David M. Peck

Genetic analysis of tolerance to Boron toxicity in the legume Medicago truncatula

BackgroundMedicago truncatula Gaertn. (barrel medic) is cultivated as a pasture legume for its high protein content and ability to improve soils through nitrogen fixation. Toxic concentrations of the micronutrient Boron (B) in agricultural soils hamper the production of cereal and leguminous crops. In cereals, the genetic analysis of B tolerance has led to the development of molecular selection tools to introgress and maintain the B tolerance trait in breeding lines. There is a comparable need for selection tools in legumes that grow on these toxic soils, often in rotation with cereals.ResultsGenetic variation for B tolerance in Medicago truncatula was utilised to generate two F2 populations from crosses between tolerant and intolerant parents. Phenotyping under B stress revealed a close correlation between B tolerance and biomass production and a segregation ratio explained by a single dominant locus. M. truncatula homologues of the Arabidopsis major intrinsic protein (MIP) gene AtNIP5;1 and the efflux-type transporter gene AtBOR1, both known for B transport, were identified and nearby molecular markers screened across F2 lines to verify linkage with the B-tolerant phenotype. Most (95%) of the phenotypic variation could be explained by the SSR markers h2_6e22a and h2_21b19a, which flank a cluster of five predicted MIP genes on chromosome 4. Three CAPS markers (MtBtol-1,-2,-3) were developed to dissect the region further. Expression analysis of the five predicted MIPs indicated that only MtNIP3 was expressed when leaf tissue and roots were assessed. MtNIP3 showed low and equal expression in the roots of tolerant and intolerant lines but a 4-fold higher expression level in the leaves of B-tolerant cultivars. The expression profile correlates closely with the B concentration measured in the leaves and roots of tolerant and intolerant plants. Whereas no significant difference in B concentration exists between roots of tolerant and intolerant plants, the B concentration in the leaves of tolerant plants is less than half that of intolerant plants, which further supports MtNIP3 as the best candidate for the tolerance trait-defining gene in Medicago truncatula.ConclusionThe close linkage of the MtNIP3 locus to B toxicity tolerance provides a source of molecular selection tools to pasture breeding programs. The economical importance of the locus warrants further investigation of the individual members of the MIP gene cluster in other pasture and in grain legumes.

Development of an early season barrel medic (Medicago truncatula Gaertn.) with tolerance to sulfonylurea herbicide residues

Abstract. Sulfonylurea (SU) herbicides are extensively applied to crops in the cereal-livestock zones of southern Australia. In low rainfall areas with alkaline soils, SU residues can persist over summer and can severely affect sown or regenerating medic pastures. A cohort of early season barrel medics (Medicago truncatula) bred and selected for tolerance to SU herbicide residues were evaluated at multiple field sites over 3 years (year of establishment and subsequent regeneration). Two lines (Z2438 and Z2415) were identified which had dry matter production and seed yield in the establishment year equivalent to their recurrent parent, Caliph, an early maturing, aphid-tolerant, barrel medic cultivar. They also had lower levels of hardseededness than Caliph, enabling them to regenerate in greater numbers in the following year and thus produce more dry matter. The two lines demonstrated good tolerance to simulated SU herbicide residues, producing up to 10 times the dry matter of the SU-intolerant parent Caliph. We anticipate that one or both of the two lines will be commercialised soon, enabling farmers in low rainfall areas with neutral-to-alkaline soils to successfully grow barrel medic pastures in the presence of SU herbicide soil residues resulting from applications to prior crops.

A new biotype of bluegreen aphid (Acyrthosiphon kondoi Shinji) found in south-eastern Australia overcomes resistance in a broad range of pasture legumes

Abstract. A new bluegreen aphid biotype (BGA, Acyrthosiphon kondoi Shinji) has been found in south-eastern Australia that causes severe damage and mortality in seedlings of previously resistant pasture legume cultivars. Populations of BGA collected at Urrbrae and Binnum, SA in 2009 caused 100% mortality in 29 cultivars of annual and perennial Medicago spp. and annual Trifolium spp. Delaying inoculation from the first trifoliate to the 6–8 trifoliate stage and removing susceptible genotypes from experiments had no impact on reducing mortality from 100% in previously resistant barrel medics. A half-sib family of lucerne from the SARDI breeding program has maintained resistance to the Urrbrae 2009 BGA. A detailed study of the virulence of BGA populations collected from Toowoomba (Qld), Tamworth, Howlong (NSW), Launceston (Tas.), Colebatch, Kimba, Urrbrae and Vivonne Bay (SA) in 2010–11 on 33 pasture legumes provides evidence of new virulent BGA being widespread, despite these populations causing less severe damage and mortality than the two populations collected in 2009.

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.

Bean leafroll virus is widespread in subterranean clover (Trifolium subterraneum L.) seed crops and can be persistently transmitted by bluegreen aphid (Acyrthosiphon kondoi Shinji)

Abstract. In the mid 2000s subterranean clover (Trifolium subterraneum) seed producers in South Australia reported symptoms of a red-leaf disease in fields with reduced seed yields. The red-leaf symptoms resembled those caused by several clover-infecting viruses. A set of molecular diagnostic tools were developed for the following viruses which are known to infect subterranean clover: Alfalfa mosaic virus; Bean leafroll virus (BLRV); Beet western yellows virus; Bean yellow mosaic virus; Cucumber mosaic virus; Pea seed-borne mosaic virus; Soybean dwarf virus and Subterranean clover stunt virus. Surveys of subterranean clover seed production fields in 2008 in the south-east of South Australia and western Victoria identified Bean leafroll virus, Alfalfa mosaic virus and Cucumber mosaic virus as present, with BLRV the most widespread. Surveys of pasture seed production fields and pasture evaluation trials in 2009 confirmed that BLRV was widespread. This result will allow seed producers to determine whether control measures directed against BLRV will overcome their seed losses. Bluegreen aphid (Acyrthosiphon kondoi) was implicated as a potential vector of BLRV because it was observed to be colonising lucerne plants adjacent to subterranean clover seed production paddocks with BLRV, and in a glasshouse trial it transmitted BLRV from an infected lucerne plant to subterranean clover in a persistent manner.

Genetic analysis of tolerance to the root lesion nematode Pratylenchus neglectus in the legume Medicago littoralis

BackgroundThe nematode Pratylenchus neglectus has a wide host range and is able to feed on the root systems of cereals, oilseeds, grain and pasture legumes. Under the Mediterranean low rainfall environments of Australia, annual Medicago pasture legumes are used in rotation with cereals to fix atmospheric nitrogen and improve soil parameters. Considerable efforts are being made in breeding programs to improve resistance and tolerance to Pratylenchus neglectus in the major crops wheat and barley, which makes it vital to develop appropriate selection tools in medics.ResultsA strong source of tolerance to root damage by the root lesion nematode (RLN) Pratylenchus neglectus had previously been identified in line RH-1 (strand medic, M. littoralis). Using RH-1, we have developed a single seed descent (SSD) population of 138 lines by crossing it to the intolerant cultivar Herald. After inoculation, RLN-associated root damage clearly segregated in the population. Genetic analysis was performed by constructing a genetic map using simple sequence repeat (SSR) and gene-based SNP markers. A highly significant quantitative trait locus (QTL), QPnTolMl.1, was identified explaining 49% of the phenotypic variation in the SSD population. All SSRs and gene-based markers in the QTL region were derived from chromosome 1 of the sequenced genome of the closely related species M. truncatula. Gene-based markers were validated in advanced breeding lines derived from the RH-1 parent and also a second RLN tolerance source, RH-2 (M. truncatula ssp. tricycla). Comparative analysis to sequenced legume genomes showed that the physical QTL interval exists as a synteny block in Lotus japonicus, common bean, soybean and chickpea. Furthermore, using the sequenced genome information of M. truncatula, the QTL interval contains 55 genes out of which five are discussed as potential candidate genes responsible for the mapped tolerance.ConclusionThe closely linked set of SNP-based PCR markers is directly applicable to select for two different sources of RLN tolerance in breeding programs. Moreover, genome sequence information has allowed proposing candidate genes for further functional analysis and nominates QPnTolMl.1 as a target locus for RLN tolerance in economically important grain legumes, e.g. chickpea.

Progress in development of spotted medics (Medicago arabica L. Huds.) for Mediterranean farming systems

Spotted medics (Medicago arabica) have become naturalised in Australia, but the spiny nature of their pods has prevented commercial release of any cultivar. Fifty-eight accessions representing Medicago arabica in the Australian Medicago Genetic Resources Collection were grown as spaced plants at Turretfield, South Australia, and the variation for important agronomic traits was studied. There was large variation for traits including days to flowering, dry matter production, pod and seed yield, and pod spininess. Principal component and cluster analyses conducted for 13 traits revealed 5 clusters. One of the clusters identified comprised accessions originating from Greece and Cyprus, which were found to have high agronomic potential. The study has helped in identifying the relationship among traits, namely pod spininess, days to flowering, dry matter yield, and pod and seed yield, which would be useful to breeders for future breeding and selection programs. A sward trial at Moree, New South Wales, comprising a selected cohort of spotted medic accessions, enabled the identification of 2 early flowering and high dry matter yielding accessions; however, both exhibited spiny pods. These 2 accessions were crossed with a smooth-podded accession, and the F1 plants were confirmed using a microsatellite marker. Days to flowering showed a continuous pattern of variation in the F2, suggesting that the trait is quantitatively inherited, whereas segregation ratio revealed that a single recessive gene controlled the smooth pod trait. Early flowering, smooth-podded F2 plants were selected for cultivar development.

Breeding system in Trifolium glanduliferum (Fabaceae)

Abstract Trifolium glanduliferum Boiss. var. nervulosum (Boiss. & Heldr.) Zoh., glandular (or gland) clover is an annual pasture species, with Mediterranean origin, recently introduced into Australia. The species was previously reported as self‐pollinating. Studies with four accessions originating from Israel clearly suggest that Trifolium glanduliferum is a highly self‐incompatible species, which requires vectors including bees to ensure pollination. Unpollinated bagged and hand tripped florets showed no signs of pollen tube growth and no seed set, while cross‐pollinated florets showed pollen tube growth and gave high seed set. A high pollen:ovule ratio suggested that the species is between facultative outcrossing and obligate outcrossing.

Diversity for resistance to a moderately virulent bluegreen aphid (Acyrthosiphon kondoi Shinji) population in Trifolium species

Abstract. The resistance of a diverse range of Trifolium species (clovers) to a highly virulent bluegreen aphid (BGA, Acyrthosiphon kondoi Shinji) population (Urrbrae 2011) collected in South Australia was assessed in greenhouse and field experiments, with the aims of determining the potential impact of this insect pest on biomass and identifying resistant genotypes for future plant-breeding activities. Resistance to BGA was found in populations of clovers that show some level of outcrossing—white clover (T. repens L.), rose clover (T. hirtum All.), crimson clover (T. incarnatum L.) and red clover (T. pratense L.)—and in one entry of the inbreeding subspecies of subterranean clover, T. subterraneum L. subsp. subterraneum (Katzn. and Morley). Resistance was not found in T. s. brachycalycinum (Katzn. and Morley) or T. s. yanninicum (Katzn. and Morley). In a greenhouse experiment, damage from BGA resulted in forage yield penalties of 72–100% when aphids were inoculated at 14 days after sowing and 13−74% when inoculated at 42 days after sowing, showing that in optimum conditions BGA can be a serious pest of clovers. Observations of severe damage caused by BGA in two regenerating field trials in southern New South Wales confirmed that field damage could occur in seasons favourable to aphid growth and reproduction. The severe damage that BGA can cause to clovers, and the sources of resistance we found, suggest that breeding for BGA resistance in clovers is warranted and feasible.