Exploitation of conserved intron scanning as a tool for molecular marker development in the Saccharum complex

This study investigated the potential of the conserved intron scanning approach to develop molecular markers for genes involved in lignin biosynthesis among members of the Saccharum complex and a distantly related Imperata species. Five intron-flanking primer sets targeting genes encoding five lignin biosynthetic enzymes—phenylalanine ammonia lyase (PAL), 4-coumarate coenzyme A ligase (4-CL), caffeoyl-CoA 3-O-methyltransferase (CCoAOMT), cinnamoyl-CoA reductase (CCR) and peroxidase (POX)—were designed based on the sequence analysis between sugarcane expressed sequence tags (ESTs) and Sorghum bicolor orthologs. Nucleotide sequence analyses of the amplicons of PAL, 4-CL and CCoAOMT orthologs revealed the presence of single nucleotide polymorphisms (SNPs) and insertions–deletions (INDELs) in the target introns as well as (CT)-simple sequence repeats (SSRs) in CCoAOMT orthologs. The SSR marker screening against fifty-nine accessions of the Saccharum complex and an Imperata species confirmed that the identified SSR markers were highly polymorphic among Saccharum and Erianthus species. PCR-restriction fragment length polymorphism (PCR–RFLP) and cleaved amplified polymorphic sequence (CAPS) marker screening of 4-CL and CCoAOMT orthologs developed genus-specific molecular markers that confirmed the intergeneric hybridization status of Saccharum Fiji hybrids. The current study showed that the conserved intron scanning strategy is applicable to multiple copy genes of polyploid monocots. The conserved intron scanning approach provides a novel way of investigating DNA polymorphisms among species within the Saccharum complex and has the potential to help in the development of marker-assisted selection in intergeneric hybrids.