Phillip Nichols

Temperate pasture legumes in Australia—their history, current use, and future prospects

Abstract. Australian farmers and scientists have embraced the use of new pasture legume species more than those in any other country, with 36 annual and 11 perennial legumes having cultivars registered for use. Lucerne (Medicago sativa), white clover (Trifolium repens), and red clover (T. pratense) were introduced by the early European settlers and are still important species in Australia, but several other species, notably annual legumes, have been developed specifically for Australian environments, leading to the evolution of unique farming systems. Subterranean clover (T. subterraneum) and annual medics (Medicago spp.) have been the most successful species, while a suite of new annual legumes, including serradellas (Ornithopus compressus and O. sativus), biserrula (Biserrula pelecinus) and other Trifolium and Medicago species, has expanded the range of legume options. Strawberry clover (T. fragiferum) was the first non-traditional, perennial legume commercialised in Australia. Other new perennial legumes have recently been developed to overcome the soil acidity and waterlogging productivity constraints of lucerne and white clover and to reduce groundwater recharge and the spread of dryland salinity. These include birdsfoot trefoil (Lotus corniculatus), Talish clover (T. tumens), and hairy canary clover (Dorycnium hirsutum). Stoloniferous red clover cultivars and sulla (Hedysarum coronarium) cultivars adapted to southern Australia have also been released, along with a new cultivar of Caucasian clover (T. ambiguum) aimed at overcoming seed production issues of cultivars released in the 1970s. New species under development include the annual legume messina (Melilotus siculus) and the perennial legume narrowleaf lotus (L. tenuis) for saline, waterlogged soils, and the drought-tolerant perennial legume tedera (Bituminaria bituminosa var. albomarginata). Traits required in future pasture legumes include greater resilience to declining rainfall and more variable seasons, higher tolerance of soil acidity, higher phosphorous utilisation efficiency, lower potential to produce methane emissions in grazing ruminants, better integration into weed management strategies on mixed farms, and resistance to new pest and disease threats. Future opportunities include supplying new fodder markets and potential pharmaceutical and health uses for humans and livestock. New species could be considered in the future to overcome constraints of existing species, but their commercial success will depend upon perceived need, size of the seed market, ease of establishment, and management and safety of grazing animals and the environment. Molecular biology has a range of potential applications in pasture legume breeding, including marker-assisted and genomics-assisted selection and the identification of quantitative trait loci and candidate genes for important traits. Genetically modified pasture plants are unlikely to be commercialised until public concerns are allayed. Private seed companies are likely to play an increasingly important role in pasture legume development, particularly of mainstream species, but the higher risk and more innovative breakthroughs are likely to come from the public sector, provided the skills base for plant breeding and associated disciplines is maintained.

Salinity and waterlogging tolerance amongst accessions of messina (Melilotus siculus)

Melilotus siculus (common name messina) has shown potential as a productive annual forage legume in saline and waterlogged areas in temperate Australia. The salt and waterlogging tolerances of 30 M. siculus accessions were evaluated at germination and as established plants. Many accessions germinated at 240 mm NaCl, but germination was 90%, but others 20% in any accession. Root porosity (% gas volume/root volume) in both the main and lateral roots increased in all accessions when in stagnant medium, but accessions differed in root porosity. Lateral root porosity was not, however, correlated with either shoot dry weight or root dry weight in stagnant conditions. No single accession of M. siculus had the highest tolerance to saline conditions both at germination and the vegetative stage, but some accessions (e.g. SA 40002 and SA 40004) performed consistently well under saline and waterlogged conditions. Further research and selection is warranted on these accessions with the aim to release a cultivar.

New strains of rhizobia that nodulate regenerating messina (Melilotus siculus) plants in saline soils

Messina(Melilotussiculus(Turra)VitmanexB.D.Jacks(syn.M.messanensis(L.)Mill.))isthemostpromising annual pasture legume for saline waterlogged soils in southern Australia. Messina forms a symbiosis with the commercial Sinorhizobiummedicaestrain,WSM1115,usedformanyannualmedic(Medicago)species.However,WSM1115doesnot persist over the summer months in saline soils and fails to adequately nodulate regenerating messina plants, restricting its commercial development as a new species for agriculture. To overcome this symbiotic constraint, two field experiments (swards and rows) and a glasshouse symbiotic effectiveness experiment were undertaken to identify strains of S. medicae able to persist in saline soils and adequately nodulate regenerating messina plants. In the sward experiment, no rhizobia were detected in WSM 1115 plots in the first autumn following seed set, whereas 3519 rhizobia per g of soil were measured for strain SRDI 554. Compared with WSM 1115, SRDI 554 increased regenerating messina nodulation from 32 to 100%, the number of nodules per plant from 1.3 to 32.4 and shoot dry weights from 23.6 to 80.2mg/plant. The row field experiment found SRDI 554 had greater saprophytic competence than WSM 1115 and increased overall mean plant nodulation from 11 to 74%. The symbiotic effectiveness experiment found plant shoot weight of the majority of S. medicae strains under non-saline conditions was similar to WSM 1115. These experiments have identified new strains of rhizobia that overcome the symbiotic constraint in regenerating messina plants in saline soils. Further evaluation, particularly in acidic saline, waterlogged soils, is required to confirm adaptation of the most promising strains to soils within the full range of messina target environments.

The influence of NaCl salinity and hypoxia on aspects of growth in Trifolium species

The effects of salinity and hypoxia on growth, nutritive value, and ion relations were evaluated in 38 species of Trifolium and 3 check legume species (Trifolium fragiferum, Trifolium michelianum, and Medicago sativa) under glasshouse conditions, with the aim of identifying species that may be suitable for saline and/or waterlogged conditions. In the first set of experiments, plants were grown hydroponically at four NaCl concentrations (0, 40, 80, and 160 mm NaCl) and harvested after exposure to these treatments for 4 weeks. NaCl concentrations up to 160 mm reduced dry matter production in most species; however, there were differences in salt tolerance among species, with T. argutum, T. diffusum, T. hybridum, and T. ornithopodioides performing well under the saline conditions (dry matter production was reduced by less than 20%). Concentrations of Na+ and Cl− in the shoots increased with increasing salinity levels, and species again differed in their capacity to limit the uptake of these ions. Dry matter digestibility at 0 mm ranged from 49.8% (T. palaestinum) to 74.0% (T. vesiculosum) and decreased with increasing NaCl concentrations. A second set of experiments evaluated the tolerance of Trifolium species to hypoxic conditions in the glasshouse. Shoot growth, and to a lesser extent root growth, were reduced in all Trifolium species when plants were exposed to stagnant, non-aerated conditions for 28 days, but T. michelianum, T. resupinatum, T. squamosum, T. nigrescens, T. ornithopodioides, T. salmoneum, and T. fragiferum were the least affected species. All species acclimated to the oxygen-depleted conditions by increasing the gas-filled porosity in the roots. This study has provided information that will assist in the identification of forage species for saline and/or waterlogged areas.

The first genetic maps for subterranean clover (Trifolium subterraneum L.) and comparative genomics with T. pratense L. and Medicago truncatula Gaertn. to identify new molecular markers for breeding

This study reports on the construction of the first genetic maps of subterranean clover (Trifolium subterraneum L.), a diploid, inbreeding annual pasture legume, and alignment of its linkage groups with those of red clover (T. pratense L.) and Medicago truncatula Gaertn. Transferability of red and white clover (T. repens L.) simple sequence repeat (SSR) markers to subterranean clover was observed. A total of 343 SSR loci were mapped into eight subterranean clover linkage groups, with 6–31 loci per linkage group and 27 loci with similar locations between two distinct F2 mapping populations. Phenotypic data obtained for flowering time, content of three isoflavonoids (formononetin, genistein and biochanin A), hardseededness, leaf markings, calyx pigmentation and hairiness of stems were analyzed, together with genotypic data. Genomic intervals influencing each trait were assigned to one to three chromosome regions, accounting for 5.5–59.8% of the phenotypic variance. Syntenic relationships were observed among subterranean clover, red clover and Medicago truncatula genomes. Comparisons of loci shared between the three species indicated that at least two chromosomal regions have undergone duplications in the subterranean clover genome. Candidate genes for isoflavone content were identified using M. truncatula as a reference genome. Synteny-based segmentation observed in Brassicaceae chromosomes helped to account for the apparent segmental-based relationship between the clover genomes, particularly within the subterranean clover lines. The proposed segmental nature of clover genome could account for the extensive variation observed between the parental genotypes, while not preventing production of fertile intercrosses.

Variability of in vitro ruminal fermentation and methanogenic potential in the pasture legume biserrula (Biserrula pelecinus L.)

Abstract. Biserrula (Biserrula pelecinus L.) is an important annual pasture legume for the wheatbelt of southern Australia and has been found to have lower levels of methane output than other pasture legumes when fermented by rumen microbes. Thirty accessions of the biserrula core germplasm collection were grown in the glasshouse to examine intra-specific variability in in vitro rumen fermentation, including methane output. One biserrula cultivar (Casbah) was also grown at two field locations to confirm that low methanogenic potential was present in field-grown samples. All of the biserrula accessions had significantly reduced methane [range 0.5–8.4 mL/g dry matter (DM)] output compared with subterranean clover (28.4 mL/g DM) and red clover (36.1 mL/g DM). There was also significant variation in fermentability profiles (except for volatile fatty acids) among accessions of the core collection. Methanogenic potential exhibited 86% broad-sense heritability within the biserrula core collection. The anti-methanogenic and gas-suppressing effect of biserrula was also confirmed in samples grown in the field. In conclusion, biserrula showed variability in in vitro fermentation traits including reduced methane production compared with controls. This bioactivity of biserrula also persists in the field, indicating scope for further selection of biserrula as an elite methane-mitigating pasture.

An advanced reference genome of Trifolium subterraneum L. reveals genes related to agronomic performance

Summary Subterranean clover is an important annual forage legume, whose diploidy and inbreeding nature make it an ideal model for genomic analysis in Trifolium. We reported a draft genome assembly of the subterranean clover TSUd_r1.1. Here we evaluate genome mapping on nanochannel arrays and generation of a transcriptome atlas across tissues to advance the assembly and gene annotation. Using a BioNano‐based assembly spanning 512 Mb (93% genome coverage), we validated the draft assembly, anchored unplaced contigs and resolved misassemblies. Multiple contigs (264) from the draft assembly coalesced into 97 super‐scaffolds (43% of genome). Sequences longer than >1 Mb increased from 40 to 189 Mb giving 1.4‐fold increase in N50 with total genome in pseudomolecules improved from 73 to 80%. The advanced assembly was re‐annotated using transcriptome atlas data to contain 31 272 protein‐coding genes capturing >96% of the gene content. Functional characterization and GO enrichment confirmed gene expression for response to water deprivation, flavonoid biosynthesis and embryo development ending in seed dormancy, reflecting adaptation to the harsh Mediterranean environment. Comparative analyses across Papilionoideae identified 24 893 Trifolium‐specific and 6325 subterranean‐clover‐specific genes that could be mined further for traits such as geocarpy and grazing tolerance. Eight key traits, including persistence, improved livestock health by isoflavonoid production in addition to important agro‐morphological traits, were fine‐mapped on the high‐density SNP linkage map anchored to the assembly. This new genomic information is crucial to identify loci governing traits allowing marker‐assisted breeding, comparative mapping and identification of tissue‐specific gene promoters for biotechnological improvement of forage legumes.

Resistance to race 1 of Kabatiella caulivora in subterranean clover (Trifolium subterraneum L.) cultivars and breeding lines

Twenty-eight cultivars and 106 F6-derived breeding lines of subterranean clover (Trifolium subterraneum) were screened in the field for their response to clover scorch disease caused by race 1 of Kabatiella caulivora. Eleven of the cultivars, including Denmark and Goulburn, were classified as resistant. Breeding lines with Denmark parentage had 55% of progeny with resistance, while those of Goulburn had only 19% of resistant progeny, suggesting different modes of inheritance. Selection for resistance to race 2 of K. caulivora in the F4 generation markedly increased the probability of selecting F6-derived lines with resistance to race 1, suggesting linkage between genes for resistance to both races.

Draft genome sequence of subterranean clover, a reference for genus Trifolium

Clovers (genus Trifolium) are widely cultivated across the world as forage legumes and make a large contribution to livestock feed production and soil improvement. Subterranean clover (T. subterraneum L.) is well suited for genomic and genetic studies as a reference species in the Trifolium genus, because it is an annual with a simple genome structure (autogamous and diploid), unlike the other economically important perennial forage clovers, red clover (T. pratense) and white clover (T. repens). This report represents the first draft genome sequence of subterranean clover. The 471.8 Mb assembled sequence covers 85.4% of the subterranean clover genome and contains 42,706 genes. Eight pseudomolecules of 401.1 Mb in length were constructed, based on a linkage map consisting of 35,341 SNPs. The comparative genomic analysis revealed that different clover chromosomes showed different degrees of conservation with other Papilionoideae species. These results provide a reference for genetic and genomic analyses in the genus Trifolium and new insights into evolutionary divergence in Papilionoideae species.

Hotspots and gaps in the world collection of subterranean clover (Trifolium subterraneum L.)

Subterranean clover (Trifolium subterraneum L.) is the most important annual pasture legume in the winter-dominant rainfall areas of Southern Australia. Systematic germplasm collections of subterranean clover from its centre of origin have been made since the 1950s, particularly by Australian scientists, in order to broaden the genetic base of the species. The present study reports on a meta-analysis of the distribution of the world collection of subterranean clovers and their relationships to eco-geographic variables of the collection sites in their native habitat. Diversity hotspots (areas rich in number of accessions and containing a high diversity of sub-species) and also gaps (areas with particular traits un- or under-represented in collections) were identified. This was achieved using a stratified data system to evaluate eco-geographical and agro-morphological data which incorporated three tiers of information for the subterranean clover collection: (1) information from each collection site, including ecological data; (2) information on the phenotypic diversity within each collection site; and (3) plant agro-morphological data from each sample grown under controlled conditions. Correlations were found between some eco-geographic conditions and agronomic performance. These included correlations between latitude and flowering time, mean temperature in winter and winter productivity and precipitation in summer and seed dormancy. The present study concluded that subterranean clover versatility is greater than suggested in the past. The results of the current analysis provide a guide for future collecting missions to specific regions towards areas of maximum diversity (hotspots) and unknown diversity (gaps).