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.

Dual targeting properties of the N-terminal signal sequence of Arabidopsis thaliana THI1 protein to mitochondria and chloroplasts.

thi1 has been recently isolated from Arabidopsis thaliana and is probably involved in both thiamine biosynthesis and as protection of organellar DNA from damage. Studies of thiamine biosynthesis in plants suggests a plastid location for the pathway, which is in agreement with the predicted THI1 N-terminal chloroplastic transit peptide (TP). On the other hand, thiamine is synthesized in mitochondria in yeast cells. Interestingly, A. thaliana thi1 cDNA complements a yeast strain disrupted for the homologous gene. Analysis of THI1 amino acid sequence revealed the presence of a putative amphiphilic alpha-helix, which is typical for mitochondrial presequences, located downstream of the chloroplast transit peptide. To define the putative role of the two predicted targeting sequences in tandem, we produced two chimeric genes encompassing the chloroplastic THI1 TP and either 4 or 27 (including the putative mitochondrial presequence) N-terminal residues of the mature THI1, both linked to the reporter (gusA) gene. Analysis of GUS distribution in subcellular fractions of transgenic plants revealed that in the construct retaining only 4 residues of mature THI1, GUS was found in the chloroplastic fraction. Extension of the THI1 transit peptide to 27 residues of the mature protein allowed import and processing of GUS into both mitochondria and chloroplasts. Direct analysis by immunogold-labeling with an anti-THI1 polyclonal antibody identified THI1 in both organelles in Arabidopsis. We also provide evidence that the precursors of both organellar isoforms are encoded by a single nuclear transcript. Thus, THI1 is targeted simultaneously to mitochondria and chloroplasts by a post transcriptional mechanism.

Soybean leghemoglobin targeted to potato chloroplasts influences growth and development of transgenic plants.

Potato tubers were transformed with a chimeric gene made by the fusion of the soybean leghemoglobin encoding gene (lba) with the chloroplastic targeting sequence from Rubisco. This construct was placed under the control of the strong constitutive 35S promoter and the 3' nontranslated region of Rubisco from pea. Leghemoglobin expression on kanamycin-resistant plants was monitored by RT-PCR. Furthermore, immunodetection of subcellular fractions of transgenic plants revealed that leghemoglobin was imported and correctively processed inside the organelle. In addition, analysis of transgenic plants revealed reduced growth and decreased tuber production compared with the untransformed plants. It is suggested that leghemoglobin expression in potato chloroplasts interferes with aerobic metabolism, leading to physiological and morphological changes.