Development of microsatellite markers by transcriptome sequencing in two species of Amorphophallus (Araceae).

BACKGROUND: Amorphophallus is a genus of perennial plants widely distributed in the tropics or subtropics of West Africa and South Asia. Its corms contain a high level of water-soluble glucomannan; therefore, it has long been used as a medicinal herb and food source. Genetic studies of Amorphophallus have been hindered by a lack of genetic markers. A large number of molecular markers are required for genetic diversity study and improving disease resistance in Amorphophallus. Here, we report large scale of transcriptome sequencing of two species: Amorphophallus konjac and Amorphophallus bulbifer using deep sequencing technology, and microsatellite (SSR) markers were identified based on these transcriptome sequences. RESULTS: cDNAs of A. konjac and A. bulbifer were sequenced using Illumina HiSeq™ 2000 sequencing technology. A total of 135,822 non-redundant unigenes were assembled from about 9.66 gigabases, and 19,596 SSRs were identified in 16,027 non-redundant unigenes. Di-nucleotide SSRs were the most abundant motif (61.6%), followed by tri- (30.3%), tetra- (5.6%), penta- (1.5%), and hexa-nucleotides (1%) repeats. The top di- and tri-nucleotide repeat motifs included AG/CT (45.2%) and AGG/CCT (7.1%), respectively. A total of 10,754 primer pairs were designed for marker development. Of these, 320 primers were synthesized and used for validation of amplification and assessment of polymorphisms in 25 individual plants. The total of 275 primer pairs yielded PCR amplification products, of which 205 were polymorphic. The number of alleles ranged from 2 to 14 and the polymorphism information content valued ranged from 0.10 to 0.90. Genetic diversity analysis was done using 177 highly polymorphic SSR markers. A phenogram based on Jaccard's similarity coefficients was constructed, which showed a distinct cluster of 25 Amorphophallus individuals. CONCLUSION: A total of 10,754 SSR markers have been identified in Amorphophallus using transcriptome sequencing. One hundred and seventy-seven polymorphic markers were successfully validated in 25 individuals. The large number of genetic markers developed in the present study should contribute greatly to research into genetic diversity and germplasm characterization in Amorphophallus.

Genetic Diversity and DNA Fingerprints of Three Important Aquatic Vegetables by EST-SSR Markers.

Twenty-two sacred lotus (Nelumbo nucifera), 46 taros (Colocasia esculenta) and 10 arrowheads (Sagittaria trifolia) were used as materials and combined with EST-SSR (expressed sequence tag-simple sequence repeats) primers developed by our laboratory. Core primers were screened from a large number of primers that were able to distinguish all materials with a high frequency of polymorphisms. Six pairs, twenty pairs and three pairs of core primers were screened from sacred lotus, taro, and arrowhead, respectively. The SSR fingerprints of these three important aquatic vegetables, producing 17-, 87- and 14-bit binary molecular identity cards, respectively, were separately determined by using the core primers. Since there were few core primers of sacred lotus and arrowhead, 3 and 9 primer pairs with higher polymorphic information content (PIC), respectively, were selected as candidate primers. These core and candidate primers were used to identify the purities of No.36 space lotus, Shandong 8502 taro and Wuhan arrowhead, which were 93.3% (84/90), 98.9% (89/90) and 100.0% (90/90), respectively. The fingerprints, displayed as binary molecular identification cards of three important aquatic vegetables, were obtained, and their purity was successfully determined with EST-SSR labeling technology. Phylogenetic trees were also constructed to analyze the genetic diversity of 22 sacred lotus, 46 taros and 10 arrowheads. This study classifies and identifies germplasm resources and is an important reference to test the authenticity and variety purity of other aquatic vegetables in the future.