De novo assembly of transcriptome sequencing in Caragana korshinskii Kom. and characterization of EST-SSR markers

Abstract

Caragana korshinskii Kom. is widely distributed in various habitats, including gravel desert, clay desert, fixed and semi-fixed sand, and saline land in the Asian and African deserts. To date, no previous genomic information or EST-SSR marker has been reported in Caragana Fabr. genus. In this study, more than two billion bases of high-quality sequence of C. korshinskii were generated by using illumina sequencing technology and demonstrated the de novo assembly and annotation of genes without prior genome information. These reads were assembled into 86,265 unigenes (mean length = 709 bp). The similarity search indicated that 33,955 and 21,978 unigenes showed significant similarities to known proteins from NCBI non-redundant and Swissprot protein databases, respectively. Among these annotated unigenes, 26,232 a unigenes were separately assigned to Gene Ontology (GO) database. When 22,756 unigenes searched against the Kyoto Encyclopedia of Genes and Genomes Pathway (KEGG) database, 5,598 unigenes were assigned to 5 main categories including 32 KEGG pathways. Among the main KEGG categories, metabolism was the biggest category (2,862, 43.7%), suggesting the active metabolic processes in the desert tree. In addition, a total of 19,150 EST-SSRs were identified from 15,484 unigenes, and the characterizations of EST-SSRs were further compared with other four species in Fabraceae. 126 potential marker sites were randomly selected to validate the assembly quality and develop EST-SSR markers. Among the 9 germplasms in Caranaga Fabr. genus, PCR success rate were 93.7% and the phylogenic tree was constructed based on the genotypic data. This research generated a substantial fraction of transcriptome sequences, which were very useful resources for gene annotation and discovery, molecular markers development, genome assembly and annotation. The EST-SSR markers identified and developed in this study will facilitate marker-assisted selection breeding.

Fig 1. Functional classification of assembled unigenes.

Functional classification of assembled unigenes based on Gene Ontology (GO) categorisation. The results are summarised in three main GO categories: biological process, cellular component and molecular function. The x-axis indicates the subcategories, and the y-axis indicates the numbers related to the total number of GO terms present; the unigene numbers that are assigned the same GO terms are indicated on the top of the bars.

Fig 2. Pathway assignment based on the Kyoto Encyclopedia of Genes and Genomes (KEGG).

(A) Classification based on cellular process categories, (B) classification based on environmental information processing categories, (C) classification based on genetic information processing categories, (D) classification based on metabolism categories, and (E) classification based on organismal systems categories.

Fig 3. SSRs motif distribution analysis in Caragana korshinskii Kom. and 4 supplementary materials from public database.

The bars from left to right in each species represent di-, tri-, tetra-, penta- and hexa-nucleotides.

Fig 4. Graph of genetic distance among the UPGMA dendrogram of the genetic relationships among 9 species from Caragana Fabr genus.

The dendrogram was generated using the Jaccard similarity coefficient based on 118 polymorphic primer pairs.