Comparative transcriptome analysis to identify candidate genes involved in 2-methoxy-1,4-naphthoquinone (MNQ) biosynthesis in Impatiens balsamina L.

Impatiens balsamina L. is a tropical ornamental and traditional medicinal herb rich in natural compounds, especially 2-methoxy-1,4-naphthoquinone (MNQ) which is a bioactive compound with tested anticancer activities. Characterization of key genes involved in the shikimate and 1,4-dihydroxy-2-naphthoate (DHNA) pathways responsible for MNQ biosynthesis and their expression profiles in I. balsamina will facilitate adoption of genetic/metabolic engineering or synthetic biology approaches to further increase production for pre-commercialization. In this study, HPLC analysis showed that MNQ was present in significantly higher quantities in the capsule pericarps throughout three developmental stages (early-, mature- and postbreaker stages) whilst its immediate precursor, 2-hydroxy-1,4-naphthoquinone (lawsone) was mainly detected in mature leaves. Transcriptomes of I. balsamina derived from leaf, flower, and three capsule developmental stages were generated, totalling 59.643 Gb of raw reads that were assembled into 94,659 unigenes (595,828 transcripts). A total of 73.96% of unigenes were functionally annotated against seven public databases and 50,786 differentially expressed genes (DEGs) were identified. Expression profiles of 20 selected genes from four major secondary metabolism pathways were studied and validated using qRT-PCR method. Majority of the DHNA pathway genes were found to be significantly upregulated in early stage capsule compared to flower and leaf, suggesting tissue-specific synthesis of MNQ. Correlation analysis identified 11 candidate unigenes related to three enzymes (NADH-quinone oxidoreductase, UDP-glycosyltransferases and S-adenosylmethionine-dependent O-methyltransferase) important in the final steps of MNQ biosynthesis based on genes expression profiles consistent with MNQ content. This study provides the first molecular insight into the dynamics of MNQ biosynthesis and accumulation across different tissues of I. balsamina and serves as a valuable resource to facilitate further manipulation to increase production of MNQ.

Data of de novo assembly and functional annotation of the leaf transcriptome of Impatiens balsamina.

Impatiens balsamina is both an ornamental and pharmacologically important plant widely distributed in many Asian countries. The leaf of the plant contains many secondary metabolites possessing anti-microbial, anti-tumour and anti-cancer properties. Though there are many phytochemical studies done on the different natural extracts for this plant, not much of genetic information is currently available. This is the first transcriptome of I. balsamina leaf using paired-end Illumina HiSeq sequencing which generated 10.79 GB of raw data. Information of pre-processing (reads filtering), de novo assembly and functional annotation are presented. This data is accessible via NCBI BioProject (PRJNA505711).

Begoniayenyeniae (Begoniaceae), a new species from Endau Rompin National Park, Johor, Malaysia.

Begoniayenyeniae is a new species of horticultural value known only from the Endau Rompin National Park, Peninsular Malaysia. It is similar to Begoniarajah with which it had previously been confused in the number of tepals and leaf characters. The new species is compared with three similar species, B.foxworthyi, B.rajah and B.reginula and photographs of all four species and descriptions of B.yenyeniae and B.rajah are provided. Molecular analysis using the ndhF-rpl132 chloroplast marker confirms the four species as distinct. Amongst native species, the three variegated species, B.yenyeniae, B.rajah and B.reginula, are some of the most popular Malaysian begonias in cultivation. Based on its restricted distribution, Begoniayenyeniae, under the IUCN Red List Categories and Criteria, is assessed as Critically Endangered.

Gene isolation using degenerate primers targeting protein motif: A laboratory exercise.

Structures and functions of protein motifs are widely included in many biology-based course syllabi. However, little emphasis is placed to link this knowledge to applications in biotechnology to enhance the learning experience. Here, the conserved motifs of nucleotide binding site-leucine rich repeats (NBS-LRR) proteins, successfully used for the isolation and characterization of many plant resistance gene analogues (RGAs), is featured in the development of a series of laboratory experiments using important molecular biology techniques. A set of previously isolated RGA sequences is used as the model for performing sequence alignment and visualising 3D protein structure using current bioinformatics programs (Clustal Omega and Argusdock software). A pair of established degenerate primer sequences is provided for the prediction of targeted amino acids sequences in the RGAs. Reverse transcription-polymerase chain reaction (RT-PCR) is used to amplify RGAs from total RNA samples extracted from the tropical wild relative of black pepper, Piper colubrinum (Piperaceae). This laboratory exercise enables students to correlate specific DNA sequences with respective amino acid codes and the interaction between conserved motifs of resistance genes with putatively targeted proteins. © 2017 by The International Union of Biochemistry and Molecular Biology, 46(1):47-53, 2018.

Malaysian weedy rice shows its true stripes: wild Oryza and elite rice cultivars shape agricultural weed evolution in Southeast Asia.

Weedy rice is a close relative of domesticated rice (Oryza sativa) that competes aggressively with the crop and limits rice productivity worldwide. Most genetic studies of weedy rice have focused on populations in regions where no reproductively compatible wild Oryza species occur (North America, Europe and northern Asia). Here, we examined the population genetics of weedy rice in Malaysia, where wild rice (O. rufipogon) can be found growing in close proximity to cultivated and weedy rice. Using 375 accessions and a combined analysis of 24 neutral SSR loci and two rice domestication genes (sh4, controlling seed shattering, and Bh4, controlling hull colour), we addressed the following questions: (i) What is the relationship of Malaysian weedy rice to domesticated and wild rice, and to weedy rice strains in the USA? (ii) To what extent does the presence of O. rufipogon influence the genetic and phenotypic diversity of Malaysian weeds? (iii) What do the distributions of sh4 and Bh4 alleles and associated phenotypes reveal about the origin and contemporary evolution of Malaysian weedy rice? Our results reveal the following: independent evolutionary origins for Malaysian weeds and US strains, despite their very close phenotypic resemblance; wild-to-weed gene flow in Malaysian weed populations, including apparent adaptive introgression of seed-shattering alleles; and a prominent role for modern Malaysian cultivars in the origin and recent proliferation of Malaysian weeds. These findings suggest that the genetic complexity and adaptability of weedy crop relatives can be profoundly influenced by proximity to reproductively compatible wild and domesticated populations.

Conservation of eelgrass (Zostera marina) genetic diversity in a mesocosm-based restoration experiment.

Eelgrass (Zostera marina) forms the foundation of an important shallow coastal community in protected estuaries and bays. Widespread population declines have stimulated restoration efforts, but these have often overlooked the importance of maintaining the evolutionary potential of restored populations by minimizing the reduction in genetic diversity that typically accompanies restoration. In an experiment simulating a small-scale restoration, we tested the effectiveness of a buoy-deployed seeding technique to maintain genetic diversity comparable to the seed source populations. Seeds from three extant source populations in San Francisco Bay were introduced into eighteen flow-through baywater mesocosms. Following seedling establishment, we used seven polymorphic microsatellite loci to compare genetic diversity indices from 128 shoots to those found in the source populations. Importantly, allelic richness and expected heterozygosity were not significantly reduced in the mesocosms, which also preserved the strong population differentiation present among source populations. However, the inbreeding coefficient F IS was elevated in two of the three sets of mesocosms when they were grouped according to their source population. This is probably a Wahlund effect from confining all half-siblings within each spathe to a single mesocosm, elevating F IS when the mesocosms were considered together. The conservation of most alleles and preservation of expected heterozygosity suggests that this seeding technique is an improvement over whole-shoot transplantation in the conservation of genetic diversity in eelgrass restoration efforts.

Effects of suppressing the DNA mismatch repair system on homeologous recombination in tomato.

In plant breeding, the ability to manipulate genetic (meiotic) recombination would be beneficial for facilitating gene transfer from wild relatives of crop plants. The DNA mismatch repair (MMR) system helps maintain genetic integrity by correcting base mismatches that arise via DNA synthesis or damage, and antagonizes recombination between homeologous (divergent) DNA sequences. Previous studies have established that the genomes of cultivated tomato (Solanum lycopersicum) and the wild relative S. lycopersicoides are substantially diverged (homeologous) such that recombination between their chromosomes is strongly reduced. Here, we report the effects on homeologous recombination of suppressing endogenous MMR genes in S. lycopersicum via RNAi-induced silencing of SlMSH2 and SlMSH7 or overexpressing dominant negatives of Arabidopsis MSH2 (AtMSH2-DN) in an alien substitution line (SL-8) of S. lycopersicoides in tomato. We show that certain inhibitions of MMR (RNAi of SlMSH7, AtMSH2-DN) are associated with modest increases in homeologous recombination, ranging from 3.8 to 29.2% (average rate of 17.8%) compared to controls. Unexpectedly, only the AtMSH2-DN proteins but not RNAi-induced silencing of MSH2 was found to increase homeologous recombination. The ratio of single to double crossovers (SCO:DCO ratio) decreased by approximately 50% in progeny of the AtMSH2-DN parents. An increase in the frequency of heterozygous SL-8 plants was also observed in the progeny of the SlMSH7-RNAi parents. Our findings may contribute to acceleration of introgression in cultivated tomato.

Characterization and comparative sequence analysis of the DNA mismatch repair MSH2 and MSH7 genes from tomato.

DNA mismatch repair proteins play an essential role in maintaining genomic integrity during replication and genetic recombination. We successfully isolated a full length MSH2 and partial MSH7 cDNAs from tomato, based on sequence similarity between MutS and plant MSH homologues. Semi-quantitative RT-PCR reveals higher levels of mRNA expression of both genes in young leaves and floral buds. Genetic mapping placed MSH2 and MSH7 on chromosomes 6 and 7, respectively, and indicates that these genes exist as single copies in the tomato genome. Analysis of protein sequences and phylogeny of the plant MSH gene family show that these proteins are evolutionarily conserved, and follow the classical model of asymmetric protein evolution. Genetic manipulation of the expression of these MSH genes in tomato will provide a potentially useful tool for modifying genetic recombination and hybrid fertility between wide crosses.

LTR-retrotransposons Tnt1 and T135 markers reveal genetic diversity and evolutionary relationships of domesticated peppers.

Plant genetic resources often constitute the foundation of successful breeding programs. Pepper (Capsicum annuum L.) is one of the most economically important and diversely utilized Solanaceous crop species worldwide, but less studied compared to tomato and potato. We developed and used molecular markers based on two copia-type retrotransposons, Tnt1 and T135, in a set of Capsicum species and wild relatives from diverse geographical origins. Results showed that Tnt1 and T135 insertion polymorphisms are very useful for studying genetic diversity and relationships within and among pepper species. Clusters of accessions correspond to cultivar types based on fruit shape, pungency, geographic origin and pedigree. Genetic diversity values, normally reflective of past transposition activity and population dynamics, showed positive correlation with the average number of insertions per accession. Similar evolutionary relationships are observed to that inferred by previous karyosystematics studies. These observations support the possibility that retrotransposons have contributed to genome inflation during Capsicum evolution.

Comparative analyses of genetic diversities within tomato and pepper collections detected by retrotransposon-based SSAP, AFLP and SSR.

The retrotransposon-based sequence-specific amplification polymorphism (SSAP) marker system was used to assess the genetic diversities of collections of tomato and pepper industrial lines. The utility of SSAP markers was compared to that of amplified fragment length polymorphism (AFLP) and simple sequence repeat (SSR) markers. On the basis of our results, SSAP is most informative of the three systems for studying genetic diversity in tomato and pepper, with a significant correlation of genetic relationships between different SSAP datasets and between SSAP, AFLP and SSR markers. SSAP showed about four- to ninefold more diversity than AFLP and had the highest number of polymorphic bands per assay ratio and the highest marker index. For tomato, SSAP is more suitable for inferring overall genetic variation and relationships, while SSR has the ability to detect specific genetic relationships. All three marker results for pepper showed general agreement with pepper types. Additionally, retrotransposon sequences isolated from one species can be used in related Solanaceae genera. These results suggest that different marker systems are suited for studying genetic diversity in different contexts depending on the group studied, where discordance between different marker systems can be very informative for understanding genetic relationships within the study group.

Intergeneric and infrafamilial phylogeny of subfamily Monsteroideae (Araceae) revealed by chloroplast trnL-F sequences.

The chloroplast trnL-F region was used as an independent data set for phylogenetic analysis of 118 aroid taxa. We investigated the intergeneric relationships of subfamily Monsteroideae (Araceae) and used this as a basis for an interspecific phylogenetic study of Rhaphidophora Hassk., the largest genus of the Monsteroideae. Results of the molecular tree were useful for inferring subfamilial and tribal circumscription and evolution in Araceae. Our results show that family Araceae consists of five clades that correspond to the subfamilies traditionally recognized. Starting from the most basal clade, these correspond to subfamilies Gymnostachydoideae and Orontioideae (proto aroids), with Lemna sp. (Lemnaceae) embedded in the Araceae and sister to the true aroids consisting of Pothoideae sister to Monsteroideae, immediately sister to Lasioideae and Aroideae. There is less agreement with existing tribal classifications. Complex relationships exist between members of the Monsteroideae. Our results show that Rhaphidophora and Epipremnum are paraphyletic with species of Rhaphidophora sampled, forming three informal groups with other genera of the Monstereae. Phylogenetic results may be used to suggest taxonomic changes to the current systematics of the monsteroids.