Does plant –microbe’s common Inter Simple Sequence Repeat lead to a new recombination? A novel mechanism of pathogenicity

A total of eight varieties of the mango from an orchard were studied using molecular markers to understand the host-pathogen interaction. From the infected leaves of the plant, a total of the 8 bacterial pathogens (Exiguobacterium arabatum, Pseudomonas mendocina, Pantoea dispersa, Bacillus sp. Pantoea ananatis, Micrococcous luteus, Microbacterium_sp., Enterobacter cloacae) were isolated and identified. All the host varieties of mango were distinguished for the genetic diversity using the Inter Simple Sequence Repeat (ISSR) DNA markers. This set of ISSR marker primers were also used for the mango pathogens. PCR amplification of the ISSR primers showed polymorphic and monomorphic band patterns in the host plants and in their pathogens. The monomorphic band generated by PCR amplification in the host and in the pathogen, by the common primer, is selected and used for PCR hybridization technique. PCR products obtained from the host, pathogen and hybridization were cloned, sequenced and compared. A multiple sequence alignment of these sequences revealed that the product of hybridization PCR was mixture of host and pathogen sequences. On this basis, we hypothesize a possibility for the recombination of host-microbes DNA as one of the mechanisms of pathogenicity for the plant pathogens using hybrid PCR technique. The possible mechanism of recombination for plant host and its pathogen is discussed.HighlightsInter Simple Sequence Repeat markers used to (i) Fingerprint the pathogens and their host (mango) and (ii) for study of the possibilities for the recombination as mechanism of pathogenicity.

Genetic Variability of 21 Tea Genotypes [Camellia sinensis (L.) O. Kuntze] Based on RAPD Markers

<em>Knowing the genetic diversity in the tea germplasms collection is one of important conditions for assembling new superior varieties. Information of genetic diversity can be obtained through analysis using RAPD molecular markers. The study aimed to determine the genetic diversity of 21 tea genotypes based on RAPD markers. The research was conducted in Integrated Laboratory, Seameo Biotrop, Bogor, from July to September 2013. Genomic DNA was isolated from 21 tea genotypes leaf samples, then amplified with primer OPA 03, OPA 05, OPB 04, OPB 06, OPC 06, and OPD 08. Electrophoresis result was converted into binary data. The genetic similarity and cluster analysis calculation was done using NTSYS-pc version 2.10. In this research, 50 polymorphic bands (94,34%) and 3 monomorphic band (5,66%) were obtained. Cluster analysis based on Nei's genetic distance using the unweighted pair-group method with arithmatic (UPGMA) divided 21 tea genotypes into two groups at a genetic similarity value of 0,48. Group 1 consisted of 20 tea genotypes, while the second group comprised only a one genotype (Sin 27). The range of genetic similarity matrix was between 28%–92%, the lowest genetic similarity (28%) was found between GMB 4 and Sin 27 genotypes, while the highest (92%) was found between AS 2 and AS 1 genotypes. The information obtained can be utilized in breeding programs with the support of agronomic characters as well as in the conservation of tea germplasm.</em>

Use of DAF markers (DNA Amplification Fingerprint) to Assess Genetic Diversity of Rice (Oryza satival L.)

This study was carried out to assess genetic diversity of ten cultivars of Rice (Oryza sativa L.). One of DNA markers based on Polymerase Chain Reaction (PCR) was used namely DAF markers (DNA Amplification Fingerprint). Six primers were tested, the results showed, that no amplification products using the primers OPD.14 and OPM.5. Two primers (OPX.8 and OPT.2) produced monomorphic band across all cultivars, while only two primers generated polymorphic bands. The number of total bands produced from one of them (OPN.7) were sixteen. Also this primer produced ten polymorphic profiles (DAF patterns) which were unique to the ten cultivars that could be distinguished. The number of total bands generated by primer OPX.1 were thirteen and this primer produced eight polymorphic patterns which was unique for distinguishing six cultivars. This means that DAF markers were able to identify all rice cultivars using only two primers reflecting the high potentialities of these markers for their applications in fingerprinting.

Identification of Molecular Markers for Selected Wood Properties of Norway Spruce Picea abies L. (Karst.) II. Extractives Content

We describe the development of a SCAR-marker linked to low extractives content of Norway Spruce (Picea abies L [Karst.]) derived from AFLPs. In these analyses 57 different primer enzyme combinations were used in a bulked segregant analysis approach comparing individuals with high and low extractives content. A total of 14 polymorphic AFLP markers were detected between the pools. Five markers were selected for further analyses to verify their linkage to extractives content based on individuals used for pool constitution. One AFLP marker, found to be significant linked to low extractives content was converted into a SCAR marker for further validation. For this marker, a monomorphic band was obtained by using sets of nested primers or restriction site specific primers (RSS) which include the AFLP-restriction recognition site. The separation of the marker from unlinked size homologous marker-alleles was realized by a SSCP-approach. Validation of the marker on different full-sib families confirmed the usability to separate the classes for low and high extractives content of Picea abies.

Identification of Molecular Markers for Selected Wood Properties of Norway Spruce Picea abies L. (Karst.) I. Wood Density

The identification of AFLP markers and their subsequent conversion to SCAR-markers linked to wood density of Norway Spruce (Picea abies L [Karst.]) is described for the first time. In AFLP-analyses, 102 different primer enzyme combinations were screened in a bulked segregant approach comparing individuals with high and low wood density. A total of 107 polymorphic AFLP fragments were obtained between the DNA-pools. Twenty-three markers were selected for further analyses to verify their linkage to wood density based on individuals used for pool constitution and additional unrelated clonal material. For 15 markers, a significant linkage to wood density was confirmed by a two-sided Fisher’s-exact test. Four markers were converted into SCAR markers and validated for plant material assayed for wood density by X-ray microdensitometry. For each marker a monomorphic band was obtained using sets of nested primers or restriction site-specific primers (RSS), which include the AFLP-restriction recognition sites. For two markers that are linked to high wood density, a separation from unlinked size homologous marker-alleles was realized by a PCR-restriction approach. Validation of these markers in different full-sib families confirmed their usability to separate the classes for low and high wood density of Picea abies.

Sequence assessment of comigrating AFLPTM bands in Echinacea — implications for comparative biological studies

The extent of sequence identity among clones derived from monomorphic and polymorphic AFLPTM polymorphism bands was quantified. A total of 79 fragments from a monomorphic band of 273 bp and 48 fragments from a polymorphic band of 159 bp, isolated from individuals belonging to different populations, varieties, and species of Echinacea, were cloned and sequenced. The monomorphic fragments exhibited above 90% sequence identity among clones within samples. Sequence identity within variety ranged from 82.78% to 94.87% and within species from 75.82% to 98.9% and was 57.97% in the genus. The polymorphic fragments exhibited much less sequence identity. In some instances, even two clones from the same fragment were different in their size and sequence. Within sample, clone sequence identity ranged from 100% to 51.57%, within variety from 33.33% to 100% in one variety, and from 23.66% to 45% within species and was as low as 1.25% within the genus. In addition, sequences of the same size were aligned to verify the nature of their sequence dissimilarity/similarity. Within each size group, identical sequences were found across species and varieties. In general, comigrating bands cannot be considered homologous. Thus, the use of AFLPTM band data for comparative studies is appropriate only if the results emanating from such analyses are considered as approximations and are interpreted as phenotypic but not genotypic.Key words: AFLP markers, false homologies.