Easy strategy used to detect the genetic variability in chickpea (Cicer arietinum L.).

A priority in the management and use of elite plant materials for breeding has been based on molecular markers or DNA sequencing of entire genomes, in order to perform genetic differentiation which is still quite costly. Chickpea (Cicer arietinum) is one of the species with genomic monotony and very low polymorphism, and its detection even with DNA markers has not been easy. In germplasm banks, the genetic distinction is a priority in order to use properly selected lines. In this study, 57 chickpea accessions from a germplasm bank were analyzed by using nrRAMP (non-radioactive Random Amplified Microsatellite Polymorphism) markers, and their genetic variability was determined. Our results showed DNA polymorphisms, which are enough to differentiate between the accessions and between C. arietinum and Cicer reticulatum (out-group); this last wild species is closely related to chickpea. We concluded that the nrRAMP technique was an effective and a highly useful method to assess the genetic diversity and variability among closely related plants, such as chickpea; in addition, this technique can be easily implemented in laboratories.

Genetic divergence between Mexican Opuntia accessions inferred by polymerase chain reaction-restriction fragment length polymorphism analysis.

Molecular methods are powerful tools in characterizing and determining relationships between plants. The aim of this study was to study genetic divergence between 103 accessions of Mexican Opuntia. To accomplish this, polymerase chain reaction (PCR)-restriction fragment length polymorphism analysis of three chloroplast intergenic spacers (atpB-rbcL, trnL-trnF, and psbA-trnH), one chloroplast gene (ycf1), two nuclear genes (ppc and PhyC), and one mitochondrial gene (cox3) was conducted. The amplified products from all the samples had very similar molecular sizes, and there were only very small differences between the undigested PCR amplicons for all regions, with the exception of ppc. We obtained 5850 bp from the seven regions, and 136 fragments were detected with eight enzymes, 37 of which (27.2%) were polymorphic. We found that 40% of the fragments from the chloroplast regions were polymorphic, 9.8% of the bands detected in the nuclear genes were polymorphic, and 20% of the bands in the mitochondrial locus were polymorphic. trnL-trnF and psbA-trnH were the most variable regions. The Nei and Li/Dice distance was very short, and ranged from 0 to 0.12; indeed, 77 of the 103 genotypes had the same genetic profile. All the xoconostle accessions (acidic fruits) were grouped together without being separated from three genotypes of prickly pear (sweet fruits). We assume that the genetic divergence between prickly pears and xoconostles is very low, and question the number of Opuntia species currently considered in Mexico.

Assessment of genetic relationship in Persea spp by traditional molecular markers.

Currently, the reclassification of the genus Persea is under discussion with molecular techniques for DNA analysis representing an alternative for inter- and intra-specific differentiation. In the present study, the traditional random-amplified polymorphic DNA (RAPD) and the inter simple sequence repeat (ISSR) markers were used to determine the genomic relationship of different species and hybrids representative of the subgenera Eriodaphne and Persea in a population conserved in a germplasm bank. The data were analyzed statistically using multivariate methods. In the RAPD analysis, a total of 190 polymorphic bands were produced, with an average of 23.7 bands per primer, the percentage contribution of each primer was from 7.66 to 19.63; the polymorphic information content (PIC) ranged from 0.23 to 0.45, with an average of 0.35. In the ISSR analysis, a total of 111 polymorphic bands were considered, with an average of 18.5 bands per primer, the percentage contribution of each was from 11.83 to 19.57; the PIC ranged from 0.35 to 0.48, with an average of 0.42. The phenograms obtained in each technique showed the relationship among the accessions through the clusters formed. In general, both the techniques grouped representatives of the Persea americana races (P. americana var. drymifolia, P. americana var. guatemalensis, and P. americana var. americana). However, it was not possible to separate the species of Persea used as reference into independent clades. In addition, they tended to separate the representatives of subgenera Eriodaphne and Persea.

Initial assessment of natural diversity in Mexican fig landraces.

The common fig (Ficus carica L.) was introduced into Mexico by Spanish Franciscan missionaries in the 16th century. It is widely assumed that Mexican figs are the Spanish cultivar Black Mission. We collected and propagated 12 fig plants from six landraces from different states in Central Mexico that represent different climate. All of them were grown in a greenhouse at Universidad Autónoma Chapingo, in the State of Mexico. During the experimental period, the greenhouse had an average temperature and relative humidity of 29.2° ± 5.4°C (SEM) and 78.1 ± 6.7% (SEM), respectively. Morphological characterization was done following a selected set of quantitative and qualitative descriptors established by the IPGRI. DNA analysis was based on a combination of ISSR and RFLP markers. We observed great diversity mainly in fruit weight (28.1-96.2 g), fruit shape (ovoid, pyriform), and neck length (0.97-3.80 cm), which could not be explained by environmental conditions such as temperature and relative humidity. The Nei and Li/Dice similarity coefficient between landraces was determined by cluster analysis using the UPGMA method. Based on the morphological characterization and DNA fingerprinting data presented in this study, our results showed that after hundreds of years, black figs have adapted to local environmental condition in Central Mexico, yielding at least six clearly distinct landraces that represent valuable and previously undescribed genetic diversity. We also suggested names for those landraces according to their location and established a basis for further agronomic and molecular characterization of fig landraces.

First Report of 'Candidatus Phytoplasma asteris'-Related Strains Infecting Lily in Mexico.

In recent years, lily (Lilium spp.) has become an important ornamental crop in diverse regions of Mexico. Since 2005, unusual symptoms have been observed on lily plants grown from imported bulbs in both greenhouse and production plots at San Pablo Ixayo, Boyeros, and Tequexquinauac, Mexico State. Symptoms included a zigzag line pattern on leaves, dwarfism, enlargement of stems, shortened internodes, leaves without petioles growing directly from bulbs, air bulbils, death of young roots, atrophy of flower buttons, and flower abortion. Symptoms were experimentally reproduced on healthy lily plants by graft inoculation. Total DNA was extracted from 50 diseased, 10 symptomless, and 10 graft-inoculated plants by the method of Dellaporta et al. (2). DNA samples were analyzed for phytoplasma presence by two different nested PCR assays. One assay employed ribosomal RNA gene primer pair P1/P7 followed by R16F2n/R16R2 (1), whereas ribosomal protein (rp) gene primer pairs rpF1/rpR1 and rp(I)F1A/rp(I)R1A (4) were used in a second assay. A DNA fragment approximately 1.2 kb long was consistently amplified from all symptomatic plant samples only by both assays. A comparative analysis of 16S rDNA sequences (Genbank Accession Nos. EF421158-EF421160 and EU124518-EU124520) and rp gene sequences (EU277012-EU277014), derived from PCR products, revealed that phytoplasma infecting lily were most similar (99.9% to 16S rDNA and 99.7% to rp) to carrot phytoplasma sp. ca2006/5 and also were similar (99.8% to 16SrDNA and 99.2% to rp) to broccoli phytoplasma sp. br273. Both carrot and broccoli phytoplasmas were classified as members of aster yellow 16S rDNA restriction fragment length polymorphism subgroup 16SrI-B (3). Although infection of lilies by aster yellows ('Ca. phytoplasma asteris') subgroup 16SrI-B and 16SrI-C was reported from the Czech Republic and Poland, to our knowledge, this is the first report of 'Ca. phytoplasma asteris'-related strains associated with lily plants in Mexico. References: (1) R. F. Davis et al. Microbiol. Res. 158:229, 2003. (2) S. L. Dellaporta et al. Plant Mol. Biol. Rep. 1:19, 1983. (3) B. Duduk et al. Bull. Insectol. 60 2:341, 2007. (4) I.-M. Lee et al. Int. J. Syst. Evol. Microbiol. 54:337, 2004.

First Report of Pantoea agglomerans Causing Leaf Blight and Vascular Wilt in Maize and Sorghum in Mexico.

Zea mays and Sorghum bicolor are important crops for animal and human nutrition worldwide. In the Central Highland Valley of Mexico, both crops are extremely important, and research is aimed toward increasing yield, disease resistance, and crop adaptation from 1,900- to 2,700-m elevation. In a 3-year field breeding experiment (2004 to 2006), leaf blight and vascular wilt symptoms were frequently observed in contiguous plots of maize and sorghum crops in Montecillo, Mexico and maize plots in Tecamac, Mexico. To identify and characterize the causal agent of these symptoms, isolations were conducted on leaves from areas where healthy and diseased tissues converged. Leaf sections of 1 cm2 from both crops were disinfested, placed on casamino acid-peptone-glucose (CPG) medium, and incubated at 28°C. After 48 h, only yellow colonies were observed and 12 isolates were selected for further characterization. Physiological and biochemical tests indicated that the isolates were nonfluorescent on King's B medium, and API 50 CHE (bioMérieux, Marcy l'Etoile, France) revealed that they were negative for gelatin hydrolysis, indole production, acid production from raffinose and positive for utilization of glycerol, D-glucose, mannitol, arbutine, esculine, salicine, cellobiose, maltose, melibiose, D-fucose, and D-arabitol; all characteristics of Pantoea agglomerans. Further identification of these isolates was accomplished by DNA analysis. For DNA analysis, 1.4-kbp fragments of the 16S rRNA gene were amplified with primer set 8F/1492R (3) and sequenced with U514F/800R universal primers (2). Five sequences were obtained and deposited in GenBank (Accession Nos. EF050806 to EF050810). A phylogenetic tree was constructed using the UPGMA method (mega version 3.1). Results of the phylogenetic analysis grouped the species P. ananatis, P. stewartti, and P. agglomerans into three clusters. The five unknown sequences were grouped into the P. agglomerans cluster. There was a 98 to 99% similarity of the five 16S rRNA gene sequences with P. agglomerans strain type ATCC 27155. Pathogenicity of the 12 isolates was confirmed by injecting 108 CFU mL-1 of inoculum into stems of 3-week-old maize cv. Triunfo and sorghum cold tolerant hybrid (A1×B5)×R1 seedlings in the greenhouse at 28°C and 80% relative humidity. Also, seedlings were inoculated with water, nonpathogenic isolates of P. agglomerans from maize (GM13, and HLA1), and not inoculated as negative controls. Three replications were included for each isolate and control. All test strains developed water-soaked lesions on juvenile leaves at 8 days postinoculation and were followed by chlorotic to straw-colored leaf streaks and then leaf blight symptoms at 3 weeks postinoculation. All negative control seedlings did not develop symptoms. In addition, the 12 isolates were infiltrated at 107 CFU mL-1 into tobacco leaves that displayed a hypersensitive response at 4 days, indicating the presence of the type III secretion system (1). Isolates were reisolated, and the 16S rRNA gene fragments were 100% similar to their original isolate sequences. P. agglomerans has been reported to affect other crops, including chinese taro in Brazil (2007), onion in the United States (2006) and South Africa (1981), and pearl millet in Zimbabwe (1997); however, to our knowledge, this is the first report of P. agglomerans associated with leaf blight and vascular wilt symptoms in maize and sorghum in the Central Highland Valley of Mexico. References: (1) J. Alfano and A. Collmer. Annu. Rev. Phytopathol 42:385, 2004. (2) Y. Anzai et al. Int. J. Syst. Evol. Microbiol. 50:1563, 2000. (3) M. Sasoh et al. Appl. Environ. Microbiol. 72:1825, 2006.