Mutant collections in
Legume functional genomics has moved many steps forward in the last two decades thanks to the improvement of genomics technologies and to the efforts of the research community. Tools for functional genomics studies are now available in
2. Why do we need a functional genomics tool for forage legumes?
Legumes are widely grown for grain and forage production, their world economic importance being second only to grasses. Legume species are unique among cultivated plants for their ability to carry out endosymbiotic nitrogen fixation with rhizobial bacteria, a process that takes place in a specialized structure known as nodule. Moreover legumes are able to establish other types of interactions such as arbuscular mycorrhyzal symbiosis with several fungi. For these outstanding biological properties legumes are considered among the most promising species for improving the sustainability of agricultural systems. In fact for farming system to remain productive and to be environmentally and economically sustainable on the long term it is necessary to replenish the reserves of nutrients which are removed or lost from the soil. Nodulating legumes have the potential to provide all nitrogen required for their growth and in this way to influence its balance in the soil and thus its availability for subsequent crops. In addition by reducing the inputs of fertilizers, legumes reduce the risk of nitrogen contamination of water resources. Furthermore, probably due to the wealth of interactions with other organisms, legumes have evolved an intricate network of secondary metabolites that, as the recent advances in the knowledge of their nutraceutical properties are proving, can be considered of great importance for livestock welfare and for the quality of their products.
Two legume species,
Medicago truncatula genome sequencing
Functional genomics is greatly aided by knowledge on genome sequence and transcriptome of the target species. A concerted effort was carried out in
Legumes are the plant family with the greatest amount of genomic data available. Three legume species,
Taken together BAC sequences and non-redundant Illumina assemblies contain 62,388 gene loci with 14,322 gene prediction annotated as transposons. The average
Another interesting feature is the presence of many amplified and somehow specialized gene families like nine leghaemoglobines, 563 Nodule Cystein-Rich Peptides (NCRs), 764 nucleotide-binding site and leucine-rich repeat (NBS-LRR) genes, genes in the flavonoid pathway such as chalcone synthases (CHS), chalcone reductases, chalcone isomerases. Many gene duplications occurred with the creation of large gene clusters .
The availability of a first draft of the
384 inbred lines of
In parallel, plant phenotyping is ongoing in greenhouse experiments for the
4. Functional genomics in
The discovery of gene function in model species is accomplished by exploiting resources such as mutant collections, using the ability to implement plant genetic transformation and analyzing gene transcription. In
Several strategies were pursued in
|(5)||EMS||Jemalong population 2828||1,500 seeds treated for 24h with 0.2%EMS. 400 M1 plants obtained. 250,000 M2 seeds harvested as a single batch.|
|(6)||γ-ray||Jemalong line J5||462 M1 plants, screened as M2 families.|
|(8)||EMS||Jemalong A17||3-7,000 M1 plants in 10-20 lots. M2 seeds bulked from each lot.|
|(9)||T-DNA||R108-1 (c3)||Test populations with 3 different T-DNAs|
|(10)||Tnt1||R108-1 (c3)||First test population with Tnt1 (~200 R0 plants)|
|(11)||FNB||Jemalong A17||80.000 M1, 460.000 M2|
|(12;13)||Tnt1||R108-1 (c3)||7,000 Tnt1 mutants, presently extended to 19,000 as reported in http://bioinfo4.noble.org/mutant/|
|(14)||Tnt1||R108-1 (c3)||~1000 R0|
|(14)||EMS||Jemalong 2HA||2500 M2 plants|
|(14)||Activation tagging||R108-1 (c3)||~100 mutant lines|
|(15)||EMS||Medicago littoralis ‘Angel’||Development of new annual medics varieties (i.e. resistant to herbicides)|
|(16)||Tnt1||Jemalong 2HA||Mutants produced in the frame of the european Grain Legumes Integrated Project (GLIP). The total number of mutants produced by 10 labs all around Europe should be several thousands (~6000). 2000 of them will integrate the Tnt1 collection at Noble.|
|(17)||FNB||Jemalong A17||31,200 M1 plants, 156,000 M2 plants|
2 populations. The first (not using single seed descent, SSD) 500 M1 produced 4500 M2. In the second (using SSD) 4350 M1 and 4350 M2.
5. Chemical-physical mutagenesis
5.1. Target Induced Local Lesion IN Genomes (TILLING)
Alkylating agents such as ethyl methanesulphonate (EMS) have been used to develop mutant collections of
|(6)||γ-ray||2||Nod-, Myc-||TR25, TR26|
|(6)||γ-ray||4||Nod±, Myc+||TR34, TR79, TR89, TRV9|
|(6)||γ-ray||9||Nod+Fix-, Myc+||TR3, TR9, TR13, TR36, TR62, TR69, TR74, TR183, TRV15|
|(6)||γ-ray||3||Nod++Nts, Myc+||TR122, TRV3, TRV8|
|(8)||EMS||1||Nod-||C71 = |
|(21)||EMS||5||Nod-||B85, B129, C54, P1, Y6 Individuation of 4 complementation groups (DMI1, DMI2, DMI3, NSP):|
dmi1-2 = B129
dmi1-3 = Y6
dmi2-1 = TR25
dmi2-2 = TR26
dmi2-3 = P1
dmi3-1 = TRV25
nsp1-1 = B85
nsp1-2 = C54
|(22)||EMS||7||Calcium oxalate defective|
|(23)||EMS||1||Nod-, root hair deformation|
|(24)||EMS||1||Blocked in the formation of nodule primordial|
|(24)||EMS||1||Blocked in the formation and/or maintenance of epidermal cell infection||lin1|
|(21)||EMS||3||Nod-, root hair deformations|
|(25)||EMS||6||Oxalate crystal morphology defective|
|(26)||Fast Neutron||2||Nod-, cortical cell division||nsp2-1|
|(27)||EMS||1||Nod-, does not respond to Nod Factors by induction of root hair deformation|
|(29)||EMS||1||Blocked in the formation and/or maintenance of epidermal cell infection||lin1|
|(30)||EMS||1||Numerous infections and polyphenolics|
|(31)||EMS||1||Nod-, defective in lateral root development|
|(36)||Fast Neutron||1||Increased nodule number|
|(37)||EMS||1||Impaired in nodule primordium invasion|
|(38)||EMS||1||Aberrant root hair curling and infection thread formation|
|(40)||Fast neutron||1||Leaf dissection|
|(42)||Tnt1||various||Leaf epidermal morphology||Various|
|(43)||Tnt1||1||Lack of lignin in the interfascicular region|
|(44)||Tnt1||1||Secondary cell wall thickening in pith|
|(45)||Fast neutron||1||Compund leaf development|
|(46)||Fast neutron||1||Root determined noudulation|
|(47;48)||Fast neutron||1||Myc-, Nod-|
|(49)||Activation tagging||1||Lack of hemolytic saponins|
|(51)||Tnt1||1||Smooth leaf margin|
|(52)||Tnt1||1||Reduced leaf blade expansion|
|(53)||Tnt1||1||Inhibition of rust germ tube differentiation|
5.2. “Delete a gene” collections
Irradiation of plant seeds to appropriate dose of fast neutrons and γ-rays results in deletion of DNA fragments of variable lengths with an average modest reduction of seed viability.
Large mutant collections by seed irradiation have been created for
Both reverse and forward genetic approaches have been successfully applied to study mutants from these collections.
Reverse screening of FNB populations have been carried out by the DeTILLING strategy described by Rogers C
Nevertheless deletion size can hamper reverse genetics screening (Chen R., personal communication) leaving forward genetics as the main choice in case of FNB populations. However map-based cloning required to discover the mutation of interest is helped by strategies such as transcriptional cloning, originally devised by Mitra R M
6. Insertional mutagenesis with DNA mobile elements
T-DNA tagging has been the strategy of choice for many mutant collections in
The collection maintained at the Noble Foundation (http://bioinfo4.noble.org/mutant/) which includes also the first mutants generated by CNRS in France, is based on the genotype R108-c3. Another collection of about 1000 lines from the same R-108 line was produced by CNR-IGV in Italy.
In the framework of the GLIP project 8000 Tnt1 mutants were produced from the Jemalong 2HA (2HA3-9-10-3) line. The GLIP collection is maintained by the various labs that participated to the project and a subset of plants were merged with the collection at the Noble Foundation.
Iantcheva and colleagues reported that Tnt1 transposition efficiency in Jemalong 2HA has a lower efficiency with only 10-15 new insertions per line and a variable percentage of regenerated plants without transposition . The adoption of 2HA line for mutagenesis instead of R108, was motivated by the highest DNA homology to the line used for genome sequencing (Jemalong A17), and for the presence of active and characterized endogenous retroelements .
Tnt1 mutant collections have been screened with both forward and reverse genetic approaches. Forward approaches have been based on cloning of host sequence flanking the insertion sites and subsequent identification of events linked to the studied mutation. Based on the duplicated Tnt1 long terminal repeats (LTR) sequences several molecular approaches including thermal asymmetric interlaced (TAIL)-PCR, Inverse-PCR have been used to recover the host sequences flanking the insertion sites . Segregation analysis of each cloned insertion site can then be used to select the event linked to the mutation. In alternative the insertion sites associated with the mutations can be selected by segregation analysis prior to host sequence cloning by employing a sequence specific amplification polymorphism (S-SAP) based protocol.
Confirmation of the identity of the mutation can be obtained by means of complementation tests based on the reintroduction of the wild type gene sequence in the mutated background. In alternative one could obtain independent alleles of the target gene and compare their similarity to the original mutant phenotype. This can be done using TILLING and Tnt1 mutant populations as demonstrated by many publications that report successful recovery of alleles by reverse screening  and Table 3. The power of the Tnt1 mutagenesis approach is also witnessed by the prevalence of publications reporting successful gene cloning based on such strategy compared to the others since 2008 (Table 2 and 3).
|(63)||AY497771, possible membrane receptor||Physical mapping|
|(64)||Ca2 and Calmodulin dependent protein kinase||Physical mapping/Transcriptional based cloning|
|(65)||GRAS Transcriptional regulator||Physical mapping|
|(66)||GRAS Transcriptional regulator||Physical mapping|
|(46)||CLV1-like LRR receptor kinase||Physical mapping and gene homology|
|(67)||MADS-box||Reverse screening on Tnt1 collection|
|(68)||Phosphate transporter||RNAi and TILLING (reverse)|
|(34)||ERF transcription factor required for nodulation (ERN)||Transcriptional based cloning|
|(35)||MtUNI (transcription factor)||Tnt1 forward|
|(36)||Ethilene responsive factor required for nodule differentiation||Fast neutron reverse|
|(38)||Putative long coiled-coil protein||Map based cloning|
|(28)||MtEIN2, ethylene signaling gene||Map based cloning and gene homology|
|(69)||E3 ubiquitin ligase containing a U-box and WD40 repeat domains||Positional cloning|
|(70)||LRR kinase||TILLING reverse and RNAi|
|(72)||Glucosyl transferase||Tnt1 reverse|
|(73)||RNAi and mutation segregation analisys|
|(40)||Cys(2)His(2)zinc finger transcription factor||Fast neutron forward and Tnt1 reverse|
|(44)||Signal peptidase complex subunit||Fast neutron microarray based cloning|
|(43)||NAC transcription factor||Forward screening and Tnt1 flanking region cloning|
|(44)||WRKY transcription factor||Forward screening and Tnt1 flanking region cloning|
|(75)||Cinnamoyl CoA Reductase||Tnt1 reverse|
|(76)||Glucosyl transferase||Tnt1 reverse|
|(45)||Class M KNOX||Fast neutron forward, map based cloning and Tnt1 reverse|
|(77)||Uncharacterized plant family||Mapping|
|(78)||MtFTa, MtFTc, protein ligands||Tnt1 reverse|
|(48)||Vapyrin||Microarray based cloning, Tnt1 reverse|
|(49)||CYP716A12, Cytochrome P450||Flanking sequence tagging and TILLING|
|(50)||Stay green gene||Tnt1 forward and flanking sequence cloning|
|(51)||Auxin efflux carrier protein||Tnt1 forward and flanking sequence cloning|
|(52)||Stenofolia, WUSCHEL-like homeobox transcription factor||Tnt1 forward and flanking sequence cloning|
|(53)||Cys(2)His(2) zinc finger transcription factor||Tnt1 forward and flanking sequence cloning|
|(79)||MYB transcription factor||Tnt1 reverse|
7. RNAi and VIGS
Reverse genetics studies in
|(75)||Lyk3||Marked reduction of nodulation when inoculated with Sm 2011ΔNodFE-GFP|
|(75)||Lyk4||Effect on infection thread morphology|
|(81)||CDPK1||Reduced root hair and root cell lengths. Diminution of both rhizobial and mycorrhizal symbiotic colonization.|
|(82)||DMI2||Reduction of organelle-like symbiosomes in nodules|
|(76)||MtHAP2-1||Alteration of nodule development|
|(84)||MtCPK3||Increased average nodule number|
|(85)||MtCRE1||Cytokinin-insensitive roots, increate number of lateral roots, strong reduction in nodulation.|
|(86)||MtPIN2, MtPIN3, MtPIN4||Reduced number of nodules|
|(87)||CHS||Reduced levels of flavonoids and subsequent inability to nodulate|
|(88)||PR10-1 (pathogenesis related)||Reduced colonization by the root pathogen A.euteiches.|
|(89)||HMGR1||Dramatic decrease in nodulation.|
|(90)||IPD3||No obvious phenotype observed|
|(63)||MtPT4||Premature death of mycorrhizal arbuscules.|
|(91)||MtSNF4b||Reduced seed longevity, alteration in non reducing sugar content.|
|(92)||ENOD40-1, ENOD40-2||Reduced nodule number and altered symbiosome development.|
|(93)||MtFNSII-1, MtFNSII-2||Reduced nodulation|
|(94)||MtCDD1||Alteration of the Arbuscular Mycorrhizal – mediated accumulation of apocarotenoids|
|(95)||MtDXS2||Reduction of AM-induced apocarotenoid accumulation.|
|(78)||MtSERF1||Strong inhibition of somatic embryogenesis|
|(73)||MtPI, MtNGL9||Altered flower development|
|(96)||MtWUS||Strong inhibition of somatic embryogenesis|
|(65)||Srlk||Transgenic root growth less inibited by salt stress.|
|(97)||FLOT2, FLOT4||Reduced nodulation and root development.|
|(98)||MtMSBP1||Aberrant mycorrhizal phenotype with thik and septated appressoria, decrease number of arbuscules and distorted arbuscule morphology.|
|(99)||MtCDC16||Decreased number of lateral roots and increased number of nodules. Reduced sensivity to auxin.|
|(100)||NPR1||Acceleration of root hair curling at the beginning of symbiosis estabilishment|
|(101)||MtSNARP2||Aberrant early senescent nodules where differentiated bacteroids degenerate rapidly.|
|(74)||MtSYMREM1||Reduced nodulation and abnormal nodule development|
|(103)||Vapyrin||Impaired passage across epidermis by AM fungi. Abolition of arbuscule formation.|
|(104)||MtAOC1||No nodulation phenotype observed|
|(105)||γECS||Lower homoglutathione content. Lower biological nitrogen fixation associated with a reduction in the expression of the leghemoglobin and thioredoxin S1 genes. Reduction in nodule size.|
|(106)||MtSAP1||Lower level of storage globulin proteins, vicilin and legumin in seeds and germination deficiency.|
|(107)||MtNR1, MtNR2||Reduced nitrate or nitrite reductase activity and NO level.|
|(108)||MtNoa/Rif1||Decrease in NO production in roots but not in nodules. Reduction of nodule number and nitrogen fixation capacity.|
|(109)||MtROPGEF2||Effect on cytosolic Ca2+ gradient and subcellular structure of root hairs. Reduced root hair growth.|
|(110)||MtROP9||Reduced growth , no ROS generation after microbial infection. Promoted mycorrhizal and A.euteiches early hyphal root colonization. Impaired rhizobial colonization.|
|(111)||MtNAC969||Improved growth under salt stress.|
Virus-induced gene silencing (VIGS) is a PTGS technique that can be used transiently by scrubbing leaves or introducing the viral vector in the plant by agro-infiltration. VIGS is being used for large scale forward genetics screening by inoculation of cDNA library and subsequent identification of the gene involved in the process of interest . Viral vectors working on
Functional genomics of forage legumes started with the aim of determining the molecular and genetic bases of nitrogen fixation and since the beginning mutant collections have been thoroughly screened also for mycorrhyzal symbiosis. These aspects are still being investigated and we expect that many more results will be published in the next years. A better understanding of nitrogen fixation and symbiosis is fundamental for the development of a sustainable agriculture aiming at a reduction of inputs and at maintaining soil fertility. Nitrogen (N) is one of the crucial nutrients for all organisms including plants. The doubling of world food production in the past four decades was contributed by a sevenfold increase of N fertilization . The anthropogenic N which is mostly lost to air, water and land affects climate, the chemistry of the atmosphere, and the composition and function of terrestrial and aquatic ecosystems . Improving the ability of plants to exploit environmental nitrogen would decrease N fertilization and its negative consequences; therefore a deep understanding of legume symbiosis with nitrogen fixing bacteria could help the long term goal of transferring the associative ability of legume species to non-symbiotic crops of agronomic relevance. As a consequence functional genomics of nodulation will have an impact on reduction of intensive agriculture practices with benefits for the preservation of environment and quality of human activities.
Another positive role for legumes in an environmental perspective is addressed by species such as
Pastures and feedstuff including forage legumes have a higher quality compared to those based only on grasses and provide an important input of protein in animal nutrition. More recently public and scientific debate has reassessed forage legumes importance for the quality of livestock nutrition and welfare has having relevant consequences on the quality of final products (meat, milk etc.) and ultimately on human health. This happened because of the occurrence of bovine spongiform encephalopathy (BSE) related to the traditional use of offal in animal feed lots as a source of protein.
Functional genomics in
Living organisms, and among them plants, can be considered as an abundant and diverse set of biofactories with the ability to synthesize an enormous variety of chemical compounds. Legumes contain chemicals that can prove useful for their anti-oxidant, anti-viral, anti-microbial, anti-diabetic, anti-allergenic and anti-inflammatory properties . These properties are related to secondary molecules such as flavonoids and saponins.
Modest levels of protoanthocyanidins (PAs) in forages reduce the occurrence of bloat and at the same time promote increased dietary protein nitrogen utilization in ruminant animals . The lack of PAs in the leaves of the major forage legume such as alfalfa has prompted studies for the understanding of the molecular and cellular biology of PA polymerization, transport, and storage helped by the functional genomics tools available for
In addition to well-known beneficial properties of flavonoids (cit) recent evidence suggests that flavonoids themselves, particularly fractions rich in PAs, can significantly reduce cognitive deterioration in animal model systems [120-122], and may more generally promote improvements in memory acquisition, consolidation, storage, and retrieval under nondegenerative conditions.
In Chinese medicine one of the oldest herbal medicine was obtained by the roots of the legume plant licorice (
In forage legumes saponins can be toxic to monogastric animals and reduce forage palatability for ruminants. Mutant analysis in
Both human and animal nutritional science are bound to profit from plant genetic analysis and nutritional genomics, opening possibilities to more personalized approaches to medicine and improvement of the quality of life.
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