Analysis of B-genome derived simple sequence repeat (SSR) markers in Musa spp.

A study was conducted to investigate the genetic variability between 40 Musa genotypes maintained at the Musa germplasm collection of the International Institute for Tropical Agriculture, Ibadan using nine B-genome derived simple sequence repeat (SSR) markers. The nine primers produced reproducible and discrete fragments and generated a total of 23 alleles with an average of 2.1. The hierarchical cluster analysis showed clusters of diploid cultivars separate from triploid ones (with the exception of TMB149 (BB) and TMB131 (AB)). Average gene diversity was He = 0.412, and differentiation, given by the fixation index (FST) was low at 0.131.Keywords: Banana, genetic diversity, gene differentiation, plantai

Field collection, preservation and large scale DNA extraction procedures for cassava (Manihot esculenta Crantz.)

Some genetic studies using molecular methods such as diversity assessment or marker-assisted selection require collection of a large number of samples from fields located in the vicinity or in remote areas, followed by isolation of good quality DNA in a short time span. In the present study, different tissue preservation methods were compared for subsequent DNA extraction using a modified CTAB method in two 96-well plates, following grinding of leaf tissues with a GenoGrinder 2000. We found that preservation of leaf tissues in NaCl-CTAB-azide buffer (as described in Rogstad, 1992) at 4°C is a better storage procedure than preservation at -20°C to obtain good quality DNA. Comparison of DNA extraction with or without use of phenol revealed that the quality of DNA was not drastically affected when non-phenol extraction protocol was used and did not affect PCR amplification. Thus, the recommended DNA extraction procedure allowed us to process 192 samples per day at a cost of $0.80 per sample, with an average yield of 1.8  g, suitable for both PCR and genotyping

Molecular Characterization of Three Cultivars of Tomato (Lycopersicon Esculentum L.) in South-West Nigeria Using SSR Markers

Molecular characterisation of local tomato cultivars – Ibadan Local (IbL), Ife and JM94/46 (JM) were assessed using simple sequence repeat (SSR) markers. Out of ten SSR primer pairs used, three primer pairs were able to differentiate amplified genomic DNA of the cultivars. Unweighted Pair Group Method Using Arithmetic Average (UPGMA) cluster analysis of the data showed a close relationship between IbL and Ife with a genetic distance (GD) of 0.067; Ife and JM had GD of 0.2 and JM and Ife had GD of 0.25

Survey of Hemileia vastatrix races from Peru to identify potential coffee mutants with disease resistance

info:eu-repo/semantics/publishedVersio

Fragmentation of pooled PCR products for highly multiplexed TILLING

Improvements to massively parallel sequencing have allowed the routine recovery of natural and induced sequence variants. A broad range of biological disciplines have benefited from this, ranging from plant breeding to cancer research. The need for high sequence coverage to accurately recover single nucleotide variants and small insertions and deletions limits the applicability of whole genome approaches. This is especially true in organisms with a large genome size or for applications requiring the screening of thousands of individuals, such as the reverse-genetic technique known as TILLING. Using PCR to target and sequence chosen genomic regions provides an attractive alternative as the vast reduction in interrogated bases means that sample size can be dramatically increased through amplicon multiplexing and multidimensional sample pooling while maintaining suitable coverage for recovery of small mutations. Direct sequencing of PCR products is limited, however, due to limitations in read lengths of many next generation sequencers. In the present study we show the optimization and use of ultrasonication for the simultaneous fragmentation of multiplexed PCR amplicons for TILLING highly pooled samples. Sequencing performance was evaluated in a total of 32 pooled PCR products produced from 4096 chemically mutagenized Hordeum vulgare DNAs pooled in three dimensions. Evaluation of read coverage and base quality across amplicons suggests this approach is suitable for high-throughput TILLING and other applications employing highly pooled complex sampling schemes. Induced mutations previously identified in a traditional TILLING screen were recovered in this dataset further supporting the efficacy of the approach

DNA SeqUeNCINg ANAlySIS Of AfRICAN Xanthomonas oryzae pv. oryzae vIRUleNCe geNe (aXaVrg) DNA MARkeR

Global rice production is constrained by bacterial leaf blight (BLB) disease caused by Xanthomonas oryzae pv. oryzae (Xoo). BLB disease incidence in West Africa was between 70–85% and yield loss in farmers’ fields was in the range of 50–90% from 2005 to 2010. In the present study, African Xoo virulence gene OPP-172000 DNA marker was identified and purified using randomly amplified polymorphic DNA polymerase chain reaction (RAPD-PCR) products from 50 Xoo isolates. Genomic DNA of 50 Xoo isolates were analyzed using OPP-17 primer in RAPD-PCR during which African Xoo virulence gene OPP-172000 DNA marker was identified, purified, cloned, and sequenced. Cloning and DNA sequencing of African Xoo virulence gene OPP-172000 DNA generated a 1953 bp nucleotide sequence consequently tagged as AXaVrg-1953. BLAST homologous analysis of the AXaVrg-1953 sequence provides comprehensive identification of the type II secretion genes and secreted proteins, type III secretion genes and secreted proteins in African Xoo virulence gene. Phylogenetic unweighted pairgroup method arithmetic (UPGMA) analysis revealed the African AXaVrg-1953 sequence was distinct from the other Xoo virulence gene sequences from China, Japan, Korea, Germany, and the United States. This information is potentially useful for effective management of BLB disease in West Africa. Bacterial leaf blight, Operon primer, RAPD-PCR products, Xoo virulence gene DNA marker, cloning, Secreted proteins, BLAST, West Afric

Two genotypes of Xanthomonas oryzae pv. oryzae virulence identified in West Africa

Bacterial leaf blight (BLB) caused by Xanthomonas oryzae pv. oryzae (Xoo), is a very destructive rice disease worldwide. The aim of the present study was to examine if the Xoo virulence pathotypes obtained using phenotypic pathotyping could be confirmed using molecular approach. After screening of 60 Operon primers with genomic DNA of two Xoo isolates (virulent pathotype, Vr and mildly virulent pathotype, MVr), 12 Operon primers that gave reproducible and useful genetic information were selected and used to analyze 50 Xoo isolates from 7 West African countries. Genetic analysis revealed two major Xoo virulence molecular type (Mt) which were Mta and Mtb with Mta having two subgroups (Mta1 and Mta2). Mta1 (Vr1) subgroup genotype has occurrence in six countries and Mta2 (Vr2) in three countries while Mtb genotype characterized mildly virulence (MVr) Xoo isolates present in five countries. The study revealed possible linkage and correlation between phenotypic pathotyping and molecular typing of Xoo virulence. Durable resistance rice cultivars would need to overcome both Mta and Mtb Xoo virulence genotypes in order to survive after their deployment into different rice ecologies in West Africa

Functional analysis and expression profiling of HcrVf1 and HcrVf2 for development of scab resistant cisgenic and intragenic apples

Apple scab resistance genes, HcrVf1 and HcrVf2, were isolated including their native promoter, coding and terminator sequences. Two fragment lengths (short and long) of the native gene promoters and the strong apple rubisco gene promoter (PMdRbc) were used for both HcrVf genes to test their effect on expression and phenotype. The scab susceptible cultivar ‘Gala’ was used for plant transformations and after selection of transformants, they were micrografted onto apple seedling rootstocks for scab disease tests. Apple transformants were also tested for HcrVf expression by quantitative RT-PCR (qRT-PCR). For HcrVf1 the long native promoter gave significantly higher expression that the short one; in case of HcrVf2 the difference between the two was not significant. The apple rubisco gene promoter proved to give the highest expression of both HcrVf1 and HcrVf2. The top four expanding leaves were used initially for inoculation with monoconidial isolate EU-B05 which belongs to race 1 of V. inaequalis. Later six other V. inaequalis isolates were used to study the resistance spectra of the individual HcrVf genes. The scab disease assays showed that HcrVf1 did not give resistance against any of the isolates tested regardless of the expression level. The HcrVf2 gene appeared to be the only functional gene for resistance against Vf avirulent isolates of V. inaequalis. HcrVf2 did not provide any resistance to Vf virulent strains, even not in case of overexpression. In conclusion, transformants carrying the apple-derived HcrVf2 gene in a cisgenic as well as in an intragenic configuration were able to reach scab resistance levels comparable to the Vf resistant control cultivar obtained by classical breeding, cv. ‘Santana’

Development of Useful Recombinant Promoter and Its Expression Analysis in Different Plant Cells Using Confocal Laser Scanning Microscopy

BACKGROUND: Designing functionally efficient recombinant promoters having reduced sequence homology and enhanced promoter activity will be an important step toward successful stacking or pyramiding of genes in a plant cell for developing transgenic plants expressing desired traits(s). Also basic knowledge regarding plant cell specific expression of a transgene under control of a promoter is crucial to assess the promoter's efficacy. METHODOLOGY/PRINCIPAL FINDINGS: We have constructed a set of 10 recombinant promoters incorporating different up-stream activation sequences (UAS) of Mirabilis mosaic virus sub-genomic transcript (MS8, -306 to +27) and TATA containing core domains of Figwort mosaic virus sub-genomic transcript promoter (FS3, -271 to +31). Efficacies of recombinant promoters coupled to GUS and GFP reporter genes were tested in tobacco protoplasts. Among these, a 369-bp long hybrid sub-genomic transcript promoter (MSgt-FSgt) showed the highest activity in both transient and transgenic systems. In a transient system, MSgt-FSgt was 10.31, 2.86 and 2.18 times more active compared to the CaMV35S, MS8 and FS3 promoters, respectively. In transgenic tobacco (Nicotiana tabaccum, var. Samsun NN) and Arabidopsis plants, the MSgt-FSgt hybrid promoter showed 14.22 and 7.16 times stronger activity compared to CaMV35S promoter respectively. The correlation between GUS activity and uidA-mRNA levels in transgenic tobacco plants were identified by qRT-PCR. Both CaMV35S and MSgt-FSgt promoters caused gene silencing but the degree of silencing are less in the case of the MSgt-FSgt promoter compared to CaMV35S. Quantification of GUS activity in individual plant cells driven by the MSgt-FSgt and the CaMV35S promoter were estimated using confocal laser scanning microscopy and compared. CONCLUSION AND SIGNIFICANCE: We propose strong recombinant promoter MSgt-FSgt, developed in this study, could be very useful for high-level constitutive expression of transgenes in a wide variety of plant cells