The Medicago truncatula nodule‐specific cysteine‐rich peptides, NCR343 and NCR‐new35 are required for the maintenance of rhizobia in nitrogen‐fixing nodules

In the nodules of IRLC legumes, including Medicago truncatula, nitrogen-fixing rhizobia undergo terminal differentiation resulting in elongated and endoreduplicated bacteroids specialized for nitrogen fixation. This irreversible transition of rhizobia is mediated by host produced nodule-specific cysteine-rich (NCR) peptides, of which c. 700 are encoded in the M. truncatula genome but only few of them have been proved to be essential for nitrogen fixation. We carried out the characterization of the nodulation phenotype of three ineffective nitrogen-fixing M. truncatula mutants using confocal and electron microscopy, monitored the expression of defence and senescence-related marker genes, and analysed the bacteroid differentiation with flow cytometry. Genetic mapping combined with microarray- or transcriptome-based cloning was used to identify the impaired genes. Mtsym19 and Mtsym20 mutants are defective in the same peptide NCR-new35 and the lack of NCR343 is responsible for the ineffective symbiosis of NF-FN9363. We found that the expression of NCR-new35 is significantly lower and limited to the transition zone of the nodule compared with other crucial NCRs. The fluorescent protein-tagged version of NCR343 and NCR-new35 localized to the symbiotic compartment. Our discovery added two additional members to the group of NCR genes essential for nitrogen-fixing symbiosis in M. truncatula

Agro-morphological and molecular characterizatuon of Turkey originated local pea (pisum sativum l.) genotypes.

TEZ9869Tez (Yüksek Lisans) -- Çukurova Üniversitesi, Adana, 2015.Kaynakça (s. 65-75) var.xi, 80 s. : res. (bzs. rnk.), tablo ; 29 cm.Bu çalışmada Türkiye’nin farklı bölgelerinden toplanmış 96 adet yerel bezelye genotipi hem agro-morfolojik olarak hem de moleküler olarak karakterize edilmiştir. Agro-morfolojik olarak bezelye genotipleri incelendiğinde; bezelye genotiplerinin bitki boyu, bitkide dal sayısı, bitkide bakla sayısı, bakla uzunluğu, baklada tane sayısı, biyolojik verim, bitkide tohum sayısı, ilk bakla yüksekliği, çiçeklenme süresi, 100 tohum ağırlığı ve bakla kıvrım derecesi bakımından geniş bir varyasyon gösterdiği saptanmıştır. Bezelye genotipleri 19 SSR primer çifti ile moleküler olarak karakterize edilmiş ve çalışmada kullanılan 19 SSR primer çiftinden 17'sinin polimorfik olduğu belirlenmiştir. Çalışma sonunda polimorfizm bilgi içeriğinin (PBİ) 0.45 ile 0.91 arasında değiştiği ve ortalama 0.62 olduğu, allel sayısının 6 ile 29 arasında değiştiği ve ortalama 12.42 olduğu saptanmıştır. SSR verileri kullanılarak UPGMA metoduna göre yapılan dendrogramda bezelye genotiplerinin A ve B olmak üzere iki ana gruba ayrıldığı, bezelye genotiplerinin toplanma yerine göre herhangi bir grup oluşturmadığı saptanmıştır. Bu çalışma sonucunda, ülkemizdeki yerel bezelye genotiplerinin genetik varyasyonunun oldukça yüksek olduğu ve bezelye ıslahında farklıamaçlar için kullanılabileceği sonucuna varılmıştır.In this study, 96 genotypes of Pisum sativum L. selected from the various geographic regions of Turkey were characterized both agro-morphologically and molecularly. According to agro-morphological examination of the pea genotypes, it wasfound that pea genotypes have a great variation in the plant height, number of branches per plant, number of pods per plant, pod length, seeds per pod, biological yield, number of seeds per plant, first pod height, flowering time, 100 seeds weight and bean fold degree. Pea genotypes were molecularly characterized by using 19 SSR primer pairs and 17 of 19 SSR primer pairs were found to be polymorphic. At the end of the study, polymorphism information content (PIC) of primers ranged from 0.45 to 0.91 with an average of 0.62. and the allele numbers of markers ranged from 6 to 29 with an average of 12.421. In the dendrogram which is prepared according to UPGMA method by using SSR markers, it was obtained the genotypes clustered in two main groups, group A and group B. The grouping was not coordinated according to geographical provenance. At the end of the this study, the Turkish pea genotypes found to have a high genetic variation and could be used for specific objectives in plant breeding programs.Bu çalışma Ç.Ü. Bilimsel Araştırma Projeleri Birimi tarafından desteklenmiştir. Proje No: FYL2014-2348 ve SMYO-007

Targeted mutagenesis of Medicago truncatula Nodule-specific Cysteine-Rich (NCR) genes using the Agrobacterium rhizogenes-mediated CRISPR/Cas9 system

Abstract The host-produced nodule specific cysteine-rich (NCR) peptides control the terminal differentiation of endosymbiotic rhizobia in the nodules of IRLC legumes. Although the Medicago truncatula genome encodes about 700 NCR peptides, only few of them have been proven to be crucial for nitrogen-fixing symbiosis. In this study, we applied the CRISPR/Cas9 gene editing technology to generate knockout mutants of NCR genes for which no genetic or functional data were previously available. We have developed a workflow to analyse the mutation and the symbiotic phenotype of individual nodules formed on Agrobacterium rhizogenes-mediated transgenic hairy roots. The selected NCR genes were successfully edited by the CRISPR/Cas9 system and nodules formed on knockout hairy roots showed wild type phenotype indicating that peptides NCR068, NCR089, NCR128 and NCR161 are not essential for symbiosis between M. truncatula Jemalong and Sinorhizobium medicae WSM419. We regenerated stable mutants edited for the NCR068 from hairy roots obtained by A. rhizogenes-mediated transformation. The analysis of the symbiotic phenotype of stable ncr068 mutants showed that peptide NCR068 is not required for symbiosis with S. meliloti strains 2011 and FSM-MA either. Our study reports that gene editing can help to elicit the role of certain NCRs in symbiotic nitrogen fixation

Targeted mutagenesis of Medicago truncatula Nodule-specific Cysteine-Rich (NCR) genes using the Agrobacterium rhizogenes-mediated CRISPR/Cas9 system

The host-produced nodule specific cysteine-rich (NCR) peptides control the terminal differentiation of endosymbiotic rhizobia in the nodules of IRLC legumes. Although the Medicago truncatula genome encodes about 700 NCR peptides, only few of them have been proven to be crucial for nitrogen-fixing symbiosis. In this study, we applied the CRISPR/Cas9 gene editing technology to generate knockout mutants of NCR genes for which no genetic or functional data were previously available. We have developed a workflow to analyse the mutation and the symbiotic phenotype of individual nodules formed on Agrobacterium rhizogenes -mediated transgenic hairy roots. The selected NCR genes were successfully edited by the CRISPR/Cas9 system and nodules formed on knockout hairy roots showed wild type phenotype indicating that peptides NCR068, NCR089, NCR128 and NCR161 are not essential for symbiosis between M. truncatula Jemalong and Sinorhizobium medicae WSM419. We regenerated stable mutants edited for the NCR068 from hairy roots obtained by A. rhizogenes -mediated transformation. The analysis of the symbiotic phenotype of stable ncr068 mutants showed that peptide NCR068 is not required for symbiosis with S. meliloti strains 2011 and FSM-MA either. Our study reports that gene editing can help to elicit the role of certain NCRs in symbiotic nitrogen fixation

Comparative genomics of the nonlegume Parasponia reveals insights into evolution of nitrogen-fixing rhizobium symbioses

Nodules harboring nitrogen-fixing rhizobia are a well-known trait of legumes, but nodules also occur in other plant lineages, with rhizobia or the actinomycete Frankia as microsymbiont. It is generally assumed that nodulation evolved independently multiple times. However, molecular-genetic support for this hypothesis is lacking, as the genetic changes underlying nodule evolution remain elusive. We conducted genetic and comparative genomics studies by using Parasponia species (Cannabaceae), the only nonlegumes that can establish nitrogen-fixing nodules with rhizobium. Intergeneric crosses between Parasponia andersonii and its nonnodulating relative Trema tomentosa demonstrated that nodule organogenesis, but not intracellular infection, is a dominant genetic trait. Comparative transcriptomics of P. andersonii and the legume Medicago truncatula revealed utilization of at least 290 orthologous symbiosis genes in nodules. Among these are key genes that, in legumes, are essential for nodulation, including NODULE INCEPTION (NIN) and RHIZOBIUM-DIRECTED POLAR GROWTH (RPG). Comparative analysis of genomes from three Parasponia species and related nonnodulating plant species show evidence of parallel loss in nonnodulating species of putative orthologs of NIN, RPG, and NOD FACTOR PERCEPTION. Parallel loss of these symbiosis genes indicates that these nonnodulating lineages lost the potential to nodulate. Taken together, our results challenge the view that nodulation evolved in parallel and raises the possibility that nodulation originated ∼100 Mya in a common ancestor of all nodulating plant species, but was subsequently lost in many descendant lineages. This will have profound implications for translational approaches aimed at engineering nitrogen-fixing nodules in crop plants

Trema orientale Genome sequencing and assembly

Genome and transcriptome sequencing of Trema orientalis RG33-

Parasponia andersonii isolate:WU1-14

Genome and transcriptome sequencing of Parasponia andersonii WU1-1

Parasponia andersonii isolate:WU1-14

Genome and transcriptome sequencing of Parasponia andersonii WU1-1

Orthogroup inference

Orthogroups were inferred with OrthoFinder version 0.4.0. Since orthogroups are defined as the set of genes that are descended from a single gene in the last common ancestor of all the species being considered, they can comprise orthologous as well as paralogous genes. Our analysis included proteomes of selected species from the Eurosid clade: Arabidopsis thaliana (Brassicaceae, Brassicales) and Eucalyptus grandis (Myrtaceae, Myrtales) from the Malvid clade; Populus trichocarpa (Salicaeae, Malpighiales), legumes Medicago truncatula, and Glycine max (Fabaceae, Fabales), Fragaria vesca (Rosaceae, Rosales), P. andersonii, and T. orientalis (Cannabaceae, Rosales) from the Fabid clade. Sequences were retrieved from phytozome (www.phytozome.net)

Trema orientale Genome sequencing and assembly

Genome and transcriptome sequencing of Trema orientalis RG33-