Single nucleotide polymorphisms in partial sequences of the gene encoding the large sub-units of ADP-glucose pyrophosphorylase within a representative collection of 10 Musa genotypes

Background ADP-glucose pyrophosphorylase (AGPase) is a rate-limiting enzyme catalyzing the first step in the starch biosynthesis pathway in higher plants. To date, there are no reported variants or isoforms of the AGPase enzyme in bananas (Musa spp. family Musaceae) as is the case of other plants. In this study, genomic DNA sequences homologous to the gene encoding one of the large subunits of the enzyme were amplified from 10 accessions of the genus Musa, including representatives of wild ancestors (AA and BB genomes), dessert bananas (AA, AAA, AB and AAB genomes), plantains (AAB genome) and cooking bananas (ABB and AAA genomes), and studied in order to find single nucleotide polymorphisms (SNP) base variations in Musa accessions. Results In the 810-base pair amplicons of the AGPase large sub-unit (LSU) gene analyzed in ten Musa accessions, a total of 36 SNPs and insertions/deletions (indels) were found. The phylogenetic analysis revealed fifteen distinct haplotypes, which were grouped into four variants. Deep examination of SNPs in the 2nd exon in the LSU of AGPase showed that at seven locations, five SNPs altered their amino acid sequence. Conclusions This work reveals the possible number of AGPase enzyme isoforms and their molecular levels in banana. Molecular markers could be designed from SNPs present in these banana accessions. This information could be useful for the development of SNP-based molecular markers for Musa germplasm, and alteration of the allosteric properties of AGPase to increase the starch content and manipulate the starch quality of banana fruits.

The in vitro secretome of Mycosphaerella fijiensis induces cell death in banana leaves.

The hemibiotrophic filamentous fungus Mycosphaerella fijiensis causes the banana foliar disease known as black Sigatoka, responsible for major worldwide losses in the banana fruit industry. In this work the in vitro secretome of M. fijiensis was characterized. Native and denaturant polyacrylamide gel protease assays showed the M. fijiensis secretome contains protease activity capable of degrading gelatin. Necrotic lesions on leaves were produced by application of the in vitro secretome to the surface of one black Sigatoka-resistant banana wild species, one susceptible cultivar and the non-host plant Carica papaya. To distinguish if necrosis by the secretome is produced by phytotoxins or proteins, the latter ones were precipitated with ammonium sulfate and applied in native or denatured forms onto leaves of the same three plant species. Proteins applied in both preparations were able to produce necrotic lesions. Application of Pronase, a commercial bacterial protease suggested that the necrosis was, at least in part, caused by protease activity from the M. fijiensis secretome. The ability to cause necrotic lesions between M. fijiensis secreted- and ammonium sulfate-precipitated proteins, and purified lipophilic or hydrophilic phytotoxins, was compared. The results suggested that leaf necrosis arises from the combined action of non-host specific hydrolytic activities from the secreted proteins and the action of phytotoxins. This is the first characterization of the M. fijiensis protein secretome produced in vitro but, more importantly, it is also the first time the M. fijiensis secretome has been shown to contain virulence factors capable of causing necrosis to its natural host.

Construction of a genetic linkage map of the fungal pathogen of banana Mycosphaerella fijiensis, causal agent of black leaf streak disease.

A genetic linkage map of the fungal plant pathogen Mycosphaerella fijiensis, causal agent of black leaf streak disease of banana was developed. A cross between the isolates CIRAD86 (from Cameroon) and CIRAD139A (from Colombia) was analyzed using molecular markers and the MAT locus. The genetic linkage map consists of 298 AFLP and 16 SSR markers with 23 linkage groups, containing five or more markers, covering 1,879 cM. Markers are separated on average by around 5.9 cM. The MAT locus was shown to segregate in a 1:1 ratio but could not be successfully mapped. An estimate of the relation between physical size and genetic distance was approximately 39.0 kb/cM. The estimated total haploid genome size was calculated using the genetic mapping data at 4,298.2 cM. This is the first genetic linkage map reported for this important foliar pathogen of banana. The great utility of the map will be for anchoring contigs in the genome sequence, evolutionary studies in comparison with other fungi, to identify quantitative trait loci (QTLs) associated with aggressiveness or oxidative stress resistance and with the recently available genome sequence, for positional cloning.