Varietal identification of tea(Camellia sinensis)using nanofluidic array of single nucleotide polymorphism(SNP)markers

Apart from water,tea is the world’s most widely consumed beverage.Tea is produced in more than 50 countries with an annual production of approximately 4.7 million tons.The market segment for specialty tea has been expanding rapidly owing to increased demand,resulting in higher revenues and profits for tea growers and the industry.Accurate varietal identification is critically important to ensure traceability and authentication of premium tea products,which in turn contribute to on-farm conservation of tea genetic diversity.Using a set of single nucleotide polymorphism(SNP)markers developed from the expressed sequence tag(EST)database of Camilla senensis,we genotyped deoxyribonucleic acid(DNA)samples extracted from a diverse group of tea varieties,including both fresh and processed commercial loose-leaf teas.The validation led to the designation of 60 SNPs that unambiguously identified all 40 tested tea varieties with high statistical rigor(p<0.0001).Varietal authenticity and genetic relationships among the analyzed cultivars were further characterized by ordination and Bayesian clustering analysis.These SNP markers,in combination with a high-throughput genotyping protocol,effectively established and verified specific DNA fingerprints for all tested tea varieties.This method provides a powerful tool for variety authentication and quality control for the tea industry.It is also highly useful for the management of tea genetic resources and breeding,where accurate and efficient genotype identification is essential.

Identification of the varietal origin of processed loose-leaf tea based on analysis of a single leaf by SNP nanofluidic array

Tea is an important cash crop, representing a $40 billion-a-year global market. Differentiation of the tea market has resulted in increasing demand for tea products that are sustainably and responsibly produced. Tea authentication is important because of growing concerns about fraud involving premium tea products. Analytical technologies are needed for protection and value enhancement of high-quality brands. For loose-leaf teas, the challenge is that the authentication needs to be established on the basis of a single leaf, so that the products can be traced back to the original varieties. A new generation of molecular markers offers an ideal solution for authentication of processed agricultural products. Using a nanofluidic array to identify variant SNP sequences, we tested genetic identities using DNA extracted from single leaves of 14 processed commercial tea products. Based on the profiles of 60 SNP markers, the genetic identity of each tea sample was unambiguously identified by multilocus matching and ordination analysis. Results for repeated samples of multiple tea leaves from the same products(using three independent DNA extractions) showed 100% concordance, showing that the nanofluidic system is a reliable platform for generating tea DNA fingerprints with high accuracy. The method worked well on green, oolong, and black teas, and can handle a large number of samples in a short period of time. It is robust and cost-effective, thus showing high potential for practical application in the value chain of the tea industry.

Developing single nucleotide polymorphism(SNP)markers from transcriptome sequences for identification of longan(Dimocarpus longan)germplasm

Longan(Dimocarpus longan Lour.)is an important tropical fruit tree crop.Accurate varietal identification is essential for germplasm management and breeding.Using longan transcriptome sequences from public databases,we developed single nucleotide polymorphism(SNP)markers;validated 60 SNPs in 50 longan germplasm accessions,including cultivated varieties and wild germplasm;and designated 25 SNP markers that unambiguously identified all tested longan varieties with high statistical rigor(P<0.0001).Multiple trees from the same clone were verified and off-type trees were identified.Diversity analysis revealed genetic relationships among analyzed accessions.Cultivated varieties differed significantly from wild populations(Fst=0.300;P<0.001),demonstrating untapped genetic diversity for germplasm conservation and utilization.Within cultivated varieties,apparent differences between varieties from China and those from Thailand and Hawaii indicated geographic patterns of genetic differentiation.These SNP markers provide a powerful tool to manage longan genetic resources and breeding,with accurate and efficient genotype identification.

Fungal endophytes in green coffee seeds

Green coffee seeds from Colombia, Guatemala, India, Kenya, Papua New Guinea, Puerto Rico and Vietnam were sampled for the presence of fungal endophytes. Stions of surface sterilized seeds were plated on yeast malt agar, and fungal growth was isolated for subsequent DNA extraction and sequencing. Several fungal genera were isolated, including Acremonium, Aspergillus, Eurotium, Fusarium, Gibberella, Penicillium, Pseudozyma and an undescribed clavicipitaceous species. The biological activities that these fungi might be playing in coffee seeds remain unknown, but in other plants some of the genera isolated have been reported to protect against plant pathogens.

Developing single nucleotide polymorphism markers for the identification of pineapple(Ananas comosus)germplasm

Pineapple(Ananas comosus[L.]Merr.)is the third most important tropical fruit in the world after banana and mango.As a crop with vegetative propagation,genetic redundancy is a major challenge for efficient genebank management and in breeding.Using expressed sequence tag and nucleotide sequences from public databases,we developed 213 single nucleotide polymorphism(SNP)markers and validated 96 SNPs by genotyping the United States Department of Agriculture-Agricultural Research Service pineapple germplasm collection,maintained in Hilo,Hawaii.The validation resulted in designation of a set of 57 polymorphic SNP markers that revealed a high rate of duplicates in this pineapple collection.Twenty-four groups of duplicates were detected,encompassing 130 of the total 170 A cosmos accessions.The results show that somatic mutation has been the main source of intra-cultivar variations in pineapple.Multivariate clustering and a model-based population stratification suggest that the modern pineapple cultivars are comprised of progenies that are derived from different wild Ananas botanical varieties.Parentage analysis further revealed that both A.comosus var.bracteatus and A.comosus var.ananassoides are likely progenitors of pineapple cultivars.However,the traditional classification of cultivated pineapple into horticultural groups(e.g.‘Cayenne’,‘Spanish’,‘Queen’)was not well supported by the present study.These SNP markers provide robust and universally comparable DNA fingerprints;thus,they can serve as an efficient genotyping tool to assist pineapple germplasm management,propagation of planting material,and pineapple cultivar protection.The high rate of genetic redundancy detected in this pineapple collection suggests the potential impact of applying this technology on other clonally propagated perennial crops.

Physiological Traits and Metabolites of Cacao Seedlings Influenced by Potassium in Growth Medium

Cacao (Theobroma cacao L.) is of significant economic importance in several tropical countries but its yield potentials are low mainly because of poor soil fertility especially low levels of potassium (K). Cacao has a high demand for K to maintain healthy growth and production. Knowledge of K use in cacao will help the development of suitable crop management practices and will aid breeding varieties adapted to environments with a limited soil K supply. Using a plant growth chamber, we investigated the growth and physiological traits among three cacao varieties at three levels of growth medium K (52, 156, and 469 mg·plant-1). Significant K effects were observed on growth traits including stem diameter, root length, chlorophyll b, and the ratio of chlorophyll a/b. Significant K effect was also found on carbohydrate metabolites, such as fructose, glucose, myo-inositol, raffinose and starch. However, no K effect was observed in other growth and physiological indicators, including biomass of seedling and net photosynthetic rate. There were significant genotype differences on seedling growth indicators, including stem diameter, stem height, total biomass, leaf biomass, leaf area, root length, chlorophyll a + b and carotenoids. Genotype difference was also found on all measured carbohydrate and starch metabolites, except maltose and raffinose. Results of this study indicate that although K plays a critical role in cacao tree growth and productivity, cacao may be less sensitive to K deficiency during the seedling stage. The present results improved our understanding about K and plants interaction in cacao seedlings, which is useful for crop management and germplasm utilization.