Are there any differences in the quality of high-mountain green tea before and after the first new leaves unfold? A comprehensive study based on E-sensors, whole metabolomics and sensory evaluation.

High-mountain green tea, where the first new leaf hasn't yet unfurled, is prized for perceived superior quality, but this hasn't yet been verified by experimentation. Electronic sensors, whole metabolomics and sensory evaluation were employed to assess the quality of yymj (tea buds with a newly unfurled leaf) and qymj (tea buds without new leaves). The qymj proved to have significant advantages in aroma, color and shape, but still had some shortcomings in umami, bitterness and sourness. Differences in the content of volatile organic compounds (including alcohols, hydrocarbons and lipids) and nonvolatile organic compounds (flavonoids, amino acids, sugars, and phenolic acids) quality of high-mountain green teas with different maturity levels and provides well explained these quality differences. This study establishes a systematic approach to study the quality of high-mountain green tea at different maturity levels, and provides important reference information for consumers, governments and tea farmers.

TILLING by sequencing to identify induced mutations in stress resistance genes of peanut (Arachis hypogaea).

BACKGROUND: Targeting Induced Local Lesions in Genomes (TILLING) is a powerful reverse genetics approach for functional genomics studies. We used high-throughput sequencing, combined with a two-dimensional pooling strategy, with either minimum read percentage with non-reference nucleotide or minimum variance multiplier as mutation prediction parameters, to detect genes related to abiotic and biotic stress resistances. In peanut, lipoxygenase genes were reported to be highly induced in mature seeds infected with Aspergillus spp., indicating their importance in plant-fungus interactions. Recent studies showed that phospholipase D (PLD) expression was elevated more quickly in drought sensitive lines than in drought tolerant lines of peanut. A newly discovered lipoxygenase (LOX) gene in peanut, along with two peanut PLD genes from previous publications were selected for TILLING. Additionally, two major allergen genes Ara h 1 and Ara h 2, and fatty acid desaturase AhFAD2, a gene which controls the ratio of oleic to linoleic acid in the seed, were also used in our study. The objectives of this research were to develop a suitable TILLING by sequencing method for this allotetraploid, and use this method to identify mutations induced in stress related genes. RESULTS: We screened a peanut root cDNA library and identified three candidate LOX genes. The gene AhLOX7 was selected for TILLING due to its high expression in seeds and roots. By screening 768 M2 lines from the TILLING population, four missense mutations were identified for AhLOX7, three missense mutations were identified for AhPLD, one missense and two silent mutations were identified for Ara h 1.01, three silent and five missense mutations were identified for Ara h 1.02, one missense mutation was identified for AhFAD2B, and one silent mutation was identified for Ara h 2.02. The overall mutation frequency was 1 SNP/1,066 kb. The SNP detection frequency for single copy genes was 1 SNP/344 kb and 1 SNP/3,028 kb for multiple copy genes. CONCLUSIONS: Our TILLING by sequencing approach is efficient to identify mutations in single and multi-copy genes. The mutations identified in our study can be used to further study gene function and have potential usefulness in breeding programs.

Confirmation of hybridity using GISH and determination of 18S rDNA copy number using FISH in interspecific F(1) hybrids of Tecoma (Bignoniaceae).

Interspecific hybridization in Tecoma Juss. was conducted to develop novel forms for the nursery industry. We report fertile hybrids from the cross T. garrocha Hieron. (pistillate parent) × T. stans (L.) Juss. ex Kunth. Leaf morphology of the F(1) hybrids of T. garrocha × T. stans was intermediate between the parents. GISH also confirmed hybridity. The F(1) hybrids were successfully backcrossed to both parents and self-pollinated to produce BC and F(2) progeny. Tecoma garrocha , T. stans, and T. guarume A. DC. 'Tangelo' were self-fertile. The F(1) hybrids also were crossed with T. capensis (Thunb.) Lindl. and T. guarume 'Tangelo', resulting in three-species hybrids. FISH conducted on F(1) hybrids identified four copies of the 18S internal transcribed spacer region. Further analysis using FISH has the potential to provide information on the evolution of Bignoniaceae and the potential role of polyploidy.

TILLING for allergen reduction and improvement of quality traits in peanut (Arachis hypogaea L.).

BACKGROUND: Allergic reactions to peanuts (Arachis hypogaea L.) can cause severe symptoms and in some cases can be fatal, but avoidance is difficult due to the prevalence of peanut-derived products in processed foods. One strategy of reducing the allergenicity of peanuts is to alter or eliminate the allergenic proteins through mutagenesis. Other seed quality traits could be improved by altering biosynthetic enzyme activities. Targeting Induced Local Lesions in Genomes (TILLING), a reverse-genetics approach, was used to identify mutations affecting seed traits in peanut. RESULTS: Two similar copies of a major allergen gene, Ara h 1, have been identified in tetraploid peanut, one in each subgenome. The same situation has been shown for major allergen Ara h 2. Due to the challenge of discriminating between homeologous genes in allotetraploid peanut, nested PCR was employed, in which both gene copies were amplified using unlabeled primers. This was followed by a second PCR using gene-specific labeled primers, heteroduplex formation, CEL1 nuclease digestion, and electrophoretic detection of labeled fragments. Using ethyl methanesulfonate (EMS) as a mutagen, a mutation frequency of 1 SNP/967 kb (3,420 M2 individuals screened) was observed. The most significant mutations identified were a disrupted start codon in Ara h 2.02 and a premature stop codon in Ara h 1.02. Homozygous individuals were recovered in succeeding generations for each of these mutations, and elimination of Ara h 2.02 protein was confirmed. Several Ara h 1 protein isoforms were eliminated or reduced according to 2D gel analyses. TILLING also was used to identify mutations in fatty acid desaturase AhFAD2 (also present in two copies), a gene which controls the ratio of oleic to linoleic acid in the seed. A frameshift mutation was identified, resulting in truncation and inactivation of AhFAD2B protein. A mutation in AhFAD2A was predicted to restore function to the normally inactive enzyme. CONCLUSIONS: This work represents the first steps toward the goal of creating a peanut cultivar with reduced allergenicity. TILLING in peanut can be extended to virtually any gene, and could be used to modify other traits such as nutritional properties of the seed, as shown in this study.

Identification of ovule transcripts from the Apospory-Specific Genomic Region (ASGR)-carrier chromosome.

BACKGROUND: Apomixis, asexual seed production in plants, holds great potential for agriculture as a means to fix hybrid vigor. Apospory is a form of apomixis where the embryo develops from an unreduced egg that is derived from a somatic nucellar cell, the aposporous initial, via mitosis. Understanding the molecular mechanism regulating aposporous initial specification will be a critical step toward elucidation of apomixis and also provide insight into developmental regulation and downstream signaling that results in apomixis. To discover candidate transcripts for regulating aposporous initial specification in P. squamulatum, we compared two transcriptomes derived from microdissected ovules at the stage of aposporous initial formation between the apomictic donor parent, P. squamulatum (accession PS26), and an apomictic derived backcross 8 (BC8) line containing only the Apospory-Specific Genomic Region (ASGR)-carrier chromosome from P. squamulatum. Toward this end, two transcriptomes derived from ovules of an apomictic donor parent and its apomictic backcross derivative at the stage of apospory initiation, were sequenced using 454-FLX technology. RESULTS: Using 454-FLX technology, we generated 332,567 reads with an average read length of 147 base pairs (bp) for the PS26 ovule transcriptome library and 363,637 reads with an average read length of 142 bp for the BC8 ovule transcriptome library. A total of 33,977 contigs from the PS26 ovule transcriptome library and 26,576 contigs from the BC8 ovule transcriptome library were assembled using the Multifunctional Inertial Reference Assembly program. Using stringent in silico parameters, 61 transcripts were predicted to map to the ASGR-carrier chromosome, of which 49 transcripts were verified as ASGR-carrier chromosome specific. One of the alien expressed genes could be assigned as tightly linked to the ASGR by screening of apomictic and sexual F1s. Only one transcript, which did not map to the ASGR, showed expression primarily in reproductive tissue. CONCLUSIONS: Our results suggest that a strategy of comparative sequencing of transcriptomes between donor parent and backcross lines containing an alien chromosome of interest can be an efficient method of identifying transcripts derived from an alien chromosome in a chromosome addition line.

[Function analysis of a wheat phosphate transporter in yeast mutant].

Phosphorus is a major nutrient acquired by plants and phosphorus availability is considered one of the major growth-limiting factors for plants in many natural ecosystems. For a better understanding of the function of the wheat phosphorus transporter gene named TaPT2, we did Southern blot analysis and studied its function with complementation test in yeast mutant strain MB192. Southern blot indicated that TaPT2 gene is a low-copy member and has several different members in wheat genome. In the function complementation study, TaPT2 has a similar function as PHO84 which is the phosphate transporter in yeast Saccharomyces cerevisiae. Both TaPT2 and PHO84 are able to complement the PHO84 mutant phenotype of yeast strain MB192 which lacks the phosphate transporter activity. Therefor, this assessment presents evidence that TaPT2 gene plays an important role in Pi acquisition.