Salinity and waterlogging interact to reduce growth of poorly adapted species by, amongst other processes, increasing the rate of Na(+) and Cl(-) transport to shoots. Xylem concentrations of these ions were measured in sap collected using xylem-feeding spittlebugs (Philaenus spumarius) from Lotus tenuis and Lotus corniculatus in saline (NaCl) and anoxic (stagnant) treatments. In aerated NaCl solution (200 mM), L. corniculatus had 50% higher Cl(-) concentrations in the xylem and shoot compared with L. tenuis, whereas concentrations of Na(+) and K(+) did not differ between the species. In stagnant-plus-NaCl solution, xylem Cl(-) and Na(+) concentrations of L. corniculatus increased to twice those of L. tenuis. These differences in xylem ion concentrations, which were not caused by variation in transpiration between the two species, contributed to lower net accumulation of Na(+) and Cl(-) in shoots of L. tenuis, indicating that ion transport mechanisms in roots of L. tenuis were contributing to better 'exclusion' of Cl(-) and Na(+) from shoots, compared with L. corniculatus. Root porosity was also higher in L. tenuis, due to constitutive aerenchyma, than in L. corniculatus, suggesting that enhanced root aeration contributed to the maintenance of Na(+) and Cl(-) 'exclusion' in L. tenuis exposed to stagnant-plus-NaCl treatment. Lotus tenuis also had greater dry mass than L. corniculatus after 56 d in NaCl or stagnant-plus-NaCl treatment. Thus, Cl(-) 'exclusion' is a key trait contributing to salt tolerance of L. tenuis, and 'exclusion' of both Cl(-) and Na(+) from the xylem enables L. tenuis to tolerate, better than L. corniculatus, the interactive stresses of salinity and waterlogging.

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Lotus tenuis (Wadst. & Kit.) is a perennial legume widely grown for pasture in the flood-prone and salt affected Pampa region of Argentina. The physiology of salt and waterlogging tolerance in L. tenuis (four cultivars) was evaluated, and compared with Lotus corniculatus (three cultivars); the most widely cultivated Lotus species. Overall, L. tenuis cultivars accumulated less Na + and Cl -, and more K + in shoots than L. corniculatus cultivars, when exposed to 200 mM NaCl for 28 days in aerated or in stagnant solutions. Root porosity was higher in L. tenuis cultivars due to greater aerenchyma formation. In a NaCl dose-response experiment (0-400 mM NaCl in aerated solution), L. tenuis (cv. Chaja) accumulated half as much Cl -in its shoots than L. corniculatus (cv. San Gabriel) at all external NaCl concentrations, and about 30% less shoot Na + in treatments above 250 mM NaCl. Ion distributions in shoots were determined for plants at 200 mM NaCl. L. tenuis (cv. Chaja) again accumulated about half as much Cl -in old leaves, young leaves and stems, compared with concentrations in L. corniculatus (cv. San Gabriel). There were not, however, significant differences between the two species for Na + concentrations in the various shoot tissues. The higher root porosity, and maintenance of lower shoot Cl -and Na + concentrations in L. tenuis, compared with L. corniculatus, contributes to the greater tolerance to combined salt and waterlogging stress in L. tenuis. Moreover, significant variation for tolerance to combined salinity and waterlogging stress was identified within both L. tenuis and L. corniculatus.

The release of effective inocula for new perennial clovers into cropping zones where subterranean clover is important might compromise N2 fixation by this valuable annual clover if symbiosis between the new inoculants and subterranean clover is not optimal. To assist our understanding of the interactions between clovers and their microsymbionts, rhizobial strains and clovers from South and equatorial Africa, North and South America, and the Euro–Mediterranean regions were tested. Glasshouse-based studies of the cross-inoculation characteristics of 38 strains of Rhizobium leguminosarum bv. trifolii associated with 38 genotypes of annual and perennial Trifolium spp. from these world centres of diversity were undertaken. Less than 7.5% of the perennial clover symbioses were effective whereas 40% of associations were effective for many of the annual clover species of Euro–Mediterranean origin. There was substantial specificity within the African clovers for effective nodulation. Rhizobial strains from the South American perennial T. polymorphum or from the African clovers were unable to nodulate subterranean clover effectively. Also, 7 of the 17 strains from these regions were unable to form nodules with the less promiscuous Mediterranean annual clovers, T. glanduliferum and T. isthmocarpum. Fifty-three of about 400 cross-inoculation treatments examined, which included annual and perennial clovers, were incapable of forming nodules, while only 65 formed effective nodules. There are 2 barriers to effective nodulation: a ‘geographic’ barrier representing the broad centres of clover diversity, across which few host-strain combinations were effective; and, within each region, a significant ‘phenological’ barrier between annual and perennial species. Clovers and their rhizobia from within the Euro–Mediterranean region of diversity were more able to cross the phenological barrier than genotypes from the other regions. It appears that only the relatively promiscuous clovers, whether annual or perennial, have been commercialised to date. The data indicate that, for perennial clovers, it will be a substantial challenge to develop inocula that do not adversely affect N2 fixation by subterranean clover and other annual clovers available commercially, especially if the perennial clovers were originally from Africa or America. Some future strategies for development of inoculants for clovers are proposed.

BackgroundBituminaria bituminosa is a perennial legume species from the Canary Islands and Mediterranean region that has potential as a drought-tolerant pasture species and as a source of pharmaceutical compounds. Three botanical varieties have previously been identified in this species: albomarginata, bituminosa and crassiuscula. B. bituminosa can be considered a genomic 'orphan' species with very few genomic resources available. New DNA sequencing technologies provide an opportunity to develop high quality molecular markers for such orphan species.Results432,306 mRNA molecules were sampled from a leaf transcriptome of a single B. bituminosa plant using Roche 454 pyrosequencing, resulting in an average read length of 345 bp (149.1 Mbp in total). Sequences were assembled into 3,838 isotigs/contigs representing putatively unique gene transcripts. Gene ontology descriptors were identified for 3,419 sequences. Raw sequence reads containing simple sequence repeat (SSR) motifs were identified, and 240 primer pairs flanking these motifs were designed. Of 87 primer pairs developed this way, 75 (86.2%) successfully amplified primarily single fragments by PCR. Fragment analysis using 20 primer pairs in 79 accessions of B. bituminosa detected 130 alleles at 21 SSR loci. Genetic diversity analyses confirmed that variation at these SSR loci accurately reflected known taxonomic relationships in original collections of B. bituminosa and provided additional evidence that a division of the botanical variety bituminosa into two according to geographical origin (Mediterranean region and Canary Islands) may be appropriate. Evidence of cross-pollination was also found between botanical varieties within a B. bituminosa breeding programme.ConclusionsB. bituminosa can no longer be considered a genomic orphan species, having now a large (albeit incomplete) repertoire of expressed gene sequences that can serve as a resource for future genetic studies. This experimental approach was effective in developing codominant and polymorphic SSR markers for application in diverse genetic studies. These markers have already given new insight into genetic variation in B. bituminosa, providing evidence that a division of the botanical variety bituminosa may be appropriate. This approach is commended to those seeking to develop useful markers for genomic orphan species.

Two perennial Trifolium, T. polymorphum Poir. and T. argentinense Speg., are American clovers unique within the genus for being amphicarpic. There is no consensus in the literature regarding the breeding system of the aerial flowers of T. polymorphum, therefore, the breeding system was studied. In 1997 T. polymorphum was collected in Uruguay and evaluated at INIA Tacuarembó. In 2001, 10 field patches were marked and in 2004, 20 plants per patch were characterized with simple sequence repeat markers. Patch J10 showed a particular molecular profile, therefore, 198 open‐pollinated progenies freely visited by honeybees were studied. In 2005, at the University of Florida, Gainesville, different hand‐pollination treatments were conducted within an accession from Paraguay. Trifolium polymorphum was able to cross‐pollinate with all the known pollen donors molecularly marked that surrounded plants from patch J10 (30%), also with some nonmarked native ones from the vicinity (10%) as well as with itself (60%), when allowed to be visited by honeybees. However, when there are no pollinators, the selfing rate is minimal. The proposed classification for the breeding system is an allogamous, self‐compatible species that benefits from pollinators to set seed.

Tedera (Bituminaria bituminosa C.H. Stirton var. albomarginata and var. crassiuscula) has been identified as one of the most productive and drought-tolerant species of herbaceous perennial legumes based on 6 years of field evaluation in Western Australia in areas with Mediterranean climate and annual rainfall ranging from 200 to 600mm. Importantly, tedera demonstrated broad adaptation to diverse soils, and some accessions have shown moderate levels of tolerance to waterlogging and salinity. Tedera exhibits minimal leaf shedding during summer and autumn. Economic modelling strongly suggests that giving livestock access to green tedera in summer and autumn will dramatically increase farm profit by reducing supplementary feeding. The breeding program (2006-12) evaluated the available genetic diversity of tedera for its field performance in seven nurseries with 6498 spaced plants in total covering a wide variation in rainfall, soils and seasons. Best overall plants were selected using a multivariate selection index generated with best linear unbiased predictors (BLUPs) of dry matter cuts and leaf retention traits. The breeding program also evaluated tedera for grazing tolerance, grazing preference by livestock, waterlogging tolerance, seed production, cold tolerance, disease susceptibility and presence of secondary compounds. Tedera is a diploid, self-pollinated species. Therefore, 28 elite parents were hand-crossed in several combinations to combine outstanding attributes of parents; F1 hybrids were confirmed with the aid of highly polymorphic, simple sequence repeat markers. The F1s were progressed to F4s by single-seed descent breeding. Elite parent plants were selfed for two generations to be progressed in the breeding program without hybridisation. Over time, selections from the crossing and selfing program will deliver cultivars of three ideotypes: (i) drought-tolerant, (ii) cold- and drought-tolerant, (iii) waterlogging- and drought-tolerant