Microsatellite Marker Development for Genes Related to Defense Responses in Musa acuminata

Bananas (Musa spp.) are cultivated across tropical and subtropical environments, contributing to global food security. Many commercial cultivars are sterile triploids or diploids lacking disease resistance. Considering the importance of molecular tools in genetic improvement, the objective of this study was to develop microsatellite (SSR) markers in Musa acuminata for unigenes related to defense responses to biotic stress. Microsatellites were mined in gene sequence data using WebSat, BEDtools and SSRIT. Data containing genes potentially involved in defense or stress responses comprised unigene sequences for M. acuminata genotypes Calcutta 4 and Cavendish Grande Naine derived from 454 transcriptome pyrosequencing. NBS-LRR family resistance gene analog domain sequences in M. acuminata Calcutta 4 and Cavendish Grande Naine BAC clones,  as well as in the M. acuminata DH Pahang whole genome assembly were also mined for SSRs. A total of 156 SSR loci flanking or within target genes were validated for polymorphism in 20 M. acuminata genotypes, initially organized into DNA bulks contrasting in resistance to Black Leaf Streak and Sigatoka disease. PCR conditions were optimized and amplicon polymorphism examined on 4% denaturing polyacrylamide gels. Over 15% of loci displayed polymorphism, with alleles for each genotype identified following analysis against individualized bulks. The number of alleles across these loci ranged from 2 to 4, with polymorphism information content values ranging from 0.31 to 0.67. Such genic SSR markers offer potential in Musa genetic improvement, through applications in genotyping and genetic diversity analysis, genetic mapping and markers for QTLs, ultimately for downstream marker assisted selection.