Plant antimicrobial peptides: an overview of SuperSAGE transcriptional profile and a functional review.

Defensin, thionin and lipid transfer protein (LTP) gene families, which antimicrobial activity has an attractive use in protein engineering and transgenic production of agronomical important plants, have been here functionally reviewed. Also, a transcriptional overview of a set of plant SuperSAGE libraries and analysis looking for 26 bp tags possibly annotated for those families is presented. Tags differentially expressed (p = 0.05) or constitutively transcribed were identified from leaves or roots SuperSAGE libraries from important Brazilian plant species [cowpea (Vigna unguiculata (L.) Walp.), soybean (Glycine max (L.) Merr.) and modern sugarcane hybrids (Saccharum spp.)] submitted to abiotic [salt (100 mM NaCl) or drought] or biotic stresses [fungus inoculation (Phakopsora pachyrhizi; Asiatic Soyben Rust phytopathogen)]. The diverse transcriptional patterns observed, probably related to the variable range of targets and functions involved, could be the first step to unravel the antimicrobial peptide world and the plant stress response relationship. Moreover, SuperSAGE opens the opportunity to find some SNPs or even rare transcript that could be important on plant stress resistance mechanisms. Putative defensin or LTP identified by SuperSAGE following a specific plant treatment or physiological condition could be useful for future use in genetic improvement of plants.

Characterization of new polymorphic functional markers for sugarcane.

Expressed sequence tags (ESTs) offer the opportunity to exploit single, low-copy, conserved sequence motifs for the development of simple sequence repeats (SSRs). The authors have examined the Sugarcane Expressed Sequence Tag database for the presence of SSRs. To test the utility of EST-derived SSR markers, a total of 342 EST-SSRs, which represent a subset of over 2005 SSR-containing sequences that were located in the sugarcane EST database, could be designed from the nonredundant SSR-positive ESTs for possible use as potential genic markers. These EST-SSR markers were used to screen 18 sugarcane (Saccharum spp.) varieties. A high proportion (65.5%) of the above EST-SSRs, which gave amplified fragments of foreseen size, detected polymorphism. The number of alleles ranged from 2 to 24 with an average of 7.55 alleles per locus, while polymorphism information content values ranged from 0.16 to 0.94, with an average of 0.73. The ability of each set of EST-SSR markers to discriminate between varieties was generally higher than the polymorphism information content analysis. When tested for functionality, 82.1% of these 224 EST-SSRs were found to be functional, showing homology to known genes. As the EST-SSRs are within the expressed portion of the genome, they are likely to be associated to a particular gene of interest, improving their utility for genetic mapping; identification of quantitative trait loci, and comparative genomics studies of sugarcane. The development of new EST-SSR markers will have important implications for the genetic analysis and exploitation of the genetic resources of sugarcane and related species and will provide a more direct estimate of functional diversity.

Detailed alignment of saccharum and sorghum chromosomes: comparative organization of closely related diploid and polyploid genomes.

The complex polyploid genomes of three Saccharum species have been aligned with the compact diploid genome of Sorghum (2n = 2x = 20). A set of 428 DNA probes from different Poaceae (grasses) detected 2460 loci in F1 progeny of the crosses Saccharum officinarum Green German x S. spontaneum IND 81-146, and S. spontaneum PIN 84-1 x S. officinarum Muntok Java. Thirty-one DNA probes detected 226 loci in S. officinarum LA Purple x S. robustum Molokai 5829. Genetic maps of the six Saccharum genotypes, including up to 72 linkage groups, were assembled into "homologous groups" based on parallel arrangements of duplicated loci. About 84% of the loci mapped by 242 common probes were homologous between Saccharum and Sorghum. Only one interchromosomal and two intrachromosomal rearrangements differentiated both S. officinarum and S. spontaneum from Sorghum, but 11 additional cases of chromosome structural polymorphism were found within Saccharum. Diploidization was advanced in S. robustum, incipient in S. officinarum, and absent in S. spontaneum, consistent with biogeographic data suggesting that S. robustum is the ancestor of S. officinarum, but raising new questions about the antiquity of S. spontaneum. The densely mapped Sorghum genome will be a valuable tool in ongoing molecular analysis of the complex Saccharum genome.

RFLP linkage map and genome analysis of Saccharum spontaneum.

An RFLP linkage map of the wild sugarcane species Saccharum spontaneum L. (2n = 8x = 40-128) was constructed, comprising 216 loci, detected by 116 DNA probes, and distributed over 44 linkage groups. At a density of at least one marker every 25-cM interval, the coverage of the genome was estimated as 86%. For the generation of RFLP markers, probes were surveyed from seven DNA libraries: three sugarcane cDNA, one oat cDNA, one rice cDNA, and one barley cDNA, as well as one sugarcane genomic. Sixty-two maize genomic clones that were previously mapped on maize were used to initiate a comparative map between the sugarcane, sorghum, and maize genomes. Based on the RFLP segregation data, we conclude that this species is an autopolyploid, with an estimated genome size of 2107 cM.

Application of molecular markers to assess genetic relationships among accessions of wild oat, Avena sterilis.

The Avena sterilis collection in the National Small Grains Collection (NSGC) is an invaluable source of genetic variation to be exploited by oat breeding programs. Prior knowledge of the structure and distribution of genetic variation within the A. sterilis collection would be useful to efficiently screen the collection for valuable traits. To determine genetic structure within a subset of the collection, restriction fragment length polymorphisms were analyzed in a stratified sample of 173 accessions originating in eight countries of Africa and Southwest Asia. Of the 48 probes used for this study 43 detected polymorphism among accessions. The average number of RFLP patterns per probe ranged from 2.9 among Ethiopian accessions to 3.7 among those from Iran. Genetic variation, as measured by genetic distances and polymorphic indexes, was highest in Iran and lowest in Ethiopia. The probability of drawing a genotype from Iran or Iraq that is not present in the more western regions was high, indicating large genetic divergence of the Iran-Iraq accessions from the other regional collections surveyed. Cluster analysis of genetic distances and probabilities of unique genotypes clearly differentiated the eastern region (Iran and Iraq) from the western region (Algeria, Ethiopia, Israel, Lebanon, Morocco, and Syria). The western region could be further subdivided into two clusters, an African cluster (Algeria, Ethiopia, and Morocco) and a southwestern Asia cluster (Israel, Lebanon, and Syria). Genetic distances were generally related to but not proportional to geographical distances.

The detection and estimation of linkage in polyploids using single-dose restriction fragments.

Restriction fragment length polymorphism (RFLP) linkage maps have been constructed in several major diploid crops. However, construction of RFLP maps directly in polyploids has lagged behind for several reasons: (1) there are a large number of possible genotypes for each DNA probe expected in a segregating population, and these genotypes cannot always be identified readily by their banding phenotypes; and (2) the genome constitutions (allopolyploidy versus autopolyploidy) in many high polyploids are not clearly understood. We present here an analysis of these problems and propose a general method for mapping polyploids based on segregation of single-dose restriction fragments (SDRFS). SDRFs segregate 1:1 (presence: absence) in gametes of heterozygous plants. Hypothetical allopolyploid and autopolyploid species with four ploidy levels of 2n = 4x, 6x, 8x, and 10x, are used to illustrate the procedures for identifying SDRFs, detecting linkages among SDRFs, and distinguishing allopolyploid versus autopolyploids from polyploids of unknown genome constitution. Family size required, probability of linkage, and attributes of different mapping populations are discussed. We estimate that a population size of 75 is required to identify SDRFs with 98% level of confidence for the four ploidy levels. This population size is also adequate for detecting and estimating linkages in the coupling phase for both allopolyploids and autopolyploids, but linkages in the repulsion phase can be estimated only in allopolyploids. For autopolyploids, it is impractical to estimate meaningful linkages in repulsion because very large family sizes (>750) are required. For high-level polyploids of unknown genome constitution, the ratio between the number of detected repulsion versus coupling linkages may provide a crude measurement of preferential chromosome pairing, which can be used to distinguish allopolyploidy from autopolyploidy. To create a mapping population, one parent (P1) should have high heterozygosity to ensure a high frequency of SDRFs, and the second parent (P2) should have a low level of heterozygosity to increase the probability of detecting polymorphic fragments. This condition could be satisfied by choosing outcrossed hybrids as one parental type and inbreds, haploids, or doubled haploids as the other parental type.