R. Varma Penmetsa

Draft genome sequence of chickpea (Cicer arietinum) provides a resource for trait improvement

Chickpea (Cicer arietinum) is the second most widely grown legume crop after soybean, accounting for a substantial proportion of human dietary nitrogen intake and playing a crucial role in food security in developing countries. We report the ∼738-Mb draft whole genome shotgun sequence of CDC Frontier, a kabuli chickpea variety, which contains an estimated 28,269 genes. Resequencing and analysis of 90 cultivated and wild genotypes from ten countries identifies targets of both breeding-associated genetic sweeps and breeding-associated balancing selection. Candidate genes for disease resistance and agronomic traits are highlighted, including traits that distinguish the two main market classes of cultivated chickpea—desi and kabuli. These data comprise a resource for chickpea improvement through molecular breeding and provide insights into both genome diversity and domestication.

Four genes of Medicago truncatula controlling components of a Nod factor transduction pathway.

Rhizobium nodulation (Nod) factors are lipo-chitooligosaccharides that act as symbiotic signals, eliciting several key developmental responses in the roots of legume hosts. Using nodulation-defective mutants of Medicago truncatula, we have started to dissect the genetic control of Nod factor transduction. Mutants in four genes (DMI1, DMI2, DMI3, and NSP) were pleiotropically affected in Nod factor responses, indicating that these genes are required for a Nod factor–activated signal transduction pathway that leads to symbiotic responses such as root hair deformations, expressions of nodulin genes, and cortical cell divisions. Mutant analysis also provides evidence that Nod factors have a dual effect on the growth of root hair: inhibition of endogenous (plant) tip growth, and elicitation of a novel tip growth dependent on (bacterial) Nod factors. dmi1, dmi2, and dmi3 mutants are also unable to establish a symbiotic association with endomycorrhizal fungi, indicating that there are at least three common steps to nodulation and endomycorrhization in M. truncatula and providing further evidence for a common signaling pathway between nodulation and mycorrhization.

A Medicago truncatula phosphate transporter indispensable for the arbuscular mycorrhizal symbiosis

The arbuscular mycorrhizal (AM) symbiosis is a mutualistic endosymbiosis formed by plant roots and AM fungi. Most vascular flowering plants have the ability to form these associations, which have a significant impact on plant health and consequently on ecosystem function. Nutrient exchange is a central feature of the AM symbiosis, and AM fungi obtain carbon from their plant host while assisting the plant with the acquisition of phosphorus (as phosphate) from the soil. In the AM symbiosis, the fungus delivers Pi to the root through specialized hyphae called arbuscules. The molecular mechanisms of Pi and carbon transfer in the symbiosis are largely unknown, as are the mechanisms by which the plant regulates the symbiosis in response to its nutrient status. Plants possess many classes of Pi transport proteins, including a unique clade (Pht1, subfamily I), members of which are expressed only in the AM symbiosis. Here, we show that MtPT4, a Medicago truncatula member of subfamily I, is essential for the acquisition of Pi delivered by the AM fungus. However, more significantly, MtPT4 function is critical for AM symbiosis. Loss of MtPT4 function leads to premature death of the arbuscules; the fungus is unable to proliferate within the root, and symbiosis is terminated. Thus, Pi transport is not only a benefit for the plant but is also a requirement for the AM symbiosis.

A Sequence-Based Genetic Map of Medicago truncatula and Comparison of Marker Colinearity with M. sativa

A core genetic map of the legume Medicago truncatula has been established by analyzing the segregation of 288 sequence-characterized genetic markers in an F2 population composed of 93 individuals. These molecular markers correspond to 141 ESTs, 80 BAC end sequence tags, and 67 resistance gene analogs, covering 513 cM. In the case of EST-based markers we used an intron-targeted marker strategy with primers designed to anneal in conserved exon regions and to amplify across intron regions. Polymorphisms were significantly more frequent in intron vs. exon regions, thus providing an efficient mechanism to map transcribed genes. Genetic and cytogenetic analysis produced eight well-resolved linkage groups, which have been previously correlated with eight chromosomes by means of FISH with mapped BAC clones. We anticipated that mapping of conserved coding regions would have utility for comparative mapping among legumes; thus 60 of the EST-based primer pairs were designed to amplify orthologous sequences across a range of legume species. As an initial test of this strategy, we used primers designed against M. truncatula exon sequences to rapidly map genes in M. sativa. The resulting comparative map, which includes 68 bridging markers, indicates that the two Medicago genomes are highly similar and establishes the basis for a Medicago composite map.

An ERF Transcription Factor in Medicago truncatula That Is Essential for Nod Factor Signal Transduction

Rhizobial bacteria activate the formation of nodules on the appropriate host legume plant, and this requires the bacterial signaling molecule Nod factor. Perception of Nod factor in the plant leads to the activation of a number of rhizobial-induced genes. Putative transcriptional regulators in the GRAS family are known to function in Nod factor signaling, but these proteins have not been shown to be capable of direct DNA binding. Here, we identify an ERF transcription factor, ERF Required for Nodulation (ERN), which contains a highly conserved AP2 DNA binding domain, that is necessary for nodulation. Mutations in this gene block the initiation and development of rhizobial invasion structures, termed infection threads, and thus block nodule invasion by the bacteria. We show that ERN is necessary for Nod factor–induced gene expression and for spontaneous nodulation activated by the calcium- and calmodulin-dependent protein kinase, DMI3, which is a component of the Nod factor signaling pathway. We propose that ERN is a component of the Nod factor signal transduction pathway and functions downstream of DMI3 to activate nodulation gene expression.

The Medicago truncatula ortholog of Arabidopsis EIN2, sickle, is a negative regulator of symbiotic and pathogenic microbial associations

SUMMARY The plant hormone ethylene negatively regulates bacterial infection and nodule formation in legumes in response to symbiotic rhizobia, but the molecular mechanism(s) of ethylene action in symbiosis remain obscure. We have identified and characterized multiple mutant alleles of the MtSkl1 gene, which controls both ethylene sensitivity and nodule numbers. We show that this locus encodes the Medicago truncatula ortholog of the Arabidopsis ethylene signaling protein EIN2. In addition to the well-characterized role of MtSkl1 in rhizobial symbiosis, we show that MtSkl1 is involved in regulating early phases of the symbiotic interaction with mycorrhizal fungi, and in mediating root responses to cytokinin. MtSkl1 also functions in the defense against Rhizoctonia solani and Phytophthora medicaginis, with the latter interaction likely to involve positive feedback amplification of ethylene biosynthesis. Overexpression of the C-terminal domain of MtEIN2 is sufficient to block nodulation responses, consistent with previous reports in Arabidopsis on the activation of ethylene signaling. This same C-terminal region is uniquely conserved throughout the EIN2 homologs of angiosperms, which is consistent with its role as a higher plant-specific innovation essential to EIN2 function.

Dual Genetic Pathways Controlling Nodule Number in Medicago truncatula

We report the isolation and characterization of a newMedicago truncatula hyper-nodulation mutant, designatedsunn (super numeric nodules). Similar to the previously described ethylene-insensitive mutant sickle,sunn exhibits a 10-fold increase in the number of nodules within the primary nodulation zone. Despite this general similarity, these two mutants are readily distinguished based on anatomical, genetic, physiological, and molecular criteria. In contrast to sickle, where insensitivity to ethylene is thought to be causal to the hyper-nodulation phenotype (R.V. Penmetsa, D.R. Cook [1997] Science 275: 527–530), nodulation in sunn is normally sensitive to ethylene. Nevertheless, sunnexhibits seedling root growth that is insensitive to ethylene, although other aspects of the ethylene triple response are normal; these observations suggest that hormonal responses might condition thesunn phenotype in a manner distinct fromsickle. The two mutants also differ in the anatomy of the nodulation zone: Successful infection and nodule development insunn occur predominantly opposite xylem poles, similar to wild type. In sickle, however, both infection and nodulation occur randomly throughout the circumference of the developing root. Genetic analysis indicates that sunnand sickle correspond to separate and unlinked loci, whereas the sunn/skl double mutant exhibits a novel and additive super-nodulation phenotype. Taken together, these results suggest a working hypothesis wherein sunn andsickle define distinct genetic pathways, withskl regulating the number and distribution of successful infection events, and sunn regulating nodule organogenesis.

Novel SSR Markers from BAC-End Sequences, DArT Arrays and a Comprehensive Genetic Map with 1,291 Marker Loci for Chickpea (Cicer arietinum L.)

Chickpea (Cicer arietinum L.) is the third most important cool season food legume, cultivated in arid and semi-arid regions of the world. The goal of this study was to develop novel molecular markers such as microsatellite or simple sequence repeat (SSR) markers from bacterial artificial chromosome (BAC)-end sequences (BESs) and diversity arrays technology (DArT) markers, and to construct a high-density genetic map based on recombinant inbred line (RIL) population ICC 4958 (C. arietinum)×PI 489777 (C. reticulatum). A BAC-library comprising 55,680 clones was constructed and 46,270 BESs were generated. Mining of these BESs provided 6,845 SSRs, and primer pairs were designed for 1,344 SSRs. In parallel, DArT arrays with ca. 15,000 clones were developed, and 5,397 clones were found polymorphic among 94 genotypes tested. Screening of newly developed BES-SSR markers and DArT arrays on the parental genotypes of the RIL mapping population showed polymorphism with 253 BES-SSR markers and 675 DArT markers. Segregation data obtained for these polymorphic markers and 494 markers data compiled from published reports or collaborators were used for constructing the genetic map. As a result, a comprehensive genetic map comprising 1,291 markers on eight linkage groups (LGs) spanning a total of 845.56 cM distance was developed (http://cmap.icrisat.ac.in/cmap/sm/cp/thudi/). The number of markers per linkage group ranged from 68 (LG 8) to 218 (LG 3) with an average inter-marker distance of 0.65 cM. While the developed resource of molecular markers will be useful for genetic diversity, genetic mapping and molecular breeding applications, the comprehensive genetic map with integrated BES-SSR markers will facilitate its anchoring to the physical map (under construction) to accelerate map-based cloning of genes in chickpea and comparative genome evolution studies in legumes.

nip, a Symbiotic Medicago truncatula Mutant That Forms Root Nodules with Aberrant Infection Threads and Plant Defense-Like Response

To investigate the legume-Rhizobium symbiosis, we isolated and studied a novel symbiotic mutant of the model legume Medicago truncatula, designated nip (numerous infections and polyphenolics). When grown on nitrogen-free media in the presence of the compatible bacterium Sinorhizobium meliloti, the nip mutant showed nitrogen deficiency symptoms. The mutant failed to form pink nitrogen-fixing nodules that occur in the wild-type symbiosis, but instead developed small bump-like nodules on its roots that were blocked at an early stage of development. Examination of the nip nodules by light microscopy after staining with X-Gal for S. meliloti expressing a constitutive GUS gene, by confocal microscopy following staining with SYTO-13, and by electron microscopy revealed that nip initiated symbiotic interactions and formed nodule primordia and infection threads. The infection threads in nip proliferated abnormally and very rarely deposited rhizobia into plant host cells; rhizobia failed to differentiate further in these cases. nip nodules contained autofluorescent cells and accumulated a brown pigment. Histochemical staining of nip nodules revealed this pigment to be polyphenolic accumulation. RNA blot analyses demonstrated that nip nodules expressed only a subset of genes associated with nodule organogenesis, as well as elevated expression of a host defense-associated phenylalanine ammonia lyase gene. nip plants were observed to have abnormal lateral roots. nip plant root growth and nodulation responded normally to ethylene inhibitors and precursors. Allelism tests showed that nip complements 14 other M. truncatula nodulation mutants but not latd, a mutant with a more severe nodulation phenotype as well as primary and lateral root defects. Thus, the nip mutant defines a new locus, NIP, required for appropriate infection thread development during invasion of the nascent nodule by rhizobia, normal lateral root elongation, and normal regulation of host defense-like responses during symbiotic interactions.

Analysis of BAC-end sequences (BESs) and development of BES-SSR markers for genetic mapping and hybrid purity assessment in pigeonpea (Cajanus spp.)

BackgroundPigeonpea [Cajanus cajan (L.) Millsp.] is an important legume crop of rainfed agriculture. Despite of concerted research efforts directed to pigeonpea improvement, stagnated productivity of pigeonpea during last several decades may be accounted to prevalence of various biotic and abiotic constraints and the situation is exacerbated by availability of inadequate genomic resources to undertake any molecular breeding programme for accelerated crop improvement. With the objective of enhancing genomic resources for pigeonpea, this study reports for the first time, large scale development of SSR markers from BAC-end sequences and their subsequent use for genetic mapping and hybridity testing in pigeonpea.ResultsA set of 88,860 BAC (bacterial artificial chromosome)-end sequences (BESs) were generated after constructing two BAC libraries by using HindIII (34,560 clones) and BamHI (34,560 clones) restriction enzymes. Clustering based on sequence identity of BESs yielded a set of >52K non-redundant sequences, comprising 35 Mbp or >4% of the pigeonpea genome. These sequences were analyzed to develop annotation lists and subdivide the BESs into genome fractions (e.g., genes, retroelements, transpons and non-annotated sequences). Parallel analysis of BESs for microsatellites or simple sequence repeats (SSRs) identified 18,149 SSRs, from which a set of 6,212 SSRs were selected for further analysis. A total of 3,072 novel SSR primer pairs were synthesized and tested for length polymorphism on a set of 22 parental genotypes of 13 mapping populations segregating for traits of interest. In total, we identified 842 polymorphic SSR markers that will have utility in pigeonpea improvement. Based on these markers, the first SSR-based genetic map comprising of 239 loci was developed for this previously uncharacterized genome. Utility of developed SSR markers was also demonstrated by identifying a set of 42 markers each for two hybrids (ICPH 2671 and ICPH 2438) for genetic purity assessment in commercial hybrid breeding programme.ConclusionIn summary, while BAC libraries and BESs should be useful for genomics studies, BES-SSR markers, and the genetic map should be very useful for linking the genetic map with a future physical map as well as for molecular breeding in pigeonpea.