Development of SCAR marker in Casuarina equisetifolia for species authentication

Casuarina equisetifolia is one of the most extensively introduced tree species outside its natural range and is a true multipurpose species, providing a range of services and products for industrial and local end users. Natural hybrids of C. equisetifolia have been reported and a putative hybrid of C. junghuhniana and C. equisetifolia is commercially cultivated in Thailand and India. In Taiwan, studies have also revealed that most plants of Casuarina are the result of introgressive hybridization involving C. equisetifolia, C. glauca, and C. cunninghamiana causing difficulty in species identification. The present study was aimed at developing DNA markers for the identification of C. equisetifolia. Seven ISSR primers were amplified in 120 individuals belonging to three species of Casuarina (C. equisetifolia, C. glauca and C. junghuhniana) and two Allocasuarina species (A. littoralis and A. huegeliana). One species-specific amplicon at 650 bp amplified in all individuals of C. equisetifolia was cloned, sequenced and primer pairs were developed and designated as IFGTBCE01. The sequence characterized amplified region (SCAR) marker was multiplexed with internal SSR primer pair (positive control) and amplified in ten randomly selected individuals of C. equisetifolia, C. junghuhniana, C. glauca, A. littoralis and A. huegeliana. The species-specific band amplified only in C. equisetifolia at 500 bp. Subsequently, the SCAR marker was validated in 30 individuals collected from 10 provenances belonging to 9 countries and in 10 locally selected clones. The marker was validated in the natural male hybrid of C. equisetifolia × C. junghuhniana which is widely planted in India for fast growth, wide adaptability and good form. Hence, this marker can be used for accurate and rapid identification of the species during certification and determination of putative hybrids in breeding programs.