Developing and characterization of single locus DNA markers for the seaweeds, Kappaphycus and Eucheuma spp. in Sabah, Malaysia

• Main Author: Salahaudin Maili Mohd Rasli
• Format: Thesis
• Language: English; English
• Published: 2015
• Subjects: SB119-124 Propagation Including breeding, selection, grafting, air layering
• Online Access: https://eprints.ums.edu.my/id/eprint/39468/1/24%20PAGES.pdf; https://eprints.ums.edu.my/id/eprint/39468/2/FULLTEXT.pdf; https://eprints.ums.edu.my/id/eprint/39468/
• Institution: Universiti Malaysia Sabah

The red algae belonging to the genus of Kappaphycus and Eucheuma seaweeds are key sources of carrageenan. The genomic information of Kappaphycus and Eucheuma in public database at present is very limited. The paucity of such genomic information may become a hurdle for marker development. Different varieties of Kappaphycus and Eucheuma are known to have different agronomical value. Despite their high economic values, the genetic information of domesticated and wild varieties remains unclear. The present study was carried out to develop and characterize single locus DNA markers derived from three different partial small-insert genomic libraries as a solution for the molecular characterization of Kappaphycus and Eucheuma in Sabah, Malaysia. A genomic library was constructed and a total of 179 positive clones was selected and sequenced for their inserts. Primers were designed for 154 sequences and an array of 147 locus specific primers were selected and assayed against 13 congeners of Kappaphycus and Eucheuma seaweeds. Database searches and other bioinformatics analyses were performed on NCBI website and other online tools revealing a large proportion of 133 sequences with no BLASTn hits and several significant hits to known and unknown protein for BLASTx. A total of 117 primers amplifying single locus was observed and another 32 primers failed to produce clear and homogenous single locus amplification. Two primers amplified in all tested varieties, 18 primers amplified only with Kappaphycus and 44 primers amplified only in Eucheuma. The remaining 53 primers showed variable amplifications across the Kappaphycus varieties. Jaccard similarity coefficient revealed low genetic variations among the tested variety of Kappaphycus and Eucheuma seaweeds. Unweighted Pair Group Method with Arithmetic Mean (UPGMA) tree was used in clustering procedure based on the binary matrix scores. The clustering was found to be congruent, with some inconsistencies, with the previously reported phylogenetic tree reconstruction of the Eucheuma and Kappaphycus seaweeds by revealing four clusters including the newly found species of Kappaphycus malesianus from Sabah, Malaysia. A total of eight diagnostic markers were identified and selected for multiplex polymerase chain reaction (PCR) in the fingerprinting maps development for variety-specific identification to be used as rapid and reliable standard protocol for varietal identification of Eucheuma and Kappaphycus seaweeds. We demonstrated that the primer arrays were able to assign the varieties to their respective species and reveal inter- and intraspecific relationship between the tested varieties. This study also represents the first step in an experimental breeding programme for Kappaphycus and Eucheuma. In future, these DNA markers can be applied to the biotechnological improvement of Kappaphycus and Eucheuma seaweeds for interspecific or intergeneric hybrid development through protoplast fusion technology by using the single locus DNA marker as a verification tool and also for seaweeds variety protection purposes.