Ramakrishnan M. Nair

Aphid resistance in Medicago truncatula involves antixenosis and phloem-specific, inducible antibiosis, and maps to a single locus flanked by NBS-LRR resistance gene analogs

Aphids and related insects feed from a single cell type in plants: the phloem sieve element. Genetic resistance to Acyrthosiphon kondoi Shinji (bluegreen aphid or blue alfalfa aphid) has been identified in Medicago truncatula Gaert. (barrel medic) and backcrossed into susceptible cultivars. The status of M. truncatula as a model legume allows an in-depth study of defense against this aphid at physiological, biochemical, and molecular levels. In this study, two closely related resistant and susceptible genotypes were used to characterize the aphid-resistance phenotype. Resistance conditions antixenosis since migratory aphids were deterred from settling on resistant plants within 6 h of release, preferring to settle on susceptible plants. Analysis of feeding behavior revealed the trait affects A. kondoi at the level of the phloem sieve element. Aphid reproduction on excised shoots demonstrated that resistance requires an intact plant. Antibiosis against A. kondoi is enhanced by prior infestation, indicating induction of this phloem-specific defense. Resistance segregates as a single dominant gene, AKR (Acyrthosiphon kondoi resistance), in two mapping populations, which have been used to map the locus to a region flanked by resistance gene analogs predicted to encode the CC-NBS-LRR subfamily of resistance proteins. This work provides the basis for future molecular analysis of defense against phloem parasitism in a plant model system.

Involvement of the octadecanoid pathway in bluegreen aphid resistance in Medicago truncatula

Aphids are major insect pests of plants that feed directly from the phloem. We used the model legume Medicago truncatula Gaert. (barrel medic) to elucidate host resistance to aphids and identified a single dominant gene which confers resistance to Acyrthosiphon kondoi Shinji (bluegreen aphid). To understand how this gene conditions resistance to bluegreen aphid, transcription profiling of 23 defense-related genes representing various signaling pathways was undertaken using a pair of near-isogenic lines that are susceptible or resistant to bluegreen aphid. All salicylic acid- and ethylene-responsive genes tested were induced by bluegreen aphid in resistant and susceptible plants, although there were some differences in the magnitude and kinetics of the induction. In contrast, 10 of 13 genes associated with the octadecanoid pathway were induced exclusively in the resistant plants following bluegreen aphid infestation. These results are in contrast to plant-pathogen interactions where similar sets of defense genes typically are induced in compatible interactions, but to a lesser degree and later than in incompatible interactions. Treatment of susceptible plants with methyl jasmonate reduced bluegreen aphid infestation but not to the same levels as the resistant line. Together, these results strongly suggest that the octadecanoid pathway is important for this naturally derived aphid resistance trait.

SSR analysis of the Medicago truncatula SARDI core collection reveals substantial diversity and unusual genotype dispersal throughout the Mediterranean basin

The world’s oldest and largest Medicago truncatula collection is housed at the South Australian Research and Development Institute (SARDI). We used six simple sequence repeat (SSR) loci to analyse the genetic diversity and relationships between randomly selected individuals from 192 accessions in the core collection. M. truncatula is composed of three subspecies (ssp.): ssp. truncatula, ssp. longeaculeata, and ssp. tricycla. Analysis at the level of six SSR loci supports the concept of ssp. tricycla, all the samples of which showed unique alleles at two loci. Contingency Chi-squared tests were significant between ssp. tricycla and ssp. truncatula at four loci, suggesting a barrier to gene flow between these subspecies. In accessions defined as ssp. longeaculeata, no unique allelic distribution or diagnostic sizes were observed, suggesting this apparent ssp. is a morphological variant of ssp. truncatula. The data also suggest M. truncatula that exhibits unusually wide genotype dispersal throughout its native Mediterranean region, possibly due to animal and trade-related movements. Our results showed the collection to be highly diverse, exhibiting an average of 25 SSR alleles per locus, with over 90% of individuals showing discrete genotypes. The rich diversity of the SARDI collection provides an invaluable resource for studying natural allelic variation of M. truncatula. To efficiently exploit the variation in the SARDI collection, we have defined a subset of accessions (n=61) that maximises the diversity.

A single gene, AIN, in Medicago truncatula mediates a hypersensitive response to both bluegreen aphid and pea aphid, but confers resistance only to bluegreen aphid

Biotic stress in plants frequently induces a hypersensitive response (HR). This distinctive reaction has been studied intensively in several pathosystems and has shed light on the biology of defence signalling. Compared with microbial pathogens, relatively little is known about the role of the HR in defence against insects. Reference genotype A17 of Medicago truncatula Gaertn., a model legume, responds to aphids of the genus Acyrthosiphon with necrotic lesions resembling a HR. In this study, the biochemical nature of this response, its mode of inheritance, and its relationship with defence against aphids were investigated. The necrotic lesion phenotype and resistance to the bluegreen aphid (BGA, Acyrthosiphon kondoi Shinji) and the pea aphid (PA, Acyrthosiphon pisum (Harris)) were analysed using reference genotypes A17 and A20, their F2 progeny and recombinant inbred lines. BGA-induced necrotic lesions co-localized with the production of H2O2, consistent with an oxidative burst widely associated with hypersensitivity. This HR correlated with stronger resistance to BGA in A17 than in A20; these phenotypes cosegregated as a semi-dominant gene, AIN (Acyrthosiphon-induced necrosis). In contrast to BGA, stronger resistance to PA in A17, compared with A20, did not cosegregate with a PA-induced HR. The AIN locus resides in a cluster of sequences predicted to encode the CC-NBS-LRR subfamily of resistance proteins. AIN-mediated resistance presents a novel opportunity to use a model plant and model aphid to study the role of the HR in defence responses to phloem-feeding insects.

Characterization of resistance to multiple aphid species (Hemiptera: Aphididae) in Medicago truncatula.

Aphids are phloem-feeding insects that damage many important crops throughout the world yet, compared to plant-pathogen interactions, little is known about the mechanisms by which plants become resistant to aphids. Medicago truncatula (barrel medic) is widely considered as the pre-eminent model legume for genetic and biological research and in Australia is an important pasture species. Six cultivars of M. truncatula with varying levels of resistance to two pests of pasture and forage legumes, the bluegreen aphid Acyrthosiphon kondoi Shinji and the spotted alfalfa aphid Therioaphis trifolii f. maculata. (Buckton) are investigated. Two resistance phenotypes against T. trifolii f. maculata are described, one of which is particularly effective, killing most aphids within 24 h of infestation. Each resistance phenotype provided a similar but somewhat less effective degree of resistance to the closely-related spotted clover aphid Therioaphis trifolii (Monell). In the case of A. kondoi only one resistance phenotype was observed, which did not vary among different genetic backgrounds. None of the observed resistance against A. kondoi or T. trifolii f. maculata significantly affected the performance of green peach aphid Myzus persicae (Sulzer) or cowpea aphid Aphis craccivora Koch. The existence of multiple aphid resistance mechanisms in similar genetic backgrounds of this model plant provides a unique opportunity to characterize the fundamental basis of plant defence to these serious agricultural pests.

Developing tetraploid perennial ryegrass (Lolium perenne L.) populations

Abstract In a breeding programme aimed at developing tetraploids from Australian adapted germplasm, a root‐tip squash technique was initially used for ploidy determination. The highest recovery of tetraploids (39.7%) was obtained by treatment of 1‐week‐old seedlings with 0.1% colchicine concentration. Significant cultivar × colchicine concentration interaction was observed in seedling survival, highlighting the difference in response of the cultivars to colchicine treatment. Subsequently, flow cytometric estimation of the nuclear DNA content in young leaf tissues was used to screen the C1 and C2 generations. The speed and the efficiency of this method make it possible to examine a large number of plants. In the C1 population the percentage of tetraploids, diploids, and aneuploids was 25, 72.7, and 2.3% respectively, while in the C2 generation the percentage of tetraploids, diploids, and aneuploids was 43, 2, and 55% respectively.

First report of meiotic chromosome number and karyotype analysis of an accession of Trigonella balansae (Leguminosae)

Abstract This study represents the first report of the number of meiotic chromosomes and morphology of the mitotic chromosomes of Trigonella balansae. The haploid complement of chromosomes of T. balansae consists of eight chromosomes (2n = 16). Of these, only the satellited chromosome and possibly a subtelocentric chromosome are identifiable. The remaining six chromosomes of each haploid chromosome complement cannot be reliably identified using arm ratio or relative length measurements.

Variation in coumarin content of Melilotus species grown in South Australia.

Abstract This paper describes the range in coumarin concentrations at 90% flowering stage in 149 accessions belonging to 15 Melilotus species grown in a genetic resources field characterisation experiment in Adelaide, South Australia, and determines coumarin concentrations in Melilotus species grown at two contrasting field sites in South Australia (Kybybolite and Keith). In the genetic resources characterisation experiment, the mean coumarin content ranged from 0.06 to 0.753% of dry matter. M. segetalis, M. dentatus, M. sulcatus, M. siculus and M. infestus recorded low (0.06 to 0.113%) coumarin levels while moderate levels (0.332–0.753%) were registered in the other species. The coumarin levels at Kybybolite and Keith sites ranged from 0.095 to 0.943% amongst M. indicus accessions compared to M. siculus (0.007–0.100%) and M. sulcatus (0.007–0.810%). No coumarin was detected in a M. segetalis accession at these two sites. Accessions of M. siculus and an accession of M. segetalis (SA 36979) and of M. sulcatus (SA 40019) could be potential candidates as pasture legumes that can combine tolerance of both salinity and waterlogging, and acceptably low concentrations of coumarin.

Genetic variability and heritability estimates for hardseededness and flowering in balansa clover (Trifolium michelianum Savi) populations

Suitability of annual pasture legume species like balansa clover (Trifolium michelianum Savi) in southern Australian farming systems depends on their hardseededness and time to flowering. Late maturing varieties with increased rate of hardseed breakdown, particularly in the late summer—early autumn period are desirable to ensure reliable regeneration in permanent pastures. Two half-sib family balansa clover populations were used to estimate heritability for the rate of breakdown of hardseededness and time to flowering. High narrow-sense heritability estimates (90.5–96.1%, rate of hardseed breakdown; 85.9–94.5%, time to flowering) were obtained, and were mainly attributed to additive gene effects. There was no relationship between rate of hardseed breakdown and time to flowering in Group1 half-sib family, which indicated that selection for both traits could be undertaken independently. In half-sib family Group 2 the relationship between the two traits suggested the possibility of selecting late maturing cultivars with increased rate of hardseed breakdown. Significant differences in time to flowering and rate of hardseed breakdown both within and between the two half-sib family groups suggested that phenotypic recurrent selection would be effective in improving the traits in these populations.

Morphological characterisation and genetic analysis of a bi-pistil mutant (bip) in Medicago truncatula Gaertn.

A floral organ mutant was observed in transgenic Medicago truncatula Gaertn. plants that had two separate stigmas borne on two separate styles that emerged from a single superior carpel primordium. We propose the name bi-pistil, bip for the mutation. We believe this is the first report of such a mutation in this species. Genetic and molecular analyses of the mutant were conducted. The mutant plant was crossed to a mtapetala plant with a wild-type pistil. Expression of the mutant trait in the F1 and F2 generations indicates that the bi-pistil trait is under the control of a single recessive gene. Other modifying genes may influence its expression. The mutation was associated with the presence of a T-DNA insert consisting of the Alfalfa mosaic virus (AMV) coat protein gene in antisense orientation and the nptII selectable marker gene. It is suggested that the mutation is due to gene disruption because multiple copies of the T-DNA were observed in the mutant. The bi-pistil gene was found to be independent of the male-sterile gene, tap. This novel mutant may assist in understanding pistil development in legumes.