Next-generation sequencing of the Chrysanthemum nankingense (Asteraceae) transcriptome permits large-scale unigene assembly and SSR marker discovery

Abstract

Background: Simple sequence repeats (SSRs) are ubiquitous in eukaryotic genomes. Chrysanthemum is one of the largest genera in the Asteraceae family. Only few Chrysanthemum expressed sequence tag (EST) sequences have been acquired to date, so the number of available EST-SSR markers is very low.

Methodology/principal findings: Illumina paired-end sequencing technology produced over 53 million sequencing reads from C. nankingense mRNA. The subsequent de novo assembly yielded 70,895 unigenes, of which 45,789 (64.59%) unigenes showed similarity to the sequences in NCBI database. Out of 45,789 sequences, 107 have hits to the Chrysanthemum Nr protein database; 679 and 277 sequences have hits to the database of Helianthus and Lactuca species, respectively. MISA software identified a large number of putative EST-SSRs, allowing 1,788 primer pairs to be designed from the de novo transcriptome sequence and a further 363 from archival EST sequence. Among 100 primer pairs randomly chosen, 81 markers have amplicons and 20 are polymorphic for genotypes analysis in Chrysanthemum. The results showed that most (but not all) of the assays were transferable across species and that they exposed a significant amount of allelic diversity.

Conclusions/significance: SSR markers acquired by transcriptome sequencing are potentially useful for marker-assisted breeding and genetic analysis in the genus Chrysanthemum and its related genera.

Figure 1. The distribution of contig and unigene sequence lengths.

Figure 2. The distribution of E-value, sequence similarity, and species of data base hits with the C. nankingense unigenes.

Figure 3. Functional classification of the C. nankingense unigenes according to COG criteria.

Figure 4. The distribution of C. nankingense unigenes among the GO functional classes.

From left to right, “Biological process” (shown in blue): biological adhesion, biological regulation, cell killing, cellular component organization or biogenesis, cellular process, death, developmental process, establishment of localization, growth, immune system process, localization, locomotion, metabolic process, multi-organism process, multicellular organismal process, negative regulation of biological process, pigmentation, positive regulation of biological process, regulation of biological process, reproduction, reproductive process, response to stimulus, rhythmic process, signaling, viral reproduction; “Cellular components” (shown in red): cell, cell junction, cell part, extracellular region, extracellular region part, macromolecular complex, membrane-enclosed lumen, organelle, organelle part, virion; “Molecular function” (shown in green): antioxidant activity, binding, catalytic activity, enzyme regulator activity, molecular transducer activity, protein binding transcription factor activity, receptor activity, transporter activity.

Figure 5. Frequencies of the various repeat motifs present in the C. nankingense EST-SSRs.

Figure 6. SSR allelic variation among 42 commercial C. morifolium cultivars.

(A) Single product, non-polymorphic amplicon(primer #214), (B) Two product, polymorphic amplicon (primer #86), (C) Multiple product, highly polymorphic amplicon (primer #245). Lanes 1–42: cv. ‘Zhongshanzigui’, cv. ‘Zhongshanjingui’, cv. ‘Zhongshanhongying’, cv. ‘Zhongshanaihuang’, cv. ‘Zhongshanzihe’, cv. ‘Zhongshanchengguang’, cv. ‘Zhongshanhongfeng’, cv. ‘Zhongshanhuangtuogui’, cv. ‘Zhongshanguohuang’, cv. ‘Zhongshanbailu’, cv. ‘Zhongshanfenzhuang’, cv. ‘Zhongshanzilian’, cv. Zhongshanfendai’, cv. ‘Zhongshanqueyu’, cv. ‘Zhongshanhongxia’, cv. ‘Zhongshanhuangying’, cv. ‘Zhongshanhuangyu’, cv. ‘Zhongshanjinyu’, cv. ‘Zhongshanyinxing’, cv. ‘Zhongshanzaobai’, cv. ‘Zhongshanzixing’, cv. ‘Zhongshanziyu’, cv. ‘Zhongshanfengui’, cv. ‘Zhongshanjinzhen’, cv. ‘Zhongshanxuegui’, cv. ‘Zhongshanzuirong’, cv. ‘Jinlingaihuang’, cv. ‘Jinlingbaifeng’, cv. ‘Jinlingbaixue’, cv. ‘Jinlingbaiyu’, cv. ‘Jinlingbaohui’, cv. ‘Jinlingbaoxia’, cv. ‘Jinlingchixin’, cv. ‘Jinlingchunse’, cv. ‘Jinlingfanxing’, cv. ‘Jinlingfendai’, cv. ‘Jinlingguofen’, cv. ‘Jinlingguohong’, cv. ‘Jinlingguohuang’, cv. ‘Jinlingguozi’, cv. ‘Jinlinghonghe’, cv. ‘Jinlinghongpao’.

Figure 7. UPGMA-based phylogeny of Chrysanthemum spp. and species belonging to closely related genera.

The tree was derived from genotype data from 20 EST-SSR loci. (A–F) Six clades were recognized.