De Novo Assembly and Characterization of the Fruit Transcriptome of Chinese Jujube (Ziziphus jujuba Mill.) Using 454 Pyrosequencing and the Development of Novel Tri-Nucleotide SSR Markers

et al. (2014) De Novo Assembly and Characterization of the Fruit Transcriptome of Chinese Jujube (Ziziphus jujuba Mill.) Using 454 Pyrosequencing and the Development of Novel Tri-Nucleotide SSR Markers. PLoS ONE 9(9): e106438. doi:10.1371/journal.pone.0106438 De Novo Assembly and Characterization of the Fruit Transcriptome of Chinese Jujube (Ziziphus jujuba Mill.) Using 454 Pyrosequencing and the Development of Novel Tri-Nucleotide SSR Markers Yingyue Li 0 Chaoqun Xu 0 Xinggu Lin 0 Binbin Cui 0 Rongling Wu 0 Xiaoming Pang 0 Ji-Hong Liu, Key Laboratory of Horticultural Plant Biology (MOE), China 0 1 National Engineering Laboratory for Tree Breeding, Key Laboratory of Genetics and Breeding in Forest Trees and Ornamental Plants, Ministry of Education, College of Biological Sciences and Biotechnology, Beijing Forestry University , Beijing , China , 2 Center for Computational Biology, Beijing Forestry University , Beijing , China Chinese jujube (Ziziphus jujuba Mill.) is an economically important deciduous tree that has high therapeutic value and health benefits. However, a lack of sequence data and molecular markers have constrained genetic and breeding studies for better fruit quality and other traits in Chinese jujube. In this study, two combined cDNA libraries of 'Dongzao' fruit representing the early and late stages of fruit development were constructed and sequenced on the 454 GS FLX Titanium platform. In total, 1,124,197 reads were generated and then de novo assembled into 97,479 unigenes. A total of 52,938 unigenes were homologous to genes in the NCBI non-redundant sequence database. A total of 33,123 unigenes were assigned to one or more Gene Ontology terms, and 16,693 unigenes were classified into 319 Kyoto Encyclopedia of Genes and Genomes pathways. The results showed that the Smirnoff-Wheeler pathway was the main pathway for the biosynthesis of ascorbic acid in Chinese jujube. The number of differentially expressed genes between the two stages of fruit development was 1,764, among which 974 and 790 genes were up-regulated and down-regulated, respectively. Furthermore, 9,893 sequences were identified containing SSRs. 93 primer pairs designed from the sequences with a tri-nucleotide repeat showed successful PCR amplification and could be validated in Chinese jujube accessions and Z. mauritiana Lam and Z. acidojujuba as well, of which 71 primer pairs were polymorphic. The obtained transcriptome provides a most comprehensive resource currently available for gene discovery and the development of functional markers in Z. jujuba. The newly developed microsatellite markers could be used in applications such as genetic linkage analysis and association studies, diversity analysis, and marker-assisted selection in Chinese jujube and related species. - Data Availability: The authors confirm that all data underlying the findings are fully available without restriction. The Roche 454 reads of Chinese jujube were submitted to NCBI Sequence Read Archive under the accession number of SRR1231563. Funding: This work was supported by Projects in the National Science & Technology Pillar Program (2013BAD14B0302), the National Natural Science Foundation of China (31372019), Special Fund for Forestry-Scientific Research in the Public welfare (201004017) and a Thousand-person Plan Award to Dr. Rongling Wu. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: Rongling Wu, one of the authors, is an associate editor of PLOS ONE. This does not alter the authors adherence to PLOS ONE Editorial policies and criteria. . These authors contributed equally to this work. Ziziphus species of the Rhamnaceae, the buckthorn family, are widespread in both hemispheres [1]. Chinese jujube (Ziziphus jujuba M.) and Indian jujube (Z. mauritiana Lam.) are two species that have considerable horticultural importance [1]. Approximately 170 other species exist worldwide, some of local importance. Chinese jujube (also known as Chinese date) is an important deciduous fruit tree that is grown in temperate and subtropical areas and is one of the most important fruit trees in China, where it has been cultivated and utilized for more than 4,000 years [2]. Due to its wider adaptability under adverse soil and climatic conditions and due to the multiple uses of jujube fruit, Chinese jujube is ranked first in dried fruit production in China, with an average annual production of 3.5 million tons (dried weight) [3]. Chinese jujube has a diploid genome (2n = 26 = 24) that is estimated to be about 430 Mbp [4]. Two triploid cultivars, Zanghuangdazao and Pingguozao, are also found in nature [[5 6], Pang et al, unpublished data]. The jujube fruit is an edible oval drupe, 1.5 to 6.0 centimeters long [1]. The fruit is rich in nutritive substances, including potassium, phosphorus, calcium and manganese, which are the major mineral components, as well as iron, sodium, zinc, copper and biologically active components, including vitamin C, phenolics, flavonoids, triterpenic acids, cyclic adenosine monophosphate (cAMP) and polysaccharides [78]. Furthermore, the fruits have also been used in traditional Chinese medicine to treat anorexia, fatigue, and loose stools in deficiency syndromes of the spleen and of hysteria in women [9]. In recent years, additional health benefits of Chinese jujube fruit have been reported, including anticancer, anti-inflammatory, anti-obesity, immunostimulating, antioxidant, hepatoprotective, and gastrointestinal protective activities and roles in the inhibition of foam cell formation in macrophages [9]. Chinese jujube can be consumed fresh, dehydrated, canned or processed into candy, jam, juice, wine, syrup or vinegar [10]. The nutritional and sensorial attributes of the fruit are determined throughout the successive phases of fruit development, which includes cell division and the expansion of the ovary tissues in a highly coordinated, complex genetically programmed process that involves a series of physiological, biochemical and organoleptic changes [1113]. The continuing development of genomics tools for important fruit crops has accelerated research in fruit development. Many genes regulating fruit development, such as fw 2.2 and SUN, have been identified, and the underlying mechanisms of fruit development have been extensively studied in several species, such as tomato, strawberry and apple [14]. A wide variety of Chinese jujube cultivars are grown in China that differ in their fruit size, shape, texture, taste and nutritional content [1,15]. However, few studies have addressed the gene expression patterns in the fruit development of Chinese jujube. Recently, Liu et al. reported a fruit cDNA library with 965 unigenes and successfully developed 119 gene-derived SSR markers [16]. Lin et al. obtained 216 genes that are related to fruit softening using suppression subtractive hybridization (SSH) technolo (...truncated)