Development of expressed sequence tag-simple sequence repeat markers for Chrysanthemum morifolium and closely related species

Abstract

ABSTRACT. With the development of chrysanthemum breeding in recent years, an increasing number of wild species in genera related to Chrysanthemum were introduced to extend the genetic resources and facilitate the genetic improvement of chrysanthemums via hybridization. However, few simple sequence repeat (SSR) markers are available for marker-assisted breeding and population genetic studies of chrysanthemum and closely related species. Expressed sequence tags (ESTs) in public databases and cross-species transferable markers are considered to be a cost-effective means for developing sequencebased markers. In this study, 25 EST-SSRs were successfully developed from Chrysanthemum EST sequences for Chrysanthemum morifolium and closely related species. In total, 4164 unigene sequences were assembled from 7180 ESTs of chrysanthemum in GenBank, which were subsequently used to screen for the presence of microsatellites with the Development of EST-SSR markers for chrysanthemum SSRIT software. The screening criteria were 8, 5, 4, and 3 repeating units for di-, tri-, tetra-, and penta-and higher-order nucleotides, respectively. Moreover, 310 SSR loci from 296 sequences were identified, and 198 primer pairs for SSR amplification were designed with the Primer Premier 5.0 software, of which 25 SSR loci showed polymorphic amplification in 52 species and varieties belonging to Chrysanthemum, Ajania, and Opisthopappus. The application of EST-SSR markers to the identification of intergeneric hybrids between Chrysanthemum and Ajania was demonstrated. Therefore, EST-SSRs can be developed for species that lack gene sequences or ESTs by utilizing ESTs of closely related species.

Linkage Map Development by EST-SSR Markers and QTL Analysis for Inflorescence and Leaf Traits in Chrysanthemum (Chrysanthemum morifolium Ramat.)

“…A total of 262 EST-SSR primer pairs were analysed, among which 245 primer pairs were developed from the “Jinbudiao” EST database [ 38 ] and 17 primer pairs were reported in [ 39 ]. All the SSR primer pairs were labelled with fluorescent dyes, and SSR genotyping was carried out using a three-primer strategy, including a forward/reverse primer labelled with FAM, HEX or TAMRA (Beijing Microread Genetics Co., Ltd, Beijing, China), as detailed in the protocol of Sun et al [ 40 ].…”

Genome-Wide Identification and Analysis of Chitinase GH18 Gene Family in Mycogone perniciosa

“…To validate the RNA-seq data, five differentially expressed GH18 genes at different time points (0, 3, 4, 5, and 10 days) of M. perniciosa infection of A. bisporus were analyzed by PCR and RT-PCR. Specific primers (Table 1) were designed for the five GH18 genes using Primer Premier 5.0 software (Liu et al, 2015). Approximately 200 ng/µL of DNA and cDNA products were used as templates for the PCR and RT-PCR.…”

Isolation and characterization of microsatellite markers for Dendranthema morifolium (Asteraceae) using next-generation sequencing

“…SSRs are frequently used to survey phylogenetic relationships and select loci associated with agronomic traits in D. morifolium cultivars (Chen et al, 2016). Previous studies have reported some D. morifolium SSR markers (Moe et al, 2011;Khaing et al, 2013;Liu et al, 2015;Shim et al, 2015). However, the limited number of SSR markers available for D. morifolium fails to meet the requirements for genetic diversity evaluation, as well as linkage and association analyses.…”