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The current study is the first worldwide to assess the genetic diversity of Kadaknath poultry using amplified fragment length polymorphism (AFLP) markers. Out of total 96 accessions, four were outliers and 92 were Kadaknath accessions of which 30 were males, 62 were females. Of these, 74 were jet black, 7 penciled and 11 were golden feather color type of Kadaknath. High level of polymorphism (23.94%) was observed in 387 loci amplified using six AFLP primer combinations. The Jaccard's similarity coefficient ranged from 0.211 to 0.754 with an average dissimilarity of 0.517. Based on the neighbor-joining method of clustering, all accessions were clustered into seven major clusters which were not consistent with their respective geographical locations. The mean values of effective multiplex ratio, polymorphic information content, resolving power and marker index were 15.16, 0.38, 9.87 and 5.85 respectively. Further, the high log-likelihood score was produced when the number of populations (K) was set at 7 while carrying out the population STRUCTURE analysis, which was also congruent with clustering analysis based on genetic diversity. The extent of genetic diversity detected in this study could be used for germplasm selection and developing conservation strategies of pure breed of Kadaknath.

Vascular wilt caused by Fusarium udum Butler is the most important disease of pigeonpea throughout the world. F. udum isolate MTCC 2204 (M1) inoculated pigeonpea plants of susceptible (ICP 2376) and resistant (ICP 8863) cultivars were taken at invasion stage of pathogenesis process for transcriptomic profiling to understand defense signaling reactions that interplay at early stage of this plant–pathogen encounter. Differential transcriptomic profiles were generated through cDNA-AFLP from M1 inoculated resistant and susceptible pigeonpea root tissues. Twenty five percent of transcript derived fragments (TDFs) were found to be pathogen induced. Among them 73 TDFs were re-amplified and sequenced. Homology search of the TDFs in available databases and thorough study of scientific literature identified several pathways, which could play crucial role in defense responses of the F. udum inoculated resistant plants. Some of the defense responsive pathways identified to be active during this interaction are, jasmonic acid and salicylic acid mediated defense responses, cell wall remodeling, vascular development and pattering, abscisic acid mediated responses, effector triggered immunity, and reactive oxygen species mediated signaling. This study identified important wilt responsive regulatory pathways in pigeonpea which will be helpful for further exploration of these resistant components for pigeonpea improvement.

In the present study, we aimed to estimate the upper limit of apomixis occurring in Jatropha under field conditions. We used unlinked microsatellite and AFLP markers on progeny arrays of six indivi...

Genetic relatedness of 24 animals belonging to seven Indian cattle breeds was studied using high throughput genotyping-by-sequencing (GBS) markers. GBS produced 93.6 million reads with an average of about 3.9 million reads per animal. A total of 107 488 SNPs were identified in these individuals. When only one SNP per read was considered, a total of 60 261 SNPs representing independent reads were identified with an average SNP-to-SNP distance of 45 kb across the bovine reference genome. About 24% of the GBS-SNP markers were more than 100 kb apart. Of these, 58 322 SNPs mapped to autosomes, 1645 to the X chromosome and 28 to the Y chromosome. The average SNP-to-SNP distance on the X chromosome was 91.3 kb, whereas on the Y chromosome it was 1546.4 kb. The minor allele frequency within the Indian cattle varied from 0.103 (Ongole) to 0.177 (Siri), whereas Holstein cattle had the lowest value of 0.089. This is the first application of GBS in cattle of South Asia. The baseline information generated in this study might prompt implementation of GBS in breeding of cattle belonging to this region.

Commercially cultivated tea clones from Northeast and Southern India were analyzed using TE-AFLP markers. Analysis of molecular variance (AMOVA) and structure analysis showed high genetic variance (74%) within five distinct populations. This study also confirmed that Assam and UPASI (Nilgiri) tea clones have originated from two different tea populations introduced from imported China tea seeds by British. We evaluated the genetic variation and population structure in 30 tea clones of Tea Research Association (TRA), Tocklai Experimental station (Tockali ES), Jorhat, from North Eastern India and 30 clones of United Planters Association of South India (UPASI), Valparai, from South-India, released for commercial plantations and four C. japonica accessions (Total 64) used as outliers using Three Endonuclease-AFLP markers. Six primer combinations were selected from the best 10 primer combinations that were used on a set of 24 tea accessions as a standardization procedure. Dendrograms were constructed by using Jaccard’s similarity coefficient and Unweighted Pair Group Method of Arithmetic Averages (UPGMA) clustering method based on high coefficient correlation ratios obtained. High level of genetic diversity and clustering of accessions according to morphological proximity was established with minor aberrations. Analysis of molecular variance (AMOVA) showed highest genetic variance (79%) within five populations whereas the lowest variance (21%) was found among populations. These values change to the highest 87% and the lowest 13% when Camellia japonica accessions were removed from the analysis. Population structure analyses were performed to establish the genetic diversity and exact type each clones vis-a-vis to their molecular and morphological characteristics at present. Attempts were made to find out the population structure of Northeast and Southern India tea germplasm with their relatedness to existing morphological type. These analyses confirmed that all tea clones were genetically diverse and grouped based on their morphological characteristics and geographic regions.

Outcrossing rates in biodiesel species Pongamia pinnata were estimated using amplified fragment length polymorphism (AFLP) and microsatellite markers. Five AFLP primer combinations and eight pairs of microsatellite primers were used for fingerprinting of 55 open pollinated progenies derived from five mother trees. The multilocus out-crossing rate was quite high with AFLP (0.917; SD = 0.103) and comparatively low with microsatellite markers (0.748; SD = 0.127). The multilocus out-crossing rate was higher than single locus out-crossing rate in case of both marker systems (tm − ts = 0.203; SD = 0.090) which implies that biparental inbreeding occurred at significant levels. The estimated F was higher than the expected which also indicated towards high inbreeding in the progeny population analyzed. This is the first study on estimation of outcrossing rates using any kind of molecular marker systems in Pongamia. We expect an increasing use of such high throughput PCR-based technologies in mating-system studies in various other tree species.

Background: Sepsis is one of the most common causes of neonatal deaths globally. Most sepsis-related deaths occur in low-income and middle-income countries, where the epidemiology of neonatal sepsis remains poorly understood. Most of these countries lack proper surveillance networks, hampering accurate assessment of the burden of sepsis, implementation of preventive measures, and investment in research. We report results of neonates born in hospital from a multicentre collaboration on neonatal sepsis. Methods: In this cohort study, dedicated research teams prospectively followed up neonates born in one of three tertiary care centres in Delhi, India (Vardhaman Mahavir Medical College, Maulana Azad Medical College, and All India Institute of Medical Sciences [coordinating centre]) and subsequently admitted to the intensive care unit. Neonates were followed up daily until discharge or death. On clinical suspicion, neonates underwent sepsis work-up including blood cultures. The isolated organisms were identified and tested for antimicrobial susceptibility. We defined Gram-negative isolates resistant to any three of five antibiotic classes (extended-spectrum cephalosporins, carbapenems, aminoglycosides, fluoroquinolones, and piperacillin-tazobactam) as multidrug resistant. Findings: 13 530 neonates of 88 636 livebirths were enrolled between July 18, 2011, and Feb 28, 2014. The incidence of total sepsis was 14·3% (95% CI 13·8–14·9) and of culture-positive sepsis was 6·2% (5·8–6·6). Nearly two-thirds of total episodes occurred at or before 72 h of life (defined as early onset; 1351 [83%] of 1980). Two-thirds (645 [64%]) of 1005 isolates were Gram-negative including, Acinetobacter spp (22%), Klebsiella spp (17%), and Escherichia coli (14%). The pathogen mix in early-onset sepsis did not differ from that of late-onset sepsis (ie, after 72 h). High rates of multidrug resistance were observed in Acinetobacter spp (181/222, 82%), Klebsiella spp (91/169, 54%), and Escherichia coli (52/137, 38%) isolates. Meticillin resistance prevailed in 61% (85/140) of coagulase-negative staphylococci and 38% (43/114) of Staphylococcus aureus isolates. Nearly a quarter of the deaths were attributable to sepsis. The population-attributable risks of mortality were 8·6% in culture-negative sepsis, 15·7% in culture-positive sepsis by multidrug-resistant organisms, and 12·0% in culture-positive sepsis by non-multidrug-resistant organisms. Interpretation: The high incidence of sepsis and alarming degree of antimicrobial resistance among pathogens in neonates born in tertiary hospitals underscore the need to understand the pathogenesis of early-onset sepsis and to devise measures to prevent it in low-income and middle-income countries. Funding: Indian Council of Medical Research

The authors compared the utility of two variants of Amplified Fragment Length Polymorphism (AFLP) technique, viz. 4-selective AFLP and three Endonuclease Amplified Fragment Length Polymorphism (TE-AFLP), for accurate assessment of genetic diversity in tea. These assays were optimized to resolve the reported discrepancies in detected levels of genetic variation in tea through conventional AFLP assay. The 4-selective AFLP assay involved the use of four selective nucleotides in MseI primer instead of three, in combination with EcoRI primers with three selective nucleotides. This led to a significant reduction in the total number of bands and improved the overall band readability as compared to conventional AFLP. However, presence of a large number of “shadow bands” of varying intensities across lanes, still disallowed an error free scoring. This was effectively resolved by TE-AFLP assay where selective adapter ligation, in addition to selective amplification, contributes to reduction in the complexity of fingerprint pattern. The major advantage of TE-AFLP over 4-select AFLP was its clear and unambiguous banding profiles that simplified the scoring of bands. Both methodologies yielded comparable results with China types clearly separating from Assam types. In contrast to previous reports, the dendrogram revealed an overall tendency of the accessions to cluster based on their morphotypes and their geographic origin. Although, 4-select AFLP detected higher polymorphism information content values, the overall reliability of TE-AFLP bands was higher due to their unambiguous nature and easy scorability. A major fraction of variation (75 %) was found to be partitioned within the morphotypes indicating predominant out-crossing in this species.

North eastern (NE) India harbours a precious germplasm repository of Capsicum in the form of various landraces. The present study was undertaken to characterise the extent of genetic variation present in different Capsicum landraces from north eastern India. A set of 171 Capsicum accessions were characterised using three-endonuclease amplified fragment length polymorphism (AFLP) markers. Out of 416 bands obtained from six primer combinations, 254 (61 %) were polymorphic. The pairwise genetic dissimilarity among accessions ranged from 0.03 to 0.97. Cluster analysis based on neighbour joining showed two major clusters. Cluster I contained most of the bhut jolokia accessions whereas cluster II contained all of the Capsicum annuum genotypes. Similar grouping was observed with population STRUCTURE analysis as well as principle coordinate analysis. Analysis of molecular variance (AMOVA) revealed 45 and 54 % variation among and within populations, respectively. This information on population structure analysis and molecular characterisation will be helpful for effective utilisation of this germplasm in Capsicum improvement programs.

In this paper, we report the estimates of outcrossing rates using open-pollinated progeny arrays of 40 BC1 individuals of Jatropha developed as a result of interspecific hybridization between J. curcas and J. integerrima. For analysis PCR-based dominant AFLP and codominant SSR markers were used. The multilocus outcrossing rate (tm) estimated from AFLP markers (0.892 ± 0.112) are almost in the same range with SSR (0.884 ± 0.293) markers which indicate a high level of heterozygosity. A low value of inbreeding coefficient (F) also points out to the fact that outcrossing was the prevalent mode of reproduction in Jatropha and suggests maintenance of adequate genetic variability within families.

Hippophae rhamnoides L. belonging to family Elaeagnaceae has been a species of interest due to its high nutritional value. In the present study, a set of 26 novel microsatellites from Hippophae developed from an enriched genomic library using biotin labelled di, tri and tetra nucleotide repeats have been reported. Out of 108 clones sequenced, only 62 fragments comprised of microsatellites. All the 26 designed primers showed amplification and 14 highly polymorphic markers were selected and genotyped in 24 Hippophae accessions. The number of alleles ranged from 3 to 7 alleles per locus with an average of 5. The average genetic distance among these accessions was 0.64. Polymorphic information content ranged from 0.47 to 0.79. Mean expected heterozygosity (He) and observed heterozygosity (Ho) were 0.69 and 0.43 respectively. Hardy–Weinberg and Linkage disequilibrium showed that all 14 polymorphic loci were in HWE (P

We report on identification of core collection from 192 accessions collected from 12 Indian states and five other countries based on 109 polymorphic amplified fragment length polymorphism (AFLP) markers. Pairwise Jaccard's similarity coefficient for accessions varied from 0.25 to 1 with a maximum genetic distance of 0.75 obtained between accessions Jc428 (from Mexico) and J204 (from Madurai, Tamil Nadu). Both UPGMA (Unweighted Pair Group Method of Arithmetic Averages) clustering and principal coordinate analyses showed similar grouping of accessions in three major clusters in which Mexican accessions clustered separately from Indian, Chinese and African accessions. Results obtained from analysis of molecular variance indicated that 59% of the genetic variation was distributed among the populations, while 41% of variation was within the populations. A total of 16 (8.3% of the entire collection) core accessions were identified, which contained the entire allelic diversity of 192 accessions with respect to the sampled AFLP loci. The core accessions would be highly useful for future genetic improvement ofJatropha. To the best of our knowledge, this is the first report on identification of core accessions inJatropha.

Genetic and fatty acid variability in four datasets of accessions and prebreeding lines of Jatropha curcas was analyzed. The datasets were comprised of J. curcas accessions (13), BC1 (28), BC1F2 (12) and single seeds (12). The BC1, BC1F2 and single seed dataset were derived from an interspecific cross between J. curcas and J. integerrima. The average within-group (within dataset) polymorphism revealed by AFLP markers was 28.5 %. The average genetic similarity within the four datasets, namely, J. curcas accessions, BC1, BC1F2 and single seeds was 0.92, 0.82, 0.90 and 0.92 respectively with an overall average genetic similarity of 0.80. The values of percent polymorphism, Shannon’s information index and expected heterozygosity were highest for BC1 dataset. The total unsaturated fatty acids in the oil varied from 63.64 to 89.10 %. Levene’s test showed that within-group variance was significantly different for three fatty acids, namely, palmitic acid, oleic acid and linoleic acid. BC1F2 dataset had the highest variance for palmitic and oleic acid, whereas BC1 dataset had the highest variance for linoleic acid. The results revealed that both genetic as well as fatty acid variability was significantly higher in the newly created prebreeding lines and the level of fatty acid variability in these datasets was different and highly correlated to the level of their genetic variability as revealed by AFLP markers. This new prebreeding material, especially, the BC1 and BC1F2 lines, will be useful for development of Jatropha varieties with specific fatty acid composition for specific industrial applications.

Jatropha curcas has been a species of interest all over the world due to its potential as a renewable biodiesel crop. In the present paper, a set of 40 novel genomic microsatellites from Jatropha developed through cloning and sequencing are reported. The microsatellite markers were evaluated at intra-specific, inter-specific and inter-generic levels. All forty markers successfully amplified fragments from J. curcas and J. integerrima showing their potential in linkage mapping using inter-specific breeding populations involving these species. The average number of alleles within 12 core accessions of J. curcas was 3.083. Polymorphic information content ranged from 0.15 to 0.80 within J. curcas accessions and from 0 to 0.37 among different genera. There was no significant deviation from Hardy–Weinberg equilibrium or linkage disequilibrium. Ten of these microsatellites showed polymorphism within Emblica officinalis while thirteen were polymorphic within Phyllanthus niruri and Ricinus communis accessions. These new set of microsatellite markers are expected to be especially useful in future for linkage mapping in J. curcas and for varietal identification and genetic diversity studies in other genera mentioned above.

a b s t r a c t Level of variation in capsaicinoid content was evaluated in Capsicum accessions belonging to chilli land races from north-eastern India. Accessions from NE India showed enormous variation in morphological characters such as fruit shape, size, colour, and fruit position. Capsaicinoid content in 139 accessions var- ied from 0.02 to 72.05 mg/g. The ratio between two major capsaicinoids, capsaicin and dihydrocapsaicin, varied from 0.34 to 4.92. The capsaicinoid content among 92 accessions of Bhut Jolokia varied from 11.95 to 72.05 mg/g with corresponding pungency levels of 191,135-1,152,832 SHU. A strong positive correlation (r = 0.90) was obtained between capsaicin and dihydrocapsaicin content. © 2014 Elsevier B.V. All rights reserved.

Seed development in Jatropha curcas L. was studied with respect to morphology, oil content and lipid profiles. Seeds were collected at 8 different stages of development starting from 6 days after pollination till maturity. Seed oil content increased from 6.15% to 35.86%. Palmitic acid (16:0) decreased from 34.27-15.62, whereas linolenic acid (18:3) reduced from 26.32 to 0.65%. A sharp increase in oleic acid (18:1) content from 8.05 to 29.56% was observed at stage 5 which increased further to 44.38% at maturity. Linoleic acid (18:2) increased steadily from 33.38 to 47.19 % till stage 7 but reduced to 33.02% at maturity with a simultaneous increase in oleic acid. Remarkable changes in fatty acid composition were observed after stage 4 (27 DAP). The biodiesel quality of oil at stage 8 was better than at stage 7 as indicated by lower iodine number and higher cetane number.

Genetic diversity of Pongamia pinnata, a plant species emerging as biodiesel source, was assessed using three endonuclease (TE)-amplified fragment length polymorphism (AFLP) markers. A total of 275 accessions from different Indian geographical regions were collected. High level of polymorphism (62.6%) was observed in 334 loci amplified using six primer combinations. The Jaccard’s dissimilarity coefficient ranged from 0.17 to 0.80. Based on the neighbour joining method of clustering, accessions were grouped into six major clusters congruent with their respective geographical locations. The mean values of effective multiplex ratio, polymorphic information content, marker index and resolving power were 34.83, 0.286, 9.700 and 14.29 respectively. In addition, a core collection consisting of 20 accessions was identified. Population STRUCTURE analysis produced the highest log likelihood score when the number of populations (K) was set at 2, which was not consistent with clustering analysis based on genetic diversity. Two TE-AFLP markers, E_ac x P_a04 and E_ac x P_a39, were significantly associated with oil content (at P = 0.005). The extent of genetic diversity detected and core germplasm of CPT identified in this study could be selected and used for tree breeding, germplasm conservation and suitable feed stock for biofuel.

Phenotypic variation of important seed traits like seed length, seed breadth, seed thickness, 100 seed weight and seed oil content were recorded in a total of 157 collected accessions of Pongamia. Out of these, fatty acid profiles of 38 accessions selected based on their high and low oil content was analyzed. Fatty acid profile revealed high variability in stearic, oleic and linoleic acid which varied from 0.42 to 10.61 %, 34.34 to 74.58 %, and 7.00 to 31.28 % respectively. Variations in palmitic and linolenic acid were small. Iodine value, saponification number and cetane number (CN) of fatty acid methyl esters (FAME) of seed oil ranges from 186.99 to 201.25, 81.13 to 108.19 and 46.16 to 56.47 respectively. Fatty acid compositions, degree of unsaturation and CN are the important parameters, which are used to determine quality of FAME were used as biodiesel. Some of the Pongamia accessions identified were higher in oil content while some accessions showed higher degree of unsaturation and a few of them had CN values higher than 55. Genetic diversity analysis with six TE-AFLP primers generated a total of 334 bands out of which 174 (52.10 %) were polymorphic. The genetic similarity ranged from 0.11 to 0.47. These findings clearly showed high level of genetic diversity and all economically desirable traits were not present in a single genotype of Pongamia. All these traits could be selected from these CPTs and transfer to a single elite variety through selection and breeding programme and could be utilized for large scale multiplication and plantation to produce high quantity and quality biodiesel in future.

The most important evolutionary event in the success of commercial tea cultivation outside China in ~30 countries came about by the origin of India hybrid tea in India, derived from the extensive spontaneous hybridization that took place between the Assam type tea growing in the forest regions of Assam, North-East India and China type tea introduced from China in ~1875 to many regions of North-East India. The release of an enormous pool of vigorous and highly variable plants of India hybrid tea in North-East India was a significant step forward for the origin and evolution of tea as a highly successful crop plant. The 1,644 accessions and clones of India hybrid tea, representatives of known 15 morphotypes, were screened by 412 AFLP markers amplified by 7 AFLP primer pair combinations. All the 412 genetic loci were polymorphic across the 1,644 accessions and clones. The analysis was done with distance (PCoA and NJ) methods, and the STRUCTURE (Bayesian) model. Both PCoA and NJ analysis clustered 1,644 tea accessions and clones into six major groups with one group in each, constituted mostly by China hybrid, Assam China hybrid and Assam hybrid morphotypes, of distinct genetic identity. No group was exclusive for any particular morphotype. The accessions and clones belonging to morphotypes, Assam type, Assam hybrid, China hybrid and China Cambod were distributed in all the groups. It is the Assam type/Assam hybrid morphotypes which exhibit much broader genetic variability than in China type/China hybrid/Cambod type/Cambod hybrid morphotypes. The STRUCTURE analysis inferred 16 populations (K = 16), for which the greatest values of probability were obtained. Nine of the 16 clusters were constituted by the tea accessions and clones of ‘pure’ ancestry. The remaining clusters were of ‘mixed’ ancestry. This analysis provides evidence that the accessions and clones of the same morphotype are not always of same genetic ancestry structure. The tea accessions and clones obtained from outside North-East India shared the same groups (distance method) and clusters (STRUCTURE model) which were constituted by North-East India accessions. The present study also demonstrates very narrow genetic diversity in the commercial tea clones vis-a-vis the profound genetic diversity existing in the tea accessions. These clones were distributed in hardly two of the six groups in NJ tree. The identified 105 core accessions and clones, capturing 98% diversity, have their origin from almost all groups/subgroups of NJ tree.

Efficacy of two dominant molecular markers, namely, amplified fragment length polymorphism (AFLP) and three endonuclease (TE)-AFLP, were assessed in 20 individuals of the biodiesel species Pongamia pinnata. Four primer combinations generated a total of 254 and 194 bands in AFLP and TE-AFLP, respectively. Both techniques could unequivocally identify each accession used in this study. The Jaccard’s similarity coefficient ranged from 0.30 to 0.90 for AFLP and from 0.25 to 0.85 for TE-AFLP. The correlation coefficient between AFLP and TE-AFLP dendrogram was 0.56 which was low but significant (P

The importance of neem (Azadirachta indica A. Juss.) as a medicinal tree species has been acknowledged worldwide. Superior trees with desired traits such as high azadirachtin content have been identified and micropropagated. Somaclonal variants that may arise in vitro, however, pose limitations to large-scale micropropagation. It is, therefore, imperative to establish genetic uniformity of such plantlets by ensuring strict quality checks at various stages of in vitro culture. This is the first study that evaluates the applicability of amplified fragment length polymorphism (AFLP) markers in establishing clonal fidelity of tissue culture(TC)-raised neem plants. Seven AFLP primer combinations generated a total of 334 amplified fragments across the mother plant, TC progenies, and other neem accessions that were included as controls. Two hundred and thirty-nine amplified fragments were monomorphic across the mother tree and its TC progenies. No extra band was detected in the TC plantlets that was absent in the mother tree, indicating that the TC plantlets regenerated through nodal explants are indeed true-to-type. Ninety-five AFLP fragments were detected in the controls, which allowed their discrimination from the elite mother tree and its TC progenies. Similarity matrix based on Jaccard's coefficient revealed that the pair-wise value between the mother tree and its TC plantlets was ‘1’, indicating perfect similarity. Phenetic dendrogram based on UPGMA (unweighted pair group method of arithmetic averages) analysis further confirmed the true-to-type nature of TC progenies, since a tie was observed between the mother tree and its TC plantlets. On the contrary, the control neem accessions were distinct from the mother and its TC progenies. AFLP markers proved to be an ideal tool for routine analysis and certification of genetic fidelity of micropropagated plants prior to commercialization, especially in tree species because of their long generation time.

Withania somnifera is an important medicinal plant, and its anticancerous properties have been attributed to various classes of withanolide compounds. The objective of the present study was to investigate the inter- and intraspecific genetic variation present in 35 individuals of W. somnifera and 5 individuals of W. coagulans using AFLP (amplified fragment length polymorphism) marker technique. The information about genetic variation determined from AFLP data for 40 individuals was employed to estimate similarity matrix value based on Jaccard's coefficient. The similarity values were further used to construct a phenetic dendrogram revealing the genetic relationships. The dendrogram generated by UPGMA (unweighted pair group method of arithmetic averages) distinguished W. somnifera from W. coagulans and formed two major clusters. These two main clusters shared a similarity coefficient of 0.3, correlating with the high level of polymorphism detected. The dendrogram further separated W. somnifera into three subclasses corresponding to Kashmiri and Nagori groups and an intermediate type. The AFLP profile of Kashmiri individuals was distinct from that of the Nagori group of plants. The intermediate genotype was distinct as it shared bands with both the Kashmiri and Nagori individuals, even though it was identified as a Kashmiri morphotype. Furthermore, the intermediate type shared a similarity coefficient of 0.8 with the Kashmiri individuals. The present work revealed low levels of variation within a population though high levels of polymorphism were detected between Nagori and Kashmiri populations. The ability of AFLP markers for efficient and rapid detection of genetic variations at the species as well as intraspecific level qualifies it as an efficient tool for estimating genetic similarity in plant species and effective management of genetic resources.

Genetic diversity was estimated in 37 neem accessions from different eco-geographic regions of India and four exotic lines from Thailand using AFLP markers. Seven AFLP selective primer combinations generated a total of 422 amplification products. The average number of scorable fragments was 60 per experiment, and a high degree (69.8%) of polymorphism was obtained per assay with values ranging from 58% to 83.8%. Several rare and accession-specific bands were identified which could be effectively used to distinguish the different genotypes. Genetic relationships within the accessions were evaluated by generating a similarity matrix based on the Jaccard index. The phenetic dendrogram generated by UPGMA as well as principal correspondence analysis separated the 37 Indian genotypes from the four Thai lines. The cluster analysis indicated that neem germplasm within India constitutes a broad genetic base with the values of genetic similarity coefficient ranging from 0.74 to 0.93. Also, the Indian genotypes were more dispersed on the principal correspondence plot, indicating a wide genetic base. The four lines from Thailand, on the other hand, formed a narrow genetic base with similarity coefficients ranging from 0.88 to 0.92. The lowest genetic similarity coefficient value (0.47) was observed between an Indian and an exotic genotype. The level of genetic variation detected within the neem accessions with AFLP analysis suggests that it is an efficient marker technology for delineating genetic relationships amongst genotypes and estimating genetic diversity, thereby enabling the formulation of appropriate strategies for conservation and tree improvement programs.

Aquilaria malaccensis is one of the 15 tree species of the genus Aquilaria widely distributed in the Indo-Malaysian region. Its resin (Agar or Gaharu) is widely known for its use in the production of highly valued incense and traditional medicine throughout Asia. The species has been listed as ‘Vulnerable’ in the IUCN red list of threatened species due to its over exploitation. In the present study, for the first time, next generation sequencing platform (Ion Torrent PGM™) was used to obtain a large number of sequences containing microsatellites for A. malaccensis. A total of 527, 425 high quality reads were identified, which contained 4138 SSRs. In these SSRs, the number of dinucleotide repeats were the most frequent one (74.77%). Primers were developed for 93 loci; of these 82 loci were successfully amplified and 47 were polymorphic across a panel of 42 genotypes. Fifteen loci with at least three or more alleles were chosen for detailed characterization. The observed heterozygosity ranged from 0.25 to 0.90, whereas the expected heterozygosity ranged from 0.24 to 0.89, with 5.9 alleles per locus on average. Simple matching dissimilarity coefficients and cluster analysis revealed significant diversity among the genotypes. The study suggested that PGM™ sequencing is a rapid technique for developing microsatellite markers. The microsatellite markers developed in the present study would be useful in comprehensive genetic diversity studies aimed towards the conservation and germplasm management of Aquilaria.

Restriction enzyme analysis of the rRNA genes of Eruca sativa indicated the presence of many length variants within a single plant and also between different cultivars which is unusual for most crucifers studied so far. Two length variants of the rDNA intergenic spacer (IGS) from a single individual E. sativa (cv. Itsa) plant were cloned and characterized. The complete nucleotide sequences of both the variants (3 kb and 4 kb) were determined. The intergenic spacer contains three families of tandemly repeated DNA sequences denoted as A, B and C. However, the long (4 kb) variant shows the presence of an additional repeat, denoted as D, which is a duplication of a 224 bp sequence just upstream of the putative transcription initiation site. Repeat units belonging to the three different families (A, B and C) were in the size range of 22 to 30 bp. Such short repeat elements are present in the IGS of most of the crucifers analysed so far. Sequence analysis of the variants (3 kb and 4 kb) revealed that the length heterogeneity of the spacer is located at three different regions and is due to the varying copy numbers of repeat units belonging to families A and B. Length variation of the spacer is also due to the presence of a large duplication (D repeats) in the 4 kb variant which is absent in the 3 kb variant. The putative transcription initiation site was identified by comparisons with the rDNA sequences from other plant species.

India has a large repository of important tea accessions and, therefore, plays a major role in improving production and quality of tea across the world. Using seven AFLP primer combinations, we analyzed 123 commercially important tea accessions representing major populations in India. The overall genetic similarity recorded was 51%. No significant differences were recorded in average genetic similarity among tea populations cultivated in various geographic regions (northwest 0.60, northeast and south both 0.59). UPGMA cluster analysis grouped the tea accessions according to geographic locations, with a bias toward China or Assam/Cambod types. Cluster analysis results were congruent with principal component analysis. Further, analysis of molecular variance detected a high level of genetic variation (85%) within and limited genetic variation (15%) among the populations, suggesting their origin from a similar genetic pool.

Hybrids of Populus ciliata x maximowiczii are very vigorous and outperform both the parents in growth performance and yield. Genetic evaluation of 24 of these interspecific hybrids along with the two mother trees ( Populus ciliata), and five male-parent ( Populus maximowiczii) genotypes was carried out using the AFLP marker assay. Eight AFLP primer combinations detected 428 markers, of which 280 (66%) were polymorphic. Genetic relationships within the samples were evaluated by generating the similarity matrix based on Jaccard's coefficient. The phenetic dendrograms, as well as the PCO plots, separated the hybrids and the two parent species into three distinct clusters. The hybrids grouped closer to the P. ciliata (female parent) cluster as compared to the P. maximowiczii (male parent) cluster. The hybrid cluster contained internal groupings, which correlated to some extent with growth performance. The four best performing hybrids (42m1, 65m1, 23m2, Cm2-5-20/91) formed a distinct sub-cluster. Data from a single primer combination was sufficient for distinguishing the hybrids from the parents and assigning paternity. The hybrids showed 22 markers that were absent in P. ciliata but were monomorphically present in all the hybrids, suggesting outcrossing and common paternity. Further, these 22 markers were found in all the P. maximowiczii genotypes confirming it as the male parent. These male-specific markers can be converted to SCAR markers and used for rapid screening of the P.ciliata x maximowiczii hybrids. The primer combination E-AAC x M-CAA was identified as most suitable for ascertaining true hybridity. AFLP proves to be a useful tool for screening of P. ciliata x maximowiczii hybrids at the early stages of development.

The 5S rRNA genes and their associated non-transcribed spacer (NTS) regions are present as repeat units arranged in tandem arrays in plant genomes. Length heterogeneity in 5S rDNA repeats was previously identified in Populus deltoides and was also observed in the present study. Primers were designed to amplify the 5S rDNA NTS variants from the P. deltoides genome. The PCR-amplified products from the two accessions of P. deltoides (G3 and G48) suggested the presence of length heterogeneity of 5S rDNA units within and among accessions, and the size of the spacers ranged from 385 to 434 bp. Sequence analysis of the non-transcribed spacer (NTS) revealed two distinct classes of 5S rDNA within both accessions: class 1, which contained GAA trinucleotide microsatellite repeats, and class 2, which lacked the repeats. The class 1 spacer shows length variation owing to the microsatellite, with two clones exhibiting 10 GAA repeat units and one clone exhibiting 16 such repeat units. However, distance analysis shows that class 1 spacer sequences are highly similar inter se, yielding nucleotide diversity (π) estimates that are less than 0.15% of those obtained for class 2 spacers (π = 0.0183 vs. 0.1433, respectively). The presence of microsatellite in the NTS region leading to variation in spacer length is reported and discussed for the first time in P. deltoides.Key words: 5S rDNA, Populus, repetitive DNA, microsatellite, sequence heterogeneity.

EcoRI restriction of the B. nigra rDNA recombinants, isolated from a lambda genomic library, showed that the 3.9-kb fragment corresponded to the Intergenic Spacer (IGS), which was sequenced and found to be 3,928 bp in size. Sequence and dot-matrix analyses showed that the organization of the B. nigra rDNA IGS was typical of most rDNA spacers, consisting of a central repetitive region and flanking unique sequences on either side. The repetitive region was composed of two repeat families-RF 'A' and RF 'B.' The B. nigra RF 'A' consisted of a tandem array of three full-length copies of a 106-bp sequence element. RF 'B' was composed of 66 tandemly repeated elements. Each 'B' element was only 21-bp in size and this is the smallest repeat unit identified in plant rDNA to date. The putative transcription initiation site (TIS) was identified as nucleotide position 3,110. Based on the sequence analysis it was suggested that the present organization of the repeat families was generated by successive cycles of deletions and amplifications and was being maintained by homogenization processes such as gene conversion and crossing-over.A detailed comparison of the rDNA IGS sequences of the three diploid Brassica species-namely, B. nigra, B. campestris, and B. oleracea-was carried out. First, comparisons revealed that B. campestris and B. oleracea were close to each other as the repeat families in both showed high sequence homology between each other. Second, the repeat elements in both the species were organized in an interspersed manner. Third, a 52-bp sequence, present just downstream of the repeats in B. campestris, was found to be identical to the B. oleracea repeats, thereby suggesting a common progenitor. On the other hand, in B. nigra no interspersion pattern of organization of repeats was observed. Further, the B. nigra RF 'A' was identified as distinct from the repeat families of B. campestris and B. oleracea. Based on this analysis, it was suggested that during speciation B. campestris and B. oleracea evolved in one lineage whereas B. nigra diverged into a separate lineage. The comparative analysis of the IGS helped in identifying not only conserved ancestral sequence motifs of possible functional significance such as promoters and enhancers, but also sequences which showed variation between the three diploid species and were therefore identified as species-specific sequences.

The 459-bp HindIII (pBN-4) and the 1732-bp Eco RI (pBNE8) fragments from the Brassica nigra genome were cloned and shown to be members of a dispersed repeat family. Of the three major diploid Brassica species, the repeat pBN-4 was found to be highly specific for the B. nigra genome. The family also hybridized to Sinapis arvensis showing that B. nigra had a closer relationship with the S. arvensis genome than with B. oleracea or B. campestris. The clone pBNE8 showed homology to a number of tRNA species indicating that this family of repeats may have originated from a tRNA sequence. The species-specific 459-bp repeat pBN-4 was localized on the B. nigra chromosomes using monosomic addition lines. In addition to the localization of pBN-4, the chromosomal distribution of two other species-specific repeats, pBN34 and pBNBH35 (reported earlier), was studied. The dispersed repeats pBN-4 and pBNBH35 were found to be present on all of the chromosomes, whereas the tandem repeat pBN34 was localized on two chromosomes.