A two-step method for identification of the Chinese glutinous rice Suyunuo, based on ISSR-SCAR and allele-specific markers.

Suyunuo is a valuable glutinous rice variety cultivated mainly in the Lake Taihu area of China. Historically, Suyunuo was presented to emperors as a tribute, and, still today, enjoys a great reputation in China. This study aimed to develop a unique, specific molecular marker for the identification of Suyunuo rice. Polymerase chain reaction (PCR) amplification of inter-simple sequence repeat (ISSR) molecular markers was performed on Suyunuo and 11 other glutinous rice varieties that are mainly cultivated in the Yangtze River Delta region. A Suyunuo-specific band was detected in the PCR products generated from primer ISSR-807. A sequence characterized amplified region (SCAR) primer pair targeting a Suyunuo-specific band was subsequently designed. The SCAR primers amplified a target band in all individuals of Suyunuo and in four glutinous indica varieties, whereas no bands were found in the seven glutinous japonica varieties. Subsequently, sequences amplified by the SCAR primer pair were analyzed to facilitate the design of Suyunuo allele-specific primers. The allele-specific primer pair produced target bands in all individuals of Suyunuo rice but no bands in individuals of any of the other 11 rice varieties. This study provides a theoretical guideline for rice germplasm identification and innovation of other valuable rice landraces.

Two-step identification of taro (Colocasia esculenta cv. Xinmaoyu) using specific psbE-petL and simple sequence repeat-sequence characterized amplified regions (SSR-SCAR) markers.

Colocasia esculenta cv. Xinmaoyu is an eddoe-type taro cultivar local to Taicang, Jiangsu Province, China; it is characterized by its pure flavor, glutinous texture, and high nutritional value. Due to its excellent qualities, the Trademark Office of the State Administration for Industry and Commerce of the People's Republic of China awarded Xinmaoyu, a geographical indication certification in 2014. Therefore, there is an urgent need to develop an efficient molecular marker for the specific identification of this cultivar, which would greatly facilitate the conservation and utilization of this unique germplasm resource. In the present study, amplifying the psbE-petL fragment from two dasheen-type and seven eddoe-type taro cultivars revealed three conserved insertions/deletions among sequences from the two taro types. Based on these sequence differences, a pair of site-specific primers was designed targeting the psbE-petL sequence from the dasheen-type taro, which specifically amplified a DNA band in all individuals from cultivars of this type, but not in those from the seven eddoe-type cultivars. To discriminate Xinmaoyu from the other eddoe-type taro cultivars, a pair of simple sequence repeat-sequence characterized amplified region (SSR-SCAR) primers was further developed to specifically amplify a DNA band from all Xinmaoyu individuals, but not from individuals of other eddoe-type taro cultivars. In conclusion, through a two-step-screening procedure using psbE-petL and SSR-SCAR markers, we developed a pair of primers that could specifically discriminate Xinmaoyu from nine taro cultivars commonly cultivated in Jiangsu Province and Fujian Province.

Analysis of genetic diversity of Brassica rapa var. chinensis using ISSR markers and development of SCAR marker specific for Fragrant Bok Choy, a product of geographic indication.

Non-heading Chinese cabbage [Brassica rapa var. chinensis (Linnaeus) Kitamura] is a popular vegetable and is also used as a medicinal plant in traditional Chinese medicine. Fragrant Bok Choy is a unique accession of non-heading Chinese cabbage and a product of geographic indication certified by the Ministry of Agriculture of China, which is noted for its rich aromatic flavor. However, transitional and overlapping morphological traits can make it difficult to distinguish this accession from other non-heading Chinese cabbages. This study aimed to develop a molecular method for efficient identification of Fragrant Bok Choy. Genetic diversity analysis, based on inter-simple sequence repeat molecular markers, was conducted for 11 non-heading Chinese cabbage accessions grown in the Yangtze River Delta region. Genetic similarity coefficients between the 11 accessions ranged from 0.5455 to 0.8961, and the genetic distance ranged from 0.0755 to 0.4475. Cluster analysis divided the 11 accessions into two major groups. The primer ISSR-840 amplified a fragment specific for Fragrant Bok Choy. A pair of specific sequence-characterized amplified region (SCAR) primers based on this fragment amplified a target band in Fragrant Bok Choy individuals, but no band was detected in individuals of other accessions. In conclusion, this study has developed an efficient strategy for authentication of Fragrant Bok Choy. The SCAR marker described here will facilitate the conservation and utilization of this unique non-heading Chinese cabbage germplasm resource.

Applying DNA barcodes for identification of economically important species in Brassicaceae.

Brassicaceae is a large plant family of special interest; it includes many economically important crops, herbs, and ornamentals, as well as model organisms. The taxonomy of the Brassicaceae has long been controversial because of the poorly delimited generic boundaries and artificially circumscribed tribes. Despite great effort to delimitate species and reconstruct the phylogeny of Brassicaceae, little research has been carried out to investigate the applicability and effectiveness of different DNA regions as barcodes - a recent aid for taxonomic identification - to identify economically important species in Brassicaceae. In this study, we evaluated the feasibility of five intensively recommended regions [rbcL, matK, trnH-psbA, internal transcribed spacer (ITS), ITS2] as candidate DNA barcodes to discriminate economic species of Brassicaceae in China and try to establish a new digital identification method for economic plants of Brassicaceae. All sequences of 58 samples from 27 economic species (Brassicaceae) in China were assessed in the success rates of PCR amplifications, intra- and inter-specific divergence, DNA barcoding gaps, and efficiency of identification. Compared with other markers, ITS showed superiority in species discrimination with an accurate identification of 67.2% at the species level. Consequently, as one of the most popular phylogenetic markers, our study indicated that ITS was a powerful but not perfect barcode for Brassicaceae identification. We further discuss the discrimination power of different loci due to inheritance pattern, polyploidization and hybridization in species-specific evolution. Further screening of other nuclear genes related to species isolation as plant barcode candidates is also proposed.

Transcriptomic analysis of Camellia ptilophylla and identification of genes associated with flavonoid and caffeine biosynthesis.

Camellia ptilophylla, or cocoa tea, is naturally decaffeinated and its predominant catechins and purine alkaloids are trans-catechins and theobromine Regular tea [Camellia sinensis (L.) O. Ktze.] is evolutionarily close to cocoa tea and produces cis-catechins and caffeine. Here, the transcriptome of C. ptilophylla was sequenced using the 101-bp paired-end technique. The quality of the raw data was assessed to yield 70,227,953 cleaned reads totaling 7.09 Gbp, which were assembled de novo into 56,695 unique transcripts and then clustered into 44,749 unigenes. In catechin biosynthesis, leucoanthocyanidin reductase (LAR) catalyzes the transition of leucoanthocyanidin to trans-catechins, while anthocyanidin synthase (ANS) and anthocyanidin reductase (ANR) catalyze cis-catechin production. Our data demonstrate that two LAR genes (CpLAR1 and CpLAR2) by C. ptilophylla may be advantageous due to the combined effects of this quantitative trait, permitting increased leucoanthocyanidin consumption for the synthesis of trans-catechins. In contrast, the only ANS gene observed in C. sinensis (CsANS) shared high identity (99.2%) to one homolog from C. ptilophylla (CpANS1), but lower identity (~80%) to another (CpANS2). We hypothesized that the diverged CpANS2 might have lost its ability to synthesize cis-catechins. C. ptilophylla and C. sinensis each contain two copies of ANR, which share high identity and may share the same function. Transcriptomic sequencing captured two N-methyl nucleosidase genes named NMT1 and NMT2. NMT2 was highly identical to three orthologous genes TCS2, PCS2, and ICS2, which did not undergo methylation in vitro; in contrast, NMT1 was less identical to TCS, PCS and ICS, indicating that NMT1 may undergo neofunctionalization.

Isolation and characterization of polymorphic microsatellite loci from aerial yam (Dioscorea bulbifera L.).

Dioscorea bulbifera L. is widely distributed in pantropical regions along the equator. The taxonomic treatment of this species is ambiguous due to its extreme polymorphic morphological characters. In order to provide tools to facilitate the study of genetic diversity, population structure, patterns of gene flow, and the mating system of this species, and to assess intraspecific variability and relationships in D. bulbifera, 14 novel polymorphic microsatellite loci were developed using the dual-suppression PCR technique. The number of alleles per locus ranged from 4 to 17, with an average of 9.93. The mean observed heterozygosities were 0.7327 and 0.7223, and the mean Shannon-Wiener indices were 1.6431 and 1.811 in the Nanjing and Nanchong populations, respectively. All novel microsatellite loci showed high levels of polymorphism, indicating that these markers offer great potential significance and profound influence for future studies of this species.

DNA barcoding of populations of Fallopia multiflora, an indigenous herb in China.

Fallopia multiflora, locally known as Heshouwu, is one of the most important and widely used Chinese medicinal herbs. However, there is still considerable confusion concerning its different provenances. DNA barcoding is a recent aid to taxonomic identification and uses a short standardized DNA region to discriminate plant species. We assessed the applicability of 4 candidate DNA barcodes (matK, rbcL, psbA-trnH, and ITS2) to identify populations of F. multiflora. To our knowledge, this is the first attempt involving the plant kingdom to apply DNA barcoding at a level lower than species. Four DNA loci (matK, rbcL, psbA-trnH, and ITS2) of 105 samples, including the wild F. multiflora distributed in 17 provinces of China and 4 cultivated F. multiflora lines, were amplified by PCR and sequenced. The 4 loci were evaluated by PCR amplification for sequence quality, extent of genetic divergence, DNA barcoding gap, and the ability to discriminate between populations by BLAST1 and Nearest Distance. We found that psbA-trnH was the best barcode, with significant inter-population variability and best potential for identifying F. multiflora. The combination of loci gave better performance for distinguishing populations than a single locus. We recommend using matK + rbcL + psbA-trnH + ITS2 or psbA-trnH alone for this species. This research demonstrates the utility of DNA barcoding for geoherbalism identifications.

Development of polymorphic microsatellite markers for Dioscorea zingiberensis and cross-amplification in other Dioscorea species.

Dioscorea zingiberensis C.H. Wright (Dioscoreaceae) is an endemic species in central and southwestern China. In order to study the genetic diversity and population structure of this species, 19 novel polymorphic microsatellite loci were developed using a dual-suppression PCR technique. The number of alleles per locus ranged from 3 to 21, with an average of 9.53. All the markers showed high transferability in cross-species amplification in other species of sect. Stenophora.

Intraoral juvenile xanthogranuloma. A case report and literature review.

Juvenile xanthogranuloma rarely occurs in the oral cavity and has received little attention. A case of histologically documented juvenile xanthogranuloma of the oral cavity is described. This is the first intraoral case reported in the Oriental race and in the vestibule. Pertinent literature regarding intraoral lesions of this condition is also reviewed.