Genetic and Morphological Diversity Assessment of Five Kalanchoe Genotypes by SCoT, ISSR and RAPD-PCR Markers

Abstract

Determining the appropriate parents for breeding programs is the most important decision that plant breeders must make to maximize the genetic variability and produce excellent recombinant genotypes. Several methods are used to identify genotypes with desirable phenotypic features for breeding experiments. In this study, five kalanchoe genotypes were morphologically characterized by assessing plant height, number of inflorescences, number of flowers, flower length, flower diameter and number of petals. The analysis showed the distinction of yellow kalanchoe in the plant height trait, while the orange kalanchoe was distinguished in the number of inflorescences, the number of flowers and flower length traits, whereas the violet kalanchoe possessed the largest flower diameter and the highest number of petals. The molecular profiling was performed by random amplified polymorphism DNA (RAPD), inter-simple sequence repeats (ISSR) and start codon targeted (SCoT)-polymerase chain reaction (PCR) tools. Genomic DNA was extracted from young leaves and the PCR reactions were performed using ten primers for each SCoT, ISSR and RAPD marker. Only four out of ten primers showed amplicon profiles in all PCR markers. A total of 70 bands were generated by SCoT, ISSR and RAPD-PCR with 35 polymorphic bands and 35 monomorphic bands. The total number of bands of RAPD, ISSR and SCoT was 15, 17 and 38, respectively. The polymorphism percentages achieved by RAPD, ISSR and SCoT were 60.25%, 15% and 57%, respectively. The cluster analysis based on morphological data revealed two clusters. Cluster I consisted of violet and orange kalanchoe, and cluster II comprised red, yellow and purple kalanchoe. Whereas the cluster analysis based on molecular data revealed three clusters. Cluster I included only yellow kalanchoe, cluster II comprised orange and violet kalanchoe while cluster III comprised red, and purple kalanchoe. The study concluded that orange, violet and yellow kalanchoe are distinguished parents for breeding economically valued traits in kalanchoe. Also, the study concluded that SCoT and RAPD markers reproduced reliable banding patterns to assess the genetic polymorphism among kalanchoe genotypes that consider the basis stone for genetic improvements in ornamental plants.

Keywords: ISSR; RAPD; SCoT; diversity assessment; genetic polymorphism; molecular markers.

Figure 1

Morphological characterization of five kalanchoe (Kalanchoe blossfeldiana) genotypes. (A) Different kalanchoe plants with various colors illustrate plant architecture. (B) Flower colors polymorphism. (C) Difference in leaf shape and size.

Figure 2

Selected characteristics of five kalanchoe genotypes; each presented value is means ± standard error, and the different letters mean that values are significantly different (p ≤ 0.05) according to Fisher’sLSD_0.05. (A) plant height (B) Number of inflorescences. (C) Number of flowers (D) Flower length (E) Flower diameter (F) Number of petals. Note: (A–C) Average of 10 plants (D–F) Average of 50 flowers/plant.

Figure 3

DNA fragment patterns of RAPD, ISSR and SCoT-PCR amplification of five kalanchoe genotypes. (A) RAPD-PCR amplification using primers OPA 2, OPA 7, OPA 9 and OPA 10, respectively. (B) ISSR-PCR amplification using primers ISSR-3, ISSR-5, ISSR-8 and ISSR-10, respectively. (C) SCoT-PCR amplification using primers SCoT3, SCoT11, SCoT13 and SCoT14, respectively. M = 100 bp Plus DNA Ladder. The monomorphic loci are presented in green numbers and the polymorphic loci are presented in yellow numbers.

Figure 4

Phylogenetic tree of five kalanchoe genotypes revealed by the weighted pair group method using arithmetic average (WPGMA) method based on morphological features.

Figure 5

Phylogenetic tree of five kalanchoe genotypes revealed by the weighted pair group method using arithmetic average (WPGMA) method based on SCoT, ISSR and RAPD banding patterns.