An unexpected actor in ammonium assimilation in conifer trees

Conifers are tree species with enormous environmental and economic interests but with several characteristics that complicate their investigation (big size, secondary compounds, large long-life cycles, megagenomes…). However, they are well adapted to ammonium-rich soils being a good model to study ammonium assimilation in plants. Although they have a special feature, only two glutamine synthetase (GS, EC 6.3.1.2) genes, GS1a and GS1b, coding for cytosolic proteins, have been identified. In angiosperms and in the gymnosperm Ginkgo biloba there are two types of this enzyme responsible of the ammonium assimilation: GS1 expressed in the cytosol and GS2 in the plastids. Until the date, the searches of new GS1 and GS2 genes in conifers have been made with classical biochemical and molecular biology techniques without satisfactory results. In the present context, the emergence of the next generation sequencing (NGS) techniques has open new opportunities in the resolution of old problems. They have allowed the whole sequencing of the massive conifer genomes and the analysis of their transcriptomes. Thus, in the framework of the European project ProCoGen, a gene expression atlas of the tissues of one-month seedlings was carried out using laser capture microdissection (LCM) and massive sequencing in maritime pine (Pinus pinaster), which is a conifer tree from the Southwestern Mediterranean region1. From the analysis of this work, a new gene coding for a new putative cytosolic GS has been identified, PpGS1c. 1Cañas, RA et al. (2017). Plant J, 91. 1064-1087Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech. Project funding by Ministerio de Economía y Competitividad BIO2015-69285-R and MicroNUpE (BIO2015-73512-JIN; MINECO/AEI/FEDER, UE

EXFI: Exon and splice graph prediction without a reference genome

For population genetic studies in nonmodel organisms, it is important to use every single source of genomic information. This paper presents EXFI, a Python pipeline that predicts the splice graph and exon sequences using an assembled transcriptome and raw whole-genome sequencing reads. The main algorithm uses Bloom filters to remove reads that are not part of the transcriptome, to predict the intron-exon boundaries, to then proceed to call exons from the assembly, and to generate the underlying splice graph. The results are returned in GFA1 format, which encodes both the predicted exon sequences and how they are connected to form transcripts.Basque Government, Grant/Award Number: predoctoral grant PRE_ 2017_2_0169 and grant IT558-1

From Pine Cones to Read Clouds: Rescaffolding the Megagenome of Sugar Pine (Pinus lambertiana).

We investigate the utility and scalability of new read cloud technologies to improve the draft genome assemblies of the colossal, and largely repetitive, genomes of conifers. Synthetic long read technologies have existed in various forms as a means of reducing complexity and resolving repeats since the outset of genome assembly. Recently, technologies that combine subhaploid pools of high molecular weight DNA with barcoding on a massive scale have brought new efficiencies to sample preparation and data generation. When combined with inexpensive light shotgun sequencing, the resulting data can be used to scaffold large genomes. The protocol is efficient enough to consider routinely for even the largest genomes. Conifers represent the largest reference genome projects executed to date. The largest of these is that of the conifer Pinus lambertiana (sugar pine), with a genome size of 31 billion bp. In this paper, we report on the molecular and computational protocols for scaffolding the P. lambertiana genome using the library technology from 10× Genomics. At 247,000 bp, the NG50 of the existing reference sequence is the highest scaffold contiguity among the currently published conifer assemblies; this new assembly's NG50 is 1.94 million bp, an eightfold increase

Emerging insights into nitrogen assimilation in gymnosperms

Gymnosperms are a heterogeneous and ancient group of seed plants that includes conifers, ginkgos, cycads and gnetophytes. Molecular studies on extant gymnosperms have been constrained by some discouraging features for experimental research such as their long life cycles, large sizes, complex megagenomes and abundant phenolic compounds in their woody tissues. However, the development of high-throughput sequencing and refined multiomics technologies in the last few years has allowed to explore the molecular basis of essential processes in this ancient lineage of plants. Nitrogen is one of the main limiting factors determining vascular development and biomass production in woody plants. Therefore, nitrogen uptake, metabolism, storage and recycling are essential processes for fundamental gymnosperm biology. Here, recent progress in the molecular regulation of nitrogen assimilation in gymnosperms is reviewed and some future perspectives on this topic are outlined.This research was fnancially supported by Ministry of Science and Innovation (BIO2015-73512-JIN, RTI2018-094041-B-I00 and PID2021-125040OB-I00) and by Junta de Andalucía (P20-00036 PAIDI 2020/FEDER, UE). JMVM was supported by a Grant from the Spanish Ministry of Education (FPU17/03517). Funding for open access publishing: Universidad Málaga/CBUA

Assessing the Gene Content of the Megagenome: Sugar Pine (Pinus lambertiana).

Sugar pine (Pinus lambertiana Douglas) is within the subgenus Strobus with an estimated genome size of 31 Gbp. Transcriptomic resources are of particular interest in conifers due to the challenges presented in their megagenomes for gene identification. In this study, we present the first comprehensive survey of the P. lambertiana transcriptome through deep sequencing of a variety of tissue types to generate more than 2.5 billion short reads. Third generation, long reads generated through PacBio Iso-Seq have been included for the first time in conifers to combat the challenges associated with de novo transcriptome assembly. A technology comparison is provided here to contribute to the otherwise scarce comparisons of second and third generation transcriptome sequencing approaches in plant species. In addition, the transcriptome reference was essential for gene model identification and quality assessment in the parallel project responsible for sequencing and assembly of the entire genome. In this study, the transcriptomic data were also used to address questions surrounding lineage-specific Dicer-like proteins in conifers. These proteins play a role in the control of transposable element proliferation and the related genome expansion in conifers

Micro and Macro-Evolutionary Studies in Non-Model Species: a Transcriptomic Perspective in Teleosts

246 p.Los recursos genómicos y las herramientas bioinformáticas son muy escasas en especies no modelo. ElRNA-Seq puede ser una herramienta efectiva para generar tales recursos genómicos con el objetivo derevelar la variación genética y funciones necesarias en estudios micro- y macro-evolutivos.La primera sección de esta Tesis aborda la presencia o ausencia de los filogrupos Este y Oeste de dospoblaciones cultivadas de Tinca tinca en Europa Central. Nuestro estudio respalda la hipótesis de que losindividuos analizados resultan ser un mosaico genómico de ambos filogrupos, y que las diferencias entrelas dos razas se deben a la composición inicial de los filogrupos en el momento de su fundación.La segunda sección de esta Tesis describe EXFI, un método que utiliza algoritmos de última generaciónpara dividir tránscritos en exones.La tercera sección muestra un método de muestreo optimizado para la caracterización de tránscritos.Apliqué la estrategia multi-tejido para obtener muestras, secuenciar y ensamblar el transcriptoma másexhaustivo de la sardina europea.En la última sección, estudié las relaciones evolutivas dentro de los Clupeiformes. Recopilé un granconjunto de transcriptomas de doce especies, construí su árbol filogenético, y descubrí grupos de genesbajo selección positiva. La principal conclusión es que la evolución ha moldeado la maquinaria molecularde los Clupeiformes hacia un almacenamiento y transporte de lípidos mejorado

Transcriptome Analysis and Intraspecific Variation in Spanish Fir (Abies pinsapo Boiss.)

Spanish fir (Abies pinsapo Boiss.) is an endemic, endangered tree that has been scarcely investigated at the molecular level. In this work, the transcriptome of Spanish fir was assembled, providing a large catalog of expressed genes (22,769), within which a high proportion were full-length transcripts (12,545). This resource is valuable for functional genomics studies and genome annotation in this relict conifer species. Two intraspecific variations of A. pinsapo can be found within its largest population at the Sierra de las Nieves National Park: one with standard green needles and another with bluish-green needles. To elucidate the causes of both phenotypes, we studied different physiological and molecular markers and transcriptome profiles in the needles. “Green” trees showed higher electron transport efficiency and enhanced levels of chlorophyll, protein, and total nitrogen in the needles. In contrast, needles from “bluish” trees exhibited higher contents of carotenoids and cellulose. These results agreed with the differential transcriptomic profiles, suggesting an imbalance in the nitrogen status of “bluish” trees. Additionally, gene expression analyses suggested that these differences could be associated with different epigenomic profiles. Taken together, the reported data provide new transcriptome resources and a better understanding of the natural variation in this tree species, which can help improve guidelines for its conservation and the implementation of adaptive management strategies under climatic change.This research was funded by research projects “Genómica funcional del pinsapo” and “Genómica del pinsapo: Estudios estructurales y funcionales” (grant numbers UMA18-FEDERJA-018 and P20-00507-R, funded by Junta de Andalucía, PAIDI) and “Modulators of adaptive capacity to climate change in forests: integration from the landscape to the gene/transcriptome in relict mountain conifers (CoMoReAdapt)” (grant number CGL2013-48843-C2-1-R, funded by the Spanish Ministry of Economy and Competitiveness, National Program for R + D+i). Partial funding for open access charge: Universidad de Málag

Genomics-Assisted Crop Improvement, Vol 1: Genomics Approaches and Platforms

Genomics research has great potential to revolutionize the discipline of plant breeding. This two-volume set provides a critical assessment of genomics tools and approaches for crop breeding. Volume 1, entitled "Genomics Approaches and Platforms", illustrates state-of-the-art genomics approaches and platforms presently available for crop improvement. Volume 2, entitled "Genomics Applications in Crops", compiles crop-specific studies that summarize both the achievements and limitations of genomics research for crop improvement. We hope that these two volumes, while providing new ideas and opportunities to those working in crop breeding, will help graduate students and teachers to develop a better understanding of the applications of crop genomics to plant research and breeding

Approaches to variant discovery for conifer transcriptome sequencing.

There is a wide diversity of bioinformatic tools available for the assembly of next generation sequence and subsequence variant calling to identify genetic markers at scale. Integration of genomics tools such as genomic selection, association studies, pedigree analysis and analysis of genetic diversity, into operational breeding is a goal for New Zealand's most widely planted exotic tree species, Pinus radiata. In the absence of full reference genomes for large megagenomes such as in conifers, RNA sequencing in a range of genotypes and tissue types, offers a rich source of genetic markers for downstream application. We compared nine different assembler and variant calling software combinations in a single transcriptomic library and found that Single Nucleotide Polymorphism (SNPs) discovery could vary by as much as an order of magnitude (8,061 SNPs up to 86,815 SNPs). The assembler with the best realignment of the packages trialled, Trinity, in combination with several variant callers was then applied to a much larger multi-genotype, multi-tissue transcriptome and identified 683,135 in silico SNPs across a predicted 449,951 exons when mapped to the Pinus taeda ver 1.01e reference

Assessing the Gene Content of the Megagenome: Sugar Pine (Pinus lambertiana)

Sugar pine (Pinus lambertiana Douglas) is within the subgenus Strobus with an estimated genome size of 31 Gbp. Transcriptomic resources are of particular interest in conifers due to the challenges presented in their megagenomes for gene identification. In this study, we present the first comprehensive survey of the P. lambertiana transcriptome through deep sequencing of a variety of tissue types to generate more than 2.5 billion short reads. Third generation, long reads generated through PacBio Iso-Seq have been included for the first time in conifers to combat the challenges associated with de novo transcriptome assembly. A technology comparison is provided here to contribute to the otherwise scarce comparisons of second and third generation transcriptome sequencing approaches in plant species. In addition, the transcriptome reference was essential for gene model identification and quality assessment in the parallel project responsible for sequencing and assembly of the entire genome. In this study, the transcriptomic data were also used to address questions surrounding lineage-specific Dicer-like proteins in conifers. These proteins play a role in the control of transposable element proliferation and the related genome expansion in conifers