Volume 4 Special Issue 1 2010
Focus on Tree and Forest Genetics

ISBN 978-4-903313-54-2

Guest Editors

Madhugiri Nageswara-Rao, Jaya R. Soneji

University of Florida, IFAS, Citrus Research & Education Center, USA

www.crec.ifas.ufl.edu/

CONTENTS AND ABSTRACTS

Priyamvada Singh, Sanjay Singh, Satya Prakash Mishra, Sanjeev Kumar Bhatia (India) Molecular Characterization of Genetic Diversity in Jatropha curcas L. (pp 1-8)

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Invited Review: Jatropha curcas L. (Euphorbiaceae) is a small tree with valuable attributes, including that as a commercially important non-edible oilseed that grows naturally in the equatorial Americas and has spread to other tropical countries. J. curcas has been found to be the most suitable tree species for production of biodiesel as it can be grown as a quick yielding plant even in problem soils and adverse climatic conditions. J. curcas has been neglected in the past and little systematic work has been done on productivity aspects. It is highly cross-pollinated and is known for continued seedling propagation, it is anticipated for the existence of wide genetic variability offering significant scope for selecting superior genotypes, which will help to improve productivity and provide sound scientific base to this crop. Few attempts have been directed to improve it as a crop plant and characterize it at molecular level. However, understanding the genetic relationship and variation is important for efficient parental selection and different techniques are being used in the study of variations in J. curcas. Application of molecular markers in J. curcas studies would be the right tool to differentiate plant varieties, for choosing right parents for cross pollination and for marker assisted breeding. In the present paper, we have reviewed the studies conducted at the molecular level to characterize the genetic diversity in J. curcas.

Leela Sahijram (India), Jaya R. Soneji, Madhugiri Nageswara Rao (USA) Molecular and Genetic Characterization of Somaclonal Variation in Micropropagated Bananas (Musa spp.) (pp 9-17)

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Invited Mini-Review: Banana is an economically important fruit as well as food security crop. Though it originated in the Southeast Asian region, it is widely grown around the world. Conventionally, bananas are vegetatively propagated by suckers or corms, while the seeds are employed for propagation only in breeding programs. Vegetative propagation is the major cause of introduction and spread of diseases to non-infected areas. To overcome this problem, tissue-cultured banana plants are being used as planting material. Somaclonal variation (SV) appears to be widespread among banana plants regenerated by tissue culture. A number of factors are known to induce SV which can be problematic during banana micropropagation, although, it may be utilized as an efficient tool in plant breeding. Somaclones have been identified based on their morphological or agronomic characteristics. Early detection of SV is, therefore, very useful. A morphological, biochemical, cytogenetic and/or molecular biology based approach of analysis would help throw light on the causes and detection of variants. Factors inducing SV, their detection and analysis have been discussed in this review.

Aykkal Riju, Vadivel Arunachalam (India) Mining for Simple Sequence Repeats from Expressed Sequence Tag Libraries of Banana (pp 18-21)

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Original Research Paper: The purpose of this study was to mine the simple sequence repeats (SSRs) in expressed sequence tags (ESTs) of banana (Musa spp.). We retrieved 31,066 EST sequences of Musa spp. belonging to different tissues and conditions from dbEST of National Centre for Biotechnology Information. These were made into 4,526 contigs as well as 9596 singletons using sequence assembly program CAP3. SSRs were located by using a MISA perl script, and were classified based on size of repeat and type of the repeating motif. A total of 2,659 SSR sites were observed and 2308 primer pairs were designed. Mononucleotide repeats (44%) were found to be more abundant followed by dinucleotide (23%) and trinucleotide (21%). We annotated the EST contigs and found skp1, putative 60S ribosomal protein L39, DET2, histone H1, GSK1 (gsk3/shaggy-like protein kinase 1), heat shock protein 70, phagocytosis and cell motility protein ELMO1, universal stress protein family as functions encoded. A database was constructed and made available at http://www.riju.byethost31.com/banana/ giving the results of SSRs and primers from the study.

Gudasalamani Ravikanth (India), Madhugiri Nageswara-Rao (India/USA), Ajay Narwade, Ramanan Uma Shaanker, Kotiganahalli N Ganeshaiah (India) Do Endemic Rattans Have Lower Genetic Variability than Their Co-generic and Con-specific Non-endemic Rattans? (pp 22-27)

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Original Research Paper: Comparisons between the population genetic variability of endemic and non-endemic species provide valuable information for the conservation of the endemic species. Comparisons between the endemic and non-endemic species are seldom carried out with con-generic and co-occurring species. In this study, we have compared three pairs of con-specific endemic and non-endemic rattan species at three locations in the Central Western Ghats, one of the global biodiversity hot-spots located in India. We have analyzed the demographic structure of these species across these three sites. We found that overall the endemic rattan species suffered from poor regeneration compared to their con-specific and con-generic non-endemic rattan species. We also used molecular markers to analyze the genetic variability of these endemic and non-endemic rattan species. Our results suggest that, except in one species, the genetic variability of the endemic species was significantly lower than the con-specific non-endemic rattan species. These results indicate the lower genetic variability of the endemic species and have important implications in prioritizing species for conservation.

Lining Tian, Antonet Svircev, Hélène Sanfaçon, Daniel Brown, Karin Schneider, Susan Sibbald, Tabita Malutan (Canada) Development of Plum Pox Virus Resistance in Plum via Expression of an Intron-Spliced Hairpin HC-Pro Transgene (pp 28-31)

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Original Research Paper: Plum pox virus (PPV) is a serious viral disease of Prunus species. Few sources of natural resistance to PPV are available. In this light, biotechnology is a strong option to develop PPV resistance in plants. We have previously shown that a transgene consisting of a region of the PPV HC-Pro sequence in an intron-spliced hairpin conformation could provide high level of resistance to PPV in Nicotiana benthamiana, a herbaceous model host. We have now generated transgenic plum lines expressing the same transgene. Plants were inoculated with PPV virus using a chip bud grafting method and were subjected to three cycles of dormant/growth periods. Plants were analyzed for PPV resistance after each cold treatment (dormant period) using a real-time polymerase chain reaction (PCR) assay. After the first cold treatment, transgenic plum plants remained PPV-free whereas all the control plants were PPV positive. After three cycles of cold treatment, many transgenic plants still exhibited strong resistance to PPV. Presence of siRNA molecules corresponding to the HC-Pro transgene were confirmed in transgenic lines that were highly resistant to PPV. This study shows that induction of PPV-specific RNA silencing via expression of a intron-spliced hairpin HC-Pro transgene is an effective approach for high level and stable resistance to plum pox virus in plum.

Rajinder Kaur, Priyanka Sood, Yogesh Vikal, Krishan Kumar, Bhawna Saxena, Dile Ram Sharma (India) Genetic Characterization of Walnut (Juglans regia L.) by Random Amplified Polymorphic DNA (pp 32-36)

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Research Note: Walnut germplasm accessions representing exotic and indigenous collections, from walnut orchards of University of Horticulture and Forestry, Nauni, Himachal Pradesh, India, were used in the present investigations. A total of seven exotic and eight indigenous accessions were evaluated for randomly amplified polymorphic DNA (RAPD) variations. 21 primers out of total 36 decamerous primers generated 190 bands, out of which 157 showed polymorphism on the agarose gel, reflecting a high level of genetic diversity within the germplasms. Pairwise distances, calculated using Jaccard’s coefficient of similarity, clustered indigenous and exotic accessions into separate groups except for one accession ‘Blackmore’ which, although exotic, was placed in a cluster of predominantly indigenous accessions. The clustering based on RAPD markers agreed to large extent with the geographical origin of the studied walnut germplasm accessions.

Aykkal Riju, Vadivel Arunachalam (India) Electronic Sorting of SNP/Indel Sites in Expressed Sequence Tag Libraries of Cocoa (Theobroma cacao L.) (pp 37-40)

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Short Communication: The objective of this study was to explore the single nucleotide polymorphims (SNPs) in expressed sequence tags (ESTs) of cocoa (Theobroma cacao L.). We retrieved 6578 EST sequences consisting of seven tissues/libraries from dbEST of National Centre for Biotechnology Information. SNPs and small Indels (insertion/deletion) were located with the help of AutoSNP. We found a density of one SNP/166 bp and one Indel/360 bp in cocoa ESTs. Candidate SNPs were categorized according to nucleotide substitution as either transition (C/T or G/A) or transversion (C/G, A/T, C/A or T/G). We observed a relative increase in the proportion of transversions (1268) over transitions (950) in bean and leaves and defense related EST sequence libraries. Transversion of G/T (562) (25% of detected SNP) was predominant in cocoa ESTs. We worked out Shannon entropy to find out the distribution of ten different types of SNPs/Indels. An online database is created to enable cocoa workers to freely access the results of the study (http://www.riju.byethost31.com/cocoa/ccsnp.html).

Ahmed Mansour (Egypt), Jaime A. Teixeira da Silva (Japan), Sherif Edris, Rania A. A. Younis (Egypt) Comparative Assessment of Genetic Diversity in Tomato Cultivars using IRAP, ISSR and RAPD Molecular Markers (pp 41-47)

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Original Research Paper: Tomato (Lycopersicon esculentum Mill.) is one of the most important agricultural and industrial crops. In this report, we investigated three different molecular marker systems, namely RAPD (randomly amplified polymorphic DNA), ISSR (inter-simple sequence repeat) and IRAP (inter-retrotransposon amplified polymorphism) to detect genomic variation within 10 tomato cultivars, which have different genotypic and phenotypic characters and are widely used in most breeding programs, namely ‘Aledo VF’, ‘Carmeuco 201M’, ‘Castle-rock’, ‘Money Maker’, ‘Peto 86’, ‘Red Star’, ‘Super Marmand’, ‘Super Queen’, ‘Super Strain B’ and ‘UC97-3’. Comparative assessments for using these three markers to study genetic diversity and analysis of molecular variance (AMOVA) among the 10 tomato cultivars are described. Different dendrograms constructed for the RAPD, ISSR and IRAP results individually and collectively reveal that similarity and clustering are highly dependant on the marker system used. This is the first ever such study in tomato to superimpose the results of three marker systems to verify their individual results.

Nahuel N. Pratta, Marta Quaglino, Gustavo R. Rodríguez, Roxana Zorzoli, Guillermo R. Pratta (Argentina) A Multivariate Approach to the Proteomics of Tomato Fruit Ripening (pp 48-51)

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Research Note: Structural and functional genomics are useful approaches to better understand the biological process regarding the genetic composition of individuals and its expression at the phenotypic level. Numerous data are produced in studies of this area of research, multivariate statistics providing powerful tools for reducing their dimensionality. The general objective of this research was to apply principal component analysis (PCA) and multivariate analysis of variance (MANOVA) to analyse the proteomics of the tomato ripening and its association with agronomic quantitative traits. A PCA was applied as a non classic exploratory way to the binary data presence / absence of a given polypeptide in mature green, breaker and red ripe tomato fruits from F₂ plants. To verify results from PCA, a hierarquical cluster analysis was performed by UPGMA to the same data. Advantage of PCA is that the relative contribution of a given polypeptide for grouping can be measured. Then, polypeptides mostly contributing for grouping were introduced as the source of variation in a one-way MANOVA to detect association with quantitative traits, which were the dependent variables. Polypeptides mostly contributing to general variability were efficiently identified by PCA, while MANOVA verified that one of them is a putative molecular marker of the quantitative traits.

Abdel Hady Sayed, Shafeek El-Morsy, Mohamed Ibrahim, Ahmed El-Kot (Egypt), Jaime A. Teixeira da Silva (Japan), Mohamed Rady (Egypt) Genetic Identification and Estimation of Genetic Relationships among Some Onion Cultivars and Lines using RAPD Analysis and Morphological Characters (pp 52-57)

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Original Research Paper: Genetic differentiation of two lines and three cultivars of onion were studied by RAPD analysis. RAPD analysis revealed that 67 bands with an average of 6.7 amplicons per primer were generated by 10 decamer primers. However, the total number of polymorphic bands was 57, which represents an 83.9% level of polymorphism. RAPD-based Dice similarity coefficient analysis showed that the highest genetic similarity value was observed between cvs. ‘T.E.Y.G’ and ‘Giza 20’ (96.8% similarity), while the lowest (61.5%) genetic similarity value was between ‘Puss’ and ‘T.E.Y.G’. A dendrogram based on genetic similarities separated the two lines and the three cultivars of onion into two main groups. Using OPC-08, OPC-14 and OPD-11 primers to compare A- or B-line with other cultivars, a high level of polymorphism was shown. Primer OPD-01 was a unique marker that could characterize A- and B-lines from other cultivars. In general, RAPD can be effectively used to assess genetic variation and identification among onion lines and cultivars and can thus assist onion breeders to predict sterile and fertile onion plants.

Priti Maheshwari (Canada/India), Anil Kumar (India) RAPD Analysis of UV-B-Induced Variations in Somaclones of Vernonia cinerea (pp 58-64)

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Original Research Paper: Plantlets were regenerated through callus-mediated organogenesis after UV–B irradiation (280–320 nm, 0.863–5.28 kJ m^-2) in Vernonia cinerea and were analyzed by Random Amplified Polymorphic DNA with 10 random decamers to assess UV–B induced polymorphism. A total of 45 different somaclones were selected based on difference in morphology compared to the parent plant. Out of the 10 primers chosen for initial screening of polymorphism in these 45 somaclones and one parent plant (total 46 genotypes), 8 primers gave amplified polymorphic products. A total of 1347 scorable bands were generated. The value of PIC (polymorphic information content) for each primer ranged from 0.238 to 0.361 with an average of 0.285. Pair-wise genetic similarity coefficients generated from all possible genotypes ranged from 0.3929 to 0.9667. All the calliclones exhibited up to a mean of 77.5% variation from the parent genotype except somaclone 14 that showed 97% similarity. As indicated by the genetic similarity indices, UV-B irradiation is a good way for generation of high frequency morphological and genetic variants during callus mediated shoot regeneration in V. cinerea and can be used for selection towards desirable traits, such as salt or drought stress.

Dayananda Chandrappa, Venkatachalam Lakshmanan, Bhagyalakshmi Neelwarne, Ravishankar Gokare Aswathanarayana (India) Assessment of Genetic Polymorphism among Green Microalgae Botryococcus of Distinct Origin by RAPD (pp 65-69)

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Original Research Paper: Genetic variation and relationships among 7 strains of Botryococcus sp. belonging to different geographical locations and different chemical races were evaluated using 35 decamer RAPD primers. Several RAPD primers were selected after preliminary screening for their ability to produce clear and multiple bands. The analyses resulted in the amplification of a total of 407 bands of 100-3000 bp, 380 of which were polymorphic, corresponding to 93.3% genetic diversity. The ability to distinguish genotypes and the Resolving power (Rp) of the primer showed a linear relationship. From these data, a genetic similarity matrix and dendrogram were obtained by using the unweighted pair group method with arithmetic means (UPGMA). The RAPD analysis produced genetic similarity coefficients ranging from 0.3312 to 0.7388. The study resulted in the identification of genetic relationship among various strains of Botryococcus sp. belonging to different climatic zones and origins. The study also revealed clear genetic distances between A race strains of B. braunii and B race strains of the same species.

Othman E. Othman, Sahar Ahmed (Egypt) Genetic Polymorphism of BoLA-DRB3 Exon 2 in Egyptian Buffalo (pp 70-73)

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Original Research Paper: The role of the BoLA-DRB3 gene in the development of an immune response system and its highly polymorphic nature make it one of the most important candidate genes in disease resistance studies. PCR-RFLP was used in this study to detect the genotypic polymorphism of BoLA-DRB3 exon 2 in 50 Egyptian buffalo. The PCR amplified fragments at 284-bp were digested by two restriction enzymes, RsaI and BstYI. Digestion of the amplified fragments using RsaI endonuclease yielded six alleles (B, F, I, K, L and O) with frequencies that ranged from 6% (I allele) to 40% (L allele). The presence of six alleles resulted in 14 different genotypes from the tested buffalo. These genotypes included three homozygous genotypes (B/B, L/L and O/O) with 6, 18 and 8% frequency, respectively, while the remaining 11 genotypes were heterozygous: B/F (2%), B/K (2%), B/L (6%), F/I (2%), F/L (10%), F/O (2%), I/K (2%), I/L (8%), K/L (8%), K/O (14%) and L/O (12%). Digestion of the PCR products with BstYI yielded three alleles and five genotypes in Egyptian buffalo. The three alleles A, B and D showed 24, 62 and 14% frequency, respectively while the five genotypes included three homozygous genotypes AA (6%), BB (34%) and DD (4%) and two heterozygous genotypes AB (36%) and BD (20%). In conclusion, genetic polymorphism of BoLA-DRB3 exon 2 in Egyptian buffalo displayed a total of 9 alleles and 19 genotypes distinguished by digestion of PCR products with two endonucleases. The results suggest that absence of the mastitis susceptibility alleles C and S in tested Egyptian buffalo and the presence of mastitis resistance alleles L in 40% and B in 11% of animals.

Nariman A.H. Aly, Osama E. El-Sayed (Egypt) Nucleotide Variations and Base-pair Substitutions of 16S rRNA Gene in some Local Bacillus thuringiensis Isolates (pp 74-83)

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Original Research Paper: Three local Bacillus thuringiensis isolates; Sn-2, Ts-5 and As-3 were used in the study. They previously collected from the soil of three governorates; Sinai, Toshkey and Aswan and were characterized by their high toxicity to Spodoptera littoralis and Culex pipiens. Three primer pairs of the forward and reverse were designed from the very similar sequences within consensus regions of twelve GenBank Bacillus thuringiensis 16S rRNA gene sequences to amplify three fragments with 252, 984 and 604 bp from isolates; Sn-2, Ts-5 and As-3 respectively. Sequence alignment using the Blast search revealed 17 nucleotide positional differences with base-pair substitutions between Sn-2 isolate with 252 bp (GU062822), whereas the highest number (5) of positional differences was guanine (G) found in Sn-2 that changed to thymine (T) in all other GenBank isolates and strains based on the 16S rRNA gene similarity. Accession GU062823 with 984 bp (isolate Ts-5) showed a total number of 11 nucleotide positional differences. While, accession GU062824 with a fragment size 604 bp (isolate As-3). In addition, 10 nucleotide positional differences were obtained from the four nucleotide bases (C, G, T, A) to (¼) and four from (¼) to the four nucleotide bases. Phylogenetic analyses based on 16S rRNA gene sequences showed that each of the three local B. thuringiensis isolates (Sn-2, Ts-5 and As-3) formed a phylogenetically distinct group, separate from all other named isolates and species of B. thuringiensis. Such obtained results evidently indicated a large diversity with unique characteristics of the local Egyptian isolates from all the other isolates and strains established around the world.