Determination of specific DNA markers and glucomannan content of Amorphophallus spp. in Thailand

Abstract

The relationship between konjac glucomannan (KGM) content and genetic characters of Amorphophallus species found in Thailand was investigated Specific DNA markers for characterization of high and low KGM content species were developed by using nucleotide region sequencing and Randomly Amplified Polymorphic DNA (RAPD) analysis. Determination of KGM content indicated that the KGM content in Thai species such as A. muelleri is as high as 68.93% (dry weight) with small variation among this group. KGM contents found in Thai Amosphophallus samples can be divided into high (40-70%), medium (20-39%) and low (<20%), based on the dry weight. Phylogeny reconstructions were studied using the chloroplast trnL-trnF spacer, nuclear ribosomal internal transcribed spacer (ITS) sequences and the second intron of LEAFY (FLint2). Moreover, same data sets were analyzed with BMGE Alignment (Block Mapping and Gathering with Entropy) and PRANK multiple aware-alignment program that indicated a strong performance of phylogenetic clade. Among three nucleotide regions, ITS showed the highest resolution of in-group relationship within this species. Furthermore, RAPD technique together with thirteen primers was used to investigate the genetic variation. From RAPD result, a total of 269 polymorphic bands were generated. The average of genetic distance was varied from 0.075 to 0.949. Genetic relationship trees based on these two techniques were in agreement with each other and related to morphological characters of Amorphophallus plants. Moreover, phylogeny result showed evolutionary relationship with KGM content. This study found two monophyletic clades of high KGM content species while in RAPD analysis primer AC-10 could generate specific band at 600 bp which is specific only to high and medium KGM content. Specific band from RAPD analysis was converted to Sequence Characterized Amplified Region (SCAR) markers. In addition, nucleotide sequencing data of each region was used to generate specific marker for high KGM content group of A. muelleri, A. bulbifer and A. xiei. Seven pairs of specific primers were designed for marker development including HKGM-4F/ HKGM-595R and MUE-129F/MUE-490R from SCAR marker and FLint2 F1/ MUBX236_Flint2, FLint2 F1/ MUBX253_Flint2, MUBX520_ITS/26S-82R, MUBX551_ITS/26S-82R and P17/MUBX994_ ITS from the sequencing markers. Amplified specific PCR products of 600, 350, 200, 200, 600, 600 and 900 bp respectively were obtained for these primers. To validate the specific markers, specificity of designed primer sets was further examined against a large sample number (N = 84). The designed primers showed their specific nature and their sensitivity of detection could be as low as 0.3 ng/mL of genomic DNA of the target species. Identification of high KGM content species with samples of different parts of plant and reproducibility testing of the method were successfully carried out. Specific DNA markers developed in this study have a potential to be used as an initial screening tool for economical species. This can contribute to improve the industrial production of KGM flour in Thailand and be used in the breeding programs to maximize KGM production in the country.