserved and variable regions across the sequences. Primers were designed based on the related coding sequences to target these regions using Primer 3 software to amplify and isolate related genomic loci in chir pine genotypes. These primers were characterized for specificity using Primer Blast and the presence of hairpin loop formation using OligoCalc software. DNA was extracted from different resin yielding pine genotypes and primers were run on them for molecular studies. With the help of bioinformatics tools, we were able to associate some markers with resin yield. This study holds a key promise for the conservation of pine germplasm in natural habitat.
Association mapping is a method for detection of gene effects based on linkage disequilibrium (LD) that complements QTL analysis in the development of tools for molecular plant breeding. A total of 240 genotypes of Pinus roxburghii (Himalayan Chir Pine) from a natural population in Chakrata division (Tiunee range), Uttarakhand (India) were evaluated for resin yield. Based on the phenotypic data and stable resin production in consecutive years, 53 genotypes were selected after excluding the individuals with similar resin production. The selected 53 individuals were best representatives of the variation in resin yield in Chakrata population which varied between 0.25 and 8.0 kg/tree/year and were used for genotyping and association analysis using SSR markers. Out of 80 primers initially screened, a total of 19 polymorphic SSRs (11 cpSSR and 8 nSSR) were used in the study. Model based clustering using 19 polymorphic SSR markers identified five subpopulations among these genotypes. LD was evaluated using the entire population. The squared allele frequency correlation, r2 was estimated for each pair of SSR loci. The comparison wise significance (p-values) of SSR marker pairs was determined by performing 100,000 permutations. The genetic divergence ranged from 50 to 100%. The UPGMA based hierarchial clustering grouped the genotypes in accordance with their resin yield. Model based clustering suggested the existence of five subpopulations in the sample. However, the distribution of P. roxburghii genotypes into five subpopulations had no correlation with their resin yield thus ruling out the possibility of any ancestral relationship among the genotypes with similar resin yield. AMOVA suggested that the variation among P. roxburghii genotypes at the molecular level was related with the variation in resin yield and not their site of collection thus highlighting the genetic basis of the trait. LD based association analysis revealed two chloroplast SSRs Pt71936 and Pt87268 and one nuclear SSR pm09a to be in significant association with resin yield. The two associated chloroplast SSRs showed significant LD (p<0.01). One of the chloroplast SSR Pt87268 showing association with resin yield was also found to be in significant LD with the nuclear SSR pm07, further showing the probability of this marker also to be associated with resin yield.
A set of 19 SSR (Simple Sequence Repeats), 9 ISSR (Inter-Simple Sequence Repeats) and 5 AFLP (Amplified Fragment Length Polymorphism) primer combinations were used to evaluate the variability among 53 genotypes of Pinus roxburghii selected based on resin yield from the natural zone of occurrence of this species in Uttarakhand, India. The selected trees of pine varied in resin yield from 0.25 to 8 kg/year/tree. Based on the comparative assessment of SSR, ISSR and AFLP markers, SSR markers were found most polymorphic with an average PIC value of 0.327 and 2.42 alleles per marker, while ISSR markers showed the highest effective multiplex ratio (15.536) and marker index (4.958). AFLP markers showed the maximum resolving power (8.099) which was comparable to the resolving power (8.059) of ISSR markers. UPGMA-based dendrogram using SSR markers revealed more distinct grouping of genotypes on the basis of resin yield as compared to ISSR and AFLP markers. AMOVA by collection site revealed no significant variation among the populations. Whereas, AMOVA by resin yield using SSR, ISSR and AFLP markers revealed FST values to be 0.1096, 0.0483 and 0.2422 indicating moderate, low and great genetic differentiation among the groups. This clearly indicated that the variation at the molecular level was attributed to the resin yield and not the site of collection.
Results of a provenance trial of Acacia nilotica Willd. ex Del. laid out in 1993 at Tropical Forest Research Institute Campus, Jabalpur (23°N lat., 79°E long. and 400 m altitude) Madhya Pradesh, a semi-arid region of India are reported and discussed. Nineteen provenances from India, Pakistan, Sudan, Senegal and Yemen were evaluated in the field at age three and six years. Local source of A. nilotica (S.F.R.I., Jabalpur) was used as check material for comparison. Significant differences between the provenances (P < 0.05) were observed for height, diameter at breast height (DBH), number of branches and field survival. The provenances from Gujrat (Punjab), Pakistan, ranked first for growth traits namely height, DBH and survival. The next superior provenance was from Beihan, Yemen, which scored second highest values of height and DBH and had good survival at age six years. Results indicate that genetic differences exist between the provenances of A.nilotica. None of the traits assessed (viz. height, DBH, number of branches, inter-nodal length and survival) were found to correlate with any of the geographical coordinates of the provenances (latitude, longitude and altitude). Height, DBH and number of branches showed significant and positive correlation with each other. Fair differences between phenotypic and genotypic coefficient of variability were observed. Heritability values were found fairly good for height, DBH and number of branches as compared to inter-nodal length. The relative performance of these provenances was fairly consistent throughout the period of observation at three and six year. The two sources viz. Gujrat (Punjab), Pakistan and Beihan, Yemen, are recommended for plantation programme under semi-arid conditions.
Pinus kesiya (Khasi pine) is the principal pine species in northeast India having high commercial value. Chloroplast microsatellites (cpSSR) were used to study the genetic diversity and population genetic structure of 10 populations of P. kesiya covering entire natural range of distribution in India. A total of 33 primer pairs (cpSSRs) of P. thunberghii and P. sylvestris were tested in P. kesiya for their transferability, out of which 18 chloroplast primers showed positive amplification and 10 were found polymorphic. A total of 250 individuals from 10 different populations were genotyped using the selected 10 cpSSRs. When alleles at each of the 10 loci were jointly analysed a total of 36 size variants were discovered, which combined to designate 90 haplotypes among 250 individuals. None of the haplotype was found common among the populations as they were population specific. The cpSSR indicated that P. kesiya populations have maintained a moderately high genetic diversity (HT=0.638) which is typical in most coniferous species. However, the inter-population genetic diversity was higher than the intra population diversity and the genetic differentiation between populations was also found to be very high (FST=0.47). A Bayesian cluster analysis separated the populations into six clusters where most of the individuals were found in single population clusters with minor admixtures. The distribution of genetic diversity and sub structuring of P. kesiya reflect week pollen mediated gene flow due to geographic isolation and genetic drift. The study has revealed useful cpSSR markers for P. kesiya, which were lacking earlier and also added an insight into the state of Khasi pine forest in the region, which can be useful for the better management and future conservation programs.
H. S. Ginwal, S. S. Phartyal, P. S. Rawat and R. L. Srivastava
The aim of the study was to determine source variation in Jatropha curcas seeds collected from ten locations in Central India. A significant seed source variation was observed in seed morphology (colour, size and weight), seed germination (viability, germination percent, germination energy, germination value) and seedling growth parameters (survival percentage, seedling height, collar diameter, leave/plant, and seedling biomass). The seed source of Chhindwara (M.P.) was found as the best source in comparison to others. The phenotypic and genotypic variance, their coefficient of variability and broad sense heritability also showed a sizeable variability. This offers a breeder ample scope to undertake screening and selection of seed sources for the desired traits. Further, high percentage of heritability coupled with moderate intensity of genetic gain, was observed for seed germination traits, which signifies that germination is under strong genetic control and good amount of heritable additive genetic component can be exploited for improvement of this species.
Anita Rawat, S. Barthwal, H. S. Ginwal and S. B. Tripathi
In Pinus roxburghii, marker-trait associations were studied for resin yield using a set of 238 DNA based markers derived from 9 ISSR and 5 AFLP primer pairs. A total of 240 genotypes of P. roxburghii from a natural population in Chakrata division (Tiunee range), Uttarakhand (India) were evaluated for resin yield. Based on stable resin production in consecutive years, 53 genotypes that were best representatives of the variation in resin yield (0.25 to 8.0 kg/tree/year) were used for genotyping and association analysis. For carrying out the association studies, two approaches were used. First, using simple linear regression, resin yield was regressed on all 238 available polymorphic markers (148 ISSR markers and 90 AFLP markers) and second, evidence for association was examined applying population structure information using STRUCTURE and STRAT to avoid any spurious associations between a candidate marker and phenotype. In regression analysis, 16 ISSR and 12 AFLP markers showed significant association with resin yield. In the second approach, 18 ISSR and 18 AFLP loci were found to be associated with resin yield in the absence of population structure out of which 2 ISSR and 3 AFLP loci were common with those associated in regression analysis. After accounting for population structure, 38 ISSR, and 3 AFLP loci revealed association out of which one ISSR and one AFLP locus was found to be common with both simple linear regression and test of association in the absence of population stratification. There was no obvious correlation between population structure and resin yield.
H. S. Ginwal, P. Chauhan, S. Barthwal, A. Sharma and R. Sharma
The study reports the transferability of chloroplast microsatellite markers developed for Pinus species to Cedrus deodara. A total of 49 primer pairs (both nuclear and chloroplast) of Pinus species were tested in C. deodara out of which 21 chloroplast primers showed positive amplification and 20 were found polymorphic. The primers were screened on 100 adult trees of two natural populations of C. deodara. Using twenty cpSSR primers, a total of 64 variants were found which combined in 70 different haplotypes. The total haplotype diversity in two populations was 0.860 and 0.876 with a mean of 0.868. These sets of markers can further be used for population genetic studies and characterization in C. deodara for which no cpSSR markers have been reported till date.
Results of a seed source evaluation trial of Jatropha curcas Linn. laid out in 1996 at Jabalpur (M.P), a semi arid region of India are reported and discussed. Seeds collected from ten sources from central India representing the states Madhya Pradesh and Maharashtra viz. Gondia, Bichia, Balaghat, Niwas, Khandwa, Burhanpur, Nasik, Chindwara, Kundam and Jabalpur were evaluated for their growth performance from nursery stage (3 months) to field (two years). Variation in seed oil content of different sources were also studied and reported. Significant differences between the seed sources at age 27 months were observed for height, collar diameter, number of branches leaf area and field survival. Seeds collected from different sources also varied significantly in respect of seed and kernel weight and oil content in seed/kernel. The Chhindwara (M.P.) source performed the best and yielded a maximum oil of 39.12% from whole seed and 58.12% from kernel. The oil content ranged from 33.02 to 39.12% in whole seeds and 47.08 to 58.12% in kernel, across the seed sources. The performance of Kundam (M.P.), Jabalpur (M.P.), Bichia (M.P.), Niwas (M.P.) and Nasik (Maharashtra) sources were also found satisfactory for oil yield. Results indicate that genetic differences exist between the seed sources of J. curcas. The growth traits showed significant positive correlation with each other. Fair differences between phenotypic and genotypic coefficient of variability were observed. Heritability (broad sense) values were fairly good with regard to leaf area, height and collar diameter in comparison to survival percent. The relative performance of these sources was fairly consistent throughout the observation period.
H. S. Ginwal, Pradip Kumar, V. K. Sharma and A. K. Mandal
Results of a provenance trial of Eucalyptus camaldulensis Dehnh. laid out in 2001 at Seothi (30° N Lat., 76° E Long. and 250 m altitude) Haryana, a semi-arid region of India are reported and discussed. Six seed sources from Australia viz. Laura River, Emu Creek, Degrey River, Wiluna, Kennedy River, Morehead River were evaluated from nursery stage (3 months) to field performance (2 year). Two sources viz. E.tereticornis (Helenvele, Qld) and FRI-4 a local seed source were used as control (check material). Significant differences between the seed sources at nursery stage were observed for collar diameter (P < 0.05) and number of leaves (P < 0.01). At age 2 years, significant differences between the seed sources (P < 0.05) were observed for height and field survival. The two seed sources viz. Emu Creek Petford, QLD and Laura river, QLD ranked first and second for height and survival at this age. Results indicate that genetic differences exist between the sources of E. camaldulensis. None of the traits assessed (viz. height, collar diameter, number of leaves, number of branches and survival) was found to correlate with any of the geographical coordinates of the site (latitude, longitude and altitude). There were fair differences between phenotypic and genotypic coefficient of variability. Heritability values were fairly high for height and clear bole length in comparison to collar diameter, number of branches and survival. The relative performance of these provenances was fairly consistent throughout the period of observation. The two sources viz. Laura river, QLD and Emu Creek Petford, QLD are recommended for potential source for plantation programme under semi-arid conditions.