Characterization of the Lycium barbarum fruit transcriptome and development of EST-SSR markers.

Chunling Chen,Wenwen Wang,Tiantian Wu,Gaixia Qiao,Meilong Xu,Zhengsheng Zhang,Cuiping Wang,Zhaoyun Tan,Kunbo Wang

doi:10.1371/journal.pone.0187738

Chunling Chen, Wenwen Wang + Show 7 more

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https://doi.org/10.1371/journal.pone.0187738

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Abstract

Lycium barbarum, commonly known as goji, is important in Chinese herbal medicine and its fruit is a very important agricultural and biological product. However, the molecular mechanism of formation of its fruit and associated medicinal and nutritional components is unexplored. Moreover, this species lacks SSR markers due to lack of genomic and transcriptomic information. In this study, a total of 139,333 unigenes with average length of 1049 bp and N50 of 1579 bp are obtained by trinity assembly from Illumina sequencing reads. A total of 92,498 (66.38%) unigenes showed similarities in at least one database including Nr (46.15%), Nt (56.56%), KO (15.56%), Swiss-prot (33.34%), Pfam (33.43%), GO (33.62%) and KOG/COG (17.55%). Genes in flavonoid and taurine biosynthesis pathways were found and validated by RT-qPCR. A total of 50,093 EST-SSRs were identified from 38,922 unigenes, and 22,537 EST-SSR primer pairs were designed. Four hundred pairs of SSR markers were randomly selected to validate assembly quality, of which 352 (88%) were successful in PCR amplification of genomic DNA from 11 Lycium accessions and 210 produced polymorphisms. The polymorphic loci showed that the genetic similarity of the 11 Lycium accessions ranged from 0.50 to 0.99 and the accessions could be divided into 4 groups. These results will facilitate investigations of the molecular mechanism of formation of L. barbarum fruit and associated medicinal and nutritional components, and will be of value to novel gene discovery and functional genomic studies. The EST-SSR markers will be useful for genetic diversity evaluation, genetic mapping and marker-assisted breeding.

Highlights

Lycium barbarum belongs to the Lycium genus, which is widely distributed in northwest China and has been used as a traditional herbal medicine for thousands of years
By transcriptome sequencing of L. barbarum fruit, we aimed to provide a resource for functional gene mining, and develop expressed sequence tag (EST)-SSR markers which can be used for genetic diversity evaluation, construction of linkage maps, fine mapping of crucial genes and marker-assisted breeding
The length distribution of unigenes is showing in S2 Fig. The length distribution comparison of transcripts and unigenes is showing in S3 Fig

Summary

Introduction

Lycium barbarum belongs to the Lycium genus, which is widely distributed in northwest China and has been used as a traditional herbal medicine for thousands of years. With the development of massively-parallel (‘ generation’) sequencing, we can rapidly sequence the transcriptome of an organism by the ‘RNA-seq’ approach, which is essential for interpreting functional elements of the genome and revealing the molecular constituents of cells and tissues [14], and RNA-seq is a very good way to develop EST-SSR markers. Transcriptome studies and functional gene mining by RNA-seq were reported in many species which have no genome sequencing, such as Piper nigrum [15], Litchi chinensis Sonn [16], Arceuthobium sichuanense [17], Idesia polycarpa [18], Cinnamomum camphora L [19], and Lycium chinense Mill, a relative of L. barbarum [20]

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