Comparing de novo transcriptome assembly tools in di- and autotetraploid non-model plant species.

BACKGROUND: Polyploidy is very common in plants and can be seen as one of the key drivers in the domestication of crops and the establishment of important agronomic traits. It can be the main source of genomic repatterning and introduces gene duplications, affecting gene expression and alternative splicing. Since fully sequenced genomes are not yet available for many plant species including crops, de novo transcriptome assembly is the basis to understand molecular and functional mechanisms. However, in complex polyploid plants, de novo transcriptome assembly is challenging, leading to increased rates of fused or redundant transcripts. Since assemblers were developed mainly for diploid organisms, they may not well suited for polyploids. Also, comparative evaluations of these tools on higher polyploid plants are extremely rare. Thus, our aim was to fill this gap and to provide a basic guideline for choosing the optimal de novo assembly strategy focusing on autotetraploids, as the scientific interest in this type of polyploidy is steadily increasing. RESULTS: We present a comparison of two common (SOAPdenovo-Trans, Trinity) and one recently published transcriptome assembler (TransLiG) on diploid and autotetraploid species of the genera Acer and Vaccinium using Arabidopsis thaliana as a reference. The number of assembled transcripts was up to 11 and 14 times higher with an increased number of short transcripts for Acer and Vaccinium, respectively, compared to A. thaliana. In diploid samples, Trinity and TransLiG performed similarly good while in autotetraploids, TransLiG assembled most complete transcriptomes with an average of 1916 assembled BUSCOs vs. 1705 BUSCOs for Trinity. Of all three assemblers, SOAPdenovo-Trans performed worst (1133 complete BUSCOs). CONCLUSION: All three assembly tools produced complete assemblies when dealing with the model organism A. thaliana, independently of its ploidy level, but their performances differed extremely when it comes to non-model autotetraploids, where specifically TransLiG and Trinity produced a high number of redundant transcripts. The recently published assembler TransLiG has not been tested yet on any plant organism but showed highest completeness and full-length transcriptomes, especially in autotetraploids. Including such species during the development and testing of new assembly tools is highly appreciated and recommended as many important crops are polyploid.

Correction to: Integrative transcriptomics reveals genotypic impact on sugar beet storability.

In the above mentioned publication, part of Fig. 6B was distorted (extra diagonal lines appeared). The original article has been corrected and the proper version of Fig. 6B is also published here.

Integrative transcriptomics reveals genotypic impact on sugar beet storability.

KEY MESSAGE: An integrative comparative transcriptomic approach on six sugar beet varieties showing different amount of sucrose loss during storage revealed genotype-specific main driver genes and pathways characterizing storability. Sugar beet is next to sugar cane one of the most important sugar crops accounting for about 15% of the sucrose produced worldwide. Since its processing is increasingly centralized, storage of beet roots over an extended time has become necessary. Sucrose loss during storage is a major concern for the sugar industry because the accumulation of invert sugar and byproducts severely affect sucrose manufacturing. This loss is mainly due to ongoing respiration, but changes in cell wall composition and pathogen infestation also contribute. While some varieties can cope better during storage, the underlying molecular mechanisms are currently undiscovered. We applied integrative transcriptomics on six varieties exhibiting different levels of sucrose loss during storage. Already prior to storage, well storable varieties were characterized by a higher number of parenchyma cells, a smaller cell area, and a thinner periderm. Supporting these findings, transcriptomics identified changes in genes involved in cell wall modifications. After 13 weeks of storage, over 900 differentially expressed genes were detected between well and badly storable varieties, mainly in the category of defense response but also in carbohydrate metabolism and the phenylpropanoid pathway. These findings were confirmed by gene co-expression network analysis where hub genes were identified as main drivers of invert sugar accumulation and sucrose loss. Our data provide insight into transcriptional changes in sugar beet roots during storage resulting in the characterization of key pathways and hub genes that might be further used as markers to improve pathogen resistance and storage properties.

Elucidating Drought Stress Tolerance in European Oaks Through Cross-Species Transcriptomics.

The impact of climate change that comes with a dramatic increase of long periods of extreme summer drought associated with heat is a fundamental challenge for European forests. As a result, forests are expected to shift their distribution patterns toward north-east, which may lead to a dramatic loss in value of European forest land. Consequently, unraveling key processes that underlie drought stress tolerance is not only of great scientific but also of utmost economic importance for forests to withstand future heat and drought wave scenarios. To reveal drought stress-related molecular patterns we applied cross-species comparative transcriptomics of three major European oak species: the less tolerant deciduous pedunculate oak (Quercus robur), the deciduous but quite tolerant pubescent oak (Q. pubescens), and the very tolerant evergreen holm oak (Q. ilex). We found 415, 79, and 222 differentially expressed genes during drought stress in Q. robur, Q. pubescens, and Q. ilex, respectively, indicating species-specific response mechanisms. Further, by comparative orthologous gene family analysis, 517 orthologous genes could be characterized that may play an important role in drought stress adaptation on the genus level. New regulatory candidate pathways and genes in the context of drought stress response were identified, highlighting the importance of the antioxidant capacity, the mitochondrial respiration machinery, the lignification of the water transport system, and the suppression of drought-induced senescence - providing a valuable knowledge base that could be integrated in breeding programs in the face of climate change.

Characterization of variable EST SSR markers for Norway spruce (Picea abies L.).

BACKGROUND: Norway spruce is widely distributed across Europe and the predominant tree of the Alpine region. Fast growth and the fact that timber can be harvested cost-effectively in relatively young populations define its status as one of the economically most important tree species of Northern Europe. In this study, EST derived simple sequence repeat (SSR) markers were developed for the assessment of putative functional diversity in Austrian Norway spruce stands. RESULTS: SSR sequences were identified by analyzing 14,022 publicly available EST sequences. Tri-nucleotide repeat motifs were most abundant in the data set followed by penta- and hexa-nucleotide repeats. Specific primer pairs were designed for sixty loci. Among these, 27 displayed polymorphism in a testing population of 16 P. abies individuals sampled across Austria and in an additional screening population of 96 P. abies individuals from two geographically distinct Austrian populations. Allele numbers per locus ranged from two to 17 with observed heterozygosity ranging from 0.075 to 0.99. CONCLUSIONS: We have characterized variable EST SSR markers for Norway spruce detected in expressed genes. Due to their moderate to high degree of variability in the two tested screening populations, these newly developed SSR markers are well suited for the analysis of stress related functional variation present in Norway spruce populations.

Identification of adaptation-specific differences in mRNA expression of sessile and pedunculate oak based on osmotic-stress-induced genes.

Quercus petraea (Matt.) Liebl. and Q. robur L. hybridize frequently and occupy similar, though distinct, ecological niches. So far, genetic discrimination between these species at the molecular level has been based mainly on neutral markers. Because such markers often exhibit low species differentiation because of high genetic compatibility and exchange between Q. robur and Q. petraea at these loci, we used adaptation-related expressed genes as markers. Accordingly, we identified osmotic-stress-induced genes in a Q. petraea cell line grown under moderate osmotic stress conditions. Two subtraction libraries were established from callus cells cultured under hyperosmotic stress for 1 or 48 h. Thirty-three differentially expressed sequence tags (ESTs) (from 70 originally isolated) were classified according to their putative functions. At least five of these gene products may contribute to osmotic-stress tolerance in oak: betaine aldehyde dehydrogenase, two trans-acting transcription factors (one abscsic acid (ABA)-responsive, the other ABA-independent), a glutathione-S- transferase and a heat-shock cognate protein. Seven genes were selected based on their putative function and their expression monitored in vivo. Leaf tissue from Q. petraea and Q. robur plantlets grown hydroponically under hyperosmotic conditions was harvested after 0, 1, 6, 24 or 72 h and analyzed by real-time polymerase chain reaction (PCR). We found indications of osmotic stress adaptation in Q. petraea based on up-regulation of genes related to protective functions, whereas down-regulation of these genes was evident in Q. robur. Thus, genetic markers related to adaptive traits may be useful for differentiating Q. petraea and Q. robur genotypes.