The bacterial artificial chromosome (BAC) library of the narrow-leafed lupin (Lupinus angustifolius L.)

The narrow-leafed lupin possesses valuable traits for environment-friendly agriculture and for the production of unconventional agricultural products. Despite various genetic and environmental studies, the breeding of improved cultivars has been slow due to the limited knowledge of its genomic structure. Further advances in genomics require, among other things, the availability of a genomic DNA library with large inserts. We report here on the construction of the first DNA library cloned in a BAC (bacterial artificial chromosome) vector from diploid Lupinus angustifolius L. cv. Sonet. The high molecular weight DNA used for its preparation was isolated from interphase nuclei that were purified by flow cytometry. The library comprises 55,296 clones and is ordered in 144×384-well microtitre plates. With an average insert size of 100 kb, the library represents six haploid genome equivalents. Thanks to the purification of the nuclei by flow cytometry, contamination with chloroplast DNA and mitochondrial DNA was negligible. The availability of a BAC library opens avenues for the development of a physical contig map and positional gene cloning, as well as for the analysis of the plant’s genome structure and evolution.     

Seed-borne cucumber mosaic virus infection of narrow-leafed lupin (Lupinus angustifolius) in Western Australia

In 1986 in Western Australia, cucumber mosaic virus (CMV) infection was widespread in breeders' selections of narrow-leafed lupin (Lupinus angustifolius), and in collections of lupin cvs and wild L. angustifolius lines. When seed of some of these selections and cvs was sown, seed-borne CMV was detected in seedlings. Infection of F₁ progenies was traced to use of infected parent plants. CMV was also widespread in 25 seed crops of the new lupin cv. Wandoo but not in 42 seed crops of the new cv. Danja. When samples of the seed sown in 1986 were tested, CMV was detected in 3 - 34% of seedlings of cv. Wandoo but in none of cv. Danja. Following intensive roguing of symptom-bearing plants in the 1986 seed crop of new lupin cv. Gungurru, the level of seedling infection with CMV in seed samples after harvest was 0·1-0·2%. CMV was detected in 6 - 8%, 0·6-5% and 0 - 18% of seedlings from seed samples of established lupin cvs Chittick, Yandee and Illyarrie respectively. Highest levels of seed transmission were in seed from crops grown in high rainfall areas. When a sample of cv. Wandoo seed was graded for size by sieving, CMV was detected in seedlings grown from seed in all grades, but the smallest grade contained the highest level of infection. When seed was collected from pods at different positions on plants in a CMV-infected crop of cv. Illyarrie, seed from primary pods transmitted the virus to seedlings at a 3% rate, seed from first order lateral pods at 8% while seed from second and third order lateral pods transmitted at 13%. Examination of CMV-infected lupin crops indicated that seed-infected plants competed poorly and tended to be shaded out in dense crops but to survive in sparse crops. In 1987 during drought conditions after seeding, plant mortality was greater with seed-infected seedlings than with healthy seedlings despite wide plant spacing. An isolate of CMV from subterranean clover (Trifolium subterraneum) induced severer symptoms in lupins than four isolates from lupin; only the subterranean clover isolate prevented seed production. In tests at one lupin breeding site, CMV was found in 15 species of weeds and volunteer legumes. Fumaria officinalis, Stachys arvensis and volunteer lupins were most frequently infected.     

Biosynthesis and processing of legumin-like storage proteins in Lupinus angustifolius (lupin).

Synthesis, secretion and post-translational proteolysis of the storage proteins in cotyledons of Lupinus angustifolius L. (lupin) have been examined in vivo and in vitro by using a combination of pulse-chase experiments with [3H]- or [35S]-labelled amino acids, subcellular fractionation and cell-free translation from poly(A)+ (polyadenylylated) RNA or membrane-bound polyribosomes. Related polypeptides were identified by immunoprecipitation, separation on sodium dodecyl sulphate/polyacrylamide gels and fluorography. The synthesis and processing of two proteins were compared. Conglutin alpha, the 11 S protein, was found as a family of precursor polypeptides of Mr 68000-88000 when translated from poly(A)+ RNA under conditions where signal segments were not cleaved, and Mr 64000-85000 both when sequestered into the endoplasmic reticulum and when accumulated in the protein bodies. Pulse-chase labelling showed that cotyledons from early stages of development were completely incapable of further proteolysis of these precursors. Nevertheless, in the same juvenile cotyledons, the precursors of the minor storage protein conglutin gamma, two polypeptides with Mr 50000-51000, were proteolytically cleaved to mature subunits of Mr 32000 and 17000 within 2 h. Further cleavage of the precursors of conglutin alpha into families of mature subunits of Mr 21000-24000 and 42000-62000 was detected in more mature cotyledons. A model is proposed which suggests that the mature subunits are produced by a single proteolytic cleavage of each of the three major precursors of conglutin alpha and also suggests that a close similarity exists between these subunits and those of other legumin-like proteins. The enzyme responsible for this cleavage, which appears at a specific stage in the middle of cotyledonary development, seems to be an integral part of the programmed developmental sequence in these pods.     

Inheritance of hypersensitive resistance to Bean yellow mosaic virus in narrow-leafed lupin (Lupinus angustifolius)

In narrow‐leafed lupin (Lupinus angustifolius), segregation for the necrotic (systemic hypersensitive) response to infection with a necrotic strain of Bean yellow mosaic virus (BYMV‐N) was studied in progeny plants from six crosses. The parents were two cultivars that always developed necrosis when infected (Danja and Merrit) and two genotypes that always responded without necrosis (90L423‐07‐13 and P26697). In the four possible combinations of crosses between the different necrotic and non‐necrotically reacting genotypes, segregation for the necrotic response in F₂ progeny plants always fitted a 3:1 ratio (necrotic: non‐necrotic). All F₂ progeny plants from the cross between the two non‐cultivar genotypes became infected without necrosis while 99% of the F₂ from the cross between the two cultivars developed necrosis. These results indicate that the systemic necrotic response to infection with BYMV‐N is probably controlled by a single dominant hypersensitivity gene for which we propose the name Nbm‐1. However, its expression seemed influenced by independently segregating modifier genes in the genetic background since necrosis developed at widely different rates within affected F₂ progeny plants resulting in staggered killing.     

Genetic diversity analysis for narrow-leafed lupin (Lupinus angustifolius L.) by SSR markers

Molecular Biology Reports - Narrow-leafed lupin (Lupinus angustifolius L.) is used as grain legumes, fodder for livestock and green manure in the world and has a great potential to be developed as...     

New evidence of ancestral polyploidy in the Genistoid legume Lupinus angustifolius L. (narrow-leafed lupin)

Key message

This is the first clear evidence of duplication and/or triplication of large chromosomal regions in a genome of a Genistoid legume, the most basal clade of Papilionoid legumes.

Abstract

Lupinus angustifolius L. (narrow-leafed lupin) is the most widely cultivated species of Genistoid legume, grown for its high-protein grain. As a member of this most basal clade of Papilionoid legumes, L. angustifolius serves as a useful model for exploring legume genome evolution. Here, we report an improved reference genetic map of L. angustifolius comprising 1207 loci, including 299 newly developed Diversity Arrays Technology markers and 54 new gene-based PCR markers. A comparison between the L. angustifolius and Medicago truncatula genomes was performed using 394 sequence-tagged site markers acting as bridging points between the two genomes. The improved L. angustifolius genetic map, the updated M. truncatula genome assembly and the increased number of bridging points between the genomes together substantially enhanced the resolution of synteny and chromosomal colinearity between these genomes compared to previous reports. While a high degree of syntenic fragmentation was observed that was consistent with the large evolutionary distance between the L. angustifolius and M. truncatula genomes, there were striking examples of conserved colinearity of loci between these genomes. Compelling evidence was found of large-scale duplication and/or triplication in the L. angustifolius genome, consistent with one or more ancestral polyploidy events.     

Domestication bottlenecks limit genetic diversity and constrain adaptation in narrow-leafed lupin (Lupinus angustifolius L.)

In contrast to most widespread broad-acre crops, the narrow-leafed lupin (Lupinus angustifolius L.) was domesticated very recently, in breeding programmes isolated in both space and time. Whereas domestication was initiated in Central Europe in the early twentieth century, the crop was subsequently industrialized in Australia, which now dominates world production. To investigate the ramifications of these bottlenecks, the genetic diversity of wild (n = 1,248) and domesticated populations (n = 95) was characterized using diversity arrays technology, and adaptation studied using G × E trials (n = 31) comprising all Australian cultivars released from 1967 to 2004 (n = 23). Principal coordinates analysis demonstrates extremely limited genetic diversity in European and Australian breeding material compared to wild stocks. AMMI analysis indicates that G × E interaction is a minor, albeit significant effect, dominated by strong responses to local, Western Australian (WA) optima. Over time Australian cultivars have become increasingly responsive to warm, intermediate rainfall environments in the northern WA grainbelt, but much less so to cool vegetative phase eastern environments, which have considerably more yield potential. G × E interaction is well explained by phenology, and its interaction with seasonal climate, as a result of varying vernalization responses. Yield differences are minimized when vegetative phase temperatures fully satisfy the vernalization requirement (typical of eastern Australia), and maximized when they do not (typical of WA). In breeding for WA optima, the vernalization response has been eliminated and there has been strong selection for terminal drought avoidance through early phenology, which limits yield potential in longer season eastern environments. Conversely, vernalization-responsive cultivars are more yield-responsive in the east, where low temperatures moderately extend the vegetative phase. The confounding of phenology and vernalization response limits adaptation in narrow-leafed lupin, isolates breeding programmes, and should be eliminated by widening the flowering time range in a vernalization-unresponsive background. Concomitantly, breeding strategies that will widen the genetic base of the breeding pool in an ongoing manner should be initiated.     

Reflective mulch decreases the spread of two non-persistently aphid transmitted viruses to narrow-leafed lupin (Lupinus angustifolius)

Trials were done in 1987–1989, to investigate the effect of a reflective aluminium painted polythene mulch in protecting rows of narrow-leafed lupin ( Lupinus angustifolius ) from infection with two non-persistently aphid-transmitted viruses, bean yellow mosaic (BYMV) and cucumber mosaic (CMV). The mulch greatly decreased the rate and extent of spread of BYMV from external sources into mulch-protected rows in two trials, but was somewhat less effective in a third. The rate and extent of spread of CMV from an adjacent external source into reflective mulch-protected rows was also greatly decreased in one trial in which the mulch also decreased spread within rows and was effective even when the primary infection source was only 2.5 m away. In a trial sown with CMV-infected seed ( c . 2% seed transmission), the mulch decreased CMV spread from primary foci within rows. Reflective mulch can be used to protect breeders' single row plots of lupins from infection with CMV and BYMV.     

Alkaloid level in narrow-leafed lupin, Lupinus angustifolius, influences green peach aphid reproductive performance

Two viviparous parthenogenetic clones of the green peach aphid, Myzus persicae (Sulzer), one collected from Rydalmere, New South Wales (NSW), and the other from South Perth (SP), Western Australia, were reared on radish, Raphanus sativus L. cv. Scarlet Globe, under controlled conditions. The NSW clone was fed on simple artificial diets containing alkaloid extracted from narrow-leafed lupin, Lupinus angustifolius L. cv. Fest., and its reproductive performance monitored over 112 h. Forty (40) h into the experiment and thereafter, aphids on the control diet (sucrose solution) produced significantly more offspring (P<0.05) than those on diets containing alkaloid. In a separate experiment, apterae of each clone were caged on three lines (cv. Yorrel, cv. Danja and 84L:441) of narrow-leafed lupin, and allowed to reproduce. The first three offspring were retained, and all developed to 3rd or 4th instar stage. Two nymphs were removed, and the remaining nymph reared through. All three lines produced adults. The number of young produced were counted over 11 days. Fecundity of the SP clone was lower on line 84L:441, but there was no difference in the fecundity of the NSW clone. Phloem exudate and green tissue was concomitantly collected from all lines, and analysed by GC-MS for the alkaloids lupanine and 13-hydroxylupanine. Line 84L:441 contained the highest level of total alkaloids in both phloem and tissue. All experiments indicate that alkaloid level may suppress fecundity of green peach aphids.     

Precursors of storage proteins in Lupinus angustifolius.

The proteins that are synthesized during differentiation and development in the cotyledons of Lupinus angustifolius L. were characterized both in situ and after purification. The proteins present in situ were separated by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and subjected to 'Western'-blot analysis to identify immunologically related polypeptides. The major storage proteins of the lupin, conglutins alpha and beta, were both present in juvenile tissue only as higher Mr precursors. For conglutin beta, a family of at least three polypeptides of Mr 66 000-72 000 accumulated during the earliest phases of protein synthesis in the developing cotyledon (20-28 days after flowering). Later in development each of these polypeptides disappeared and there was the concurrent appearance in the cotyledon of the lower-Mr fragments characteristic of mature conglutin beta. For conglutin alpha, an equivalent family of precursor polypeptides of Mr 60 000-83 000 was detected. Multiple internal sites for proteolytic cleavage of all these precursors appeared to be present. However, processing of the precursors was sufficiently slow to allow them to accumulate to over 50% of total soluble protein in juvenile tissue. The precursors were purified by column chromatography under non-dissociating conditions and shown by ultracentrifugation to be multimeric proteins with Mr values in the range 150 000-200 000.     

The nutritional value of narrow-leafed lupine (Lupinus angustifolius) for fattening pigs

The aim of this study was to determine the nutrient digestibility of seeds of four varieties of narrow-leafed lupines (Lupinus angustifolius) and the possibility of soya bean meal (SBM) substitution by lupine seeds alone and in combination with rapeseed meal (RSM) in the diets of pigs. The seeds of the lupine varieties Kalif, Sonet, Zeus and Boruta were analysed. The apparent total tract digestibility (ATTD) was determined on 50 cross-bred pigs using the difference method with titanium dioxide as a marker. The substitution of SBM by lupine seeds alone (at 0 – 100%) was tested on 60 pigs (20–105 kg body weight (BW)) and by a combination of lupine seeds and RSM on 180 fattening pigs (35–80 kg BW). The chemical composition of lupine seeds differed considerably, especially in terms of crude protein and mineral content. All seeds contained less than 0.05% alkaloids and 9.3% oligosaccharides in dry matter. The ATTD of protein ranged from 70% to 74%, those of ether extract from 36% to 55% and those of gross energy from 77% to 84%. The entire replacement of SBM by lupine seeds (var. Sonet) did not have a negative effect on the performance of grower and fattener pigs. The substitution of SBM by a combination of lupines and RSM reduced the performance of growing and finishing pigs significantly.     

Application of comparative genomics to narrow-leafed lupin (Lupinus angustifolius L.) using sequence information from soybean and Arabidopsis.

The completion of genome-sequencing initiatives for model plants and EST databases for major crop species provides a large resource for gaining fundamental knowledge of complex gene interactions and the functional significance of proteins. There are increasingly numerous opportunities to transfer this information to other plant species with uncharacterized genomes and make advances in genome analysis, gene expression, and predicted protein function. In this study, we have used DNA sequences from soybean and Arabidopsis to determine the feasibility of applying comparative genomics to narrow-leafed lupin. We have used transcribed sequences from soybean and showed that a high proportion cross hybridize to lupin DNA, identifying similar genes and providing landmarks for estimating the degree of chromosomal synteny between species. To further investigate comparative relationships in this study, a detailed analysis of three lupin genes and comparison of orthologs from soybean and Arabidopsis shows that, in some cases, gene structure and expression are highly conserved and their proteins may have similar function. In other cases, genes show variation in expression profiles indicating alternative functions across species. The advantages and limitation of using soybean and Arabidopsis sequences for comparative genomics in lupins are discussed.     

Karyotyping of the narrow-leafed lupin (Lupinus angustifolius L.) by using FISH, PRINS and computer measurements of chromosomes

BAC (bacterial artificial chromosome) clones from the genomic BAC library of the narrow-leafed lupin (Lupinus angustifolius) were used for cytogenetic mapping of mitotic metaphase chromosomes of that species by the BAC-FISH technique. Location of the clones, together with cytogenetic markers localised earlier by FISH (fluorescencein situ hybridisation) and PRINS (primedin situ DNA labelling), was combined with computer-aided chromosome measurements, to construct the first idiogram of the narrow-leafed lupin. The chromosomes are meta- or submetacentric; the mean absolute chromosome lengths range from 1.9 μm to 3.8 μm, and mean relative lengths from 1.6% to 3.3%. Data concerning linkage of resistance to 2 fungal pathogens as well as assignment of the second linkage group to the appropriate chromosome are given for the first time.     

Development of sequence-specific PCR markers linked to the Tardus gene that reduces pod shattering in narrow-leafed lupin (Lupinus angustifolius L.)

Seed pods of wild-type narrow-leafed lupins (Lupinus angustifolius L.) shatter upon maturity, dispersing their seeds. Recessive alleles of the genes Tardus and Lentus that confer reduced pod shattering have been incorporated into domesticated cultivars to facilitate harvesting. Tardus was mapped in an F₈ recombinant inbred population of a cross between domesticated and wild lupins. A microsatellite–anchored fragment length polymorphism marker (TaM1), which mapped 2.1 cM from Tardus, was converted to a locus-specific PCR assay. Marker TaM2, a restriction fragment length polymorphism marker was converted to a PCR assay and mapped to 3.9 cM on the other side of Tardus. Marker TaM3, a cleaved amplified polymorphic sequence marker, was positioned along-side marker TaM1 at 3.9 cM from Tardus. One or more markers was polymorphic in 70% of possible pairwise crosses between Australian domesticated lines and wild accessions tested, indicating wide applicability of the markers in crosses between wild and domesticated germplasm.     

Development of molecular markers using MFLP linked to a gene conferring resistance to Diaporthe toxica in narrow-leafed lupin ( Lupinus angustifolius L.)

Phomopsis stem blight (PSB) caused by Diaporthe toxica is a major disease in narrow-leafed lupin ( Lupinus angustifolius L.). The F(2) progeny and the parental plants from a cross between a breeding line 75A:258 (containing a single dominant resistance gene Phr1 against the disease) and a commercial cultivar Unicrop (susceptible to the disease) were used for development of molecular markers linked to the disease resistance gene. Two pairs of co-dominant DNA polymorphisms were detected using the microsatellite-anchored fragment length polymorphism (MFLP) technique. Both pairs of polymorphisms were isolated from the MFLP gels, re-amplified by PCR, sequenced, and converted into co-dominant, sequence-specific and PCR-based markers. Linkage analysis by MAPMAKER suggested that one marker (Ph258M2) was 5.7 centiMorgans (cM) from Phr1, and the other marker (Ph258M1) was 2.1 cM from Ph258M2 but further away from Phr1. These markers are suitable for marker-assisted selection (MAS) in lupin breeding.     

Further studies on cucumber mosaic virus infection of narrow-leafed lupin (Lupinus angustifolius): seed-borne infection, aphid transmission, spread and effects on grain yield

Four field trials were done with narrow-leafed lupins (Lupinus angustifolius) in 1988 - 1989, to examine the effect of sowing seed with 5% and 0.5% cucumber mosaic virus (CMV) infection on subsequent virus spread, grain yield and percentage of infection in harvested seed. A proportion of the CM V-infected seed failed to produce established plants and thus, plots sown with 5% and 0.5% infected seed contained 1.5-2.9% and 0.2-0.3% of seed-infected plants respectively. The rate of virus spread by aphids was faster and resulted in more extensive infection at maturity in plots sown with 5% infected seed than with 0.5% infected seed. In three trials, sowing 5% infected seed resulted in yield losses of 34 - 53% and CMV infection in the seed harvested of 6 - 13%. The spread of CMV infection resulting from sowing 0.5% infected seed did not significantly decrease yield. However, late CMV spread in these plots caused > 1% seed infection. In the fourth trial, which was badly affected by drought, CMV spread only slowly, there was no significant effect of CMV on grain yield and the percentage of infected seed harvested was 3–5 times less than that in the seed sown. When CMV-infected seed was sown at different depths, target depths of 8 and 11 cm decreased the incidence of seed-infected plants by c. 15% and c. 50% respectively compared with sowing at 5 cm. However, in glasshouse tests, treatment with the pre-emergence herbicide simazine failed to selectively cull out seed-infected plants. The field trials were colonised by green peach (Myzus persicae), blue-green (Acyrthosiphon kondoi) and cowpea (Aphis craccivora) aphids. When the abilities of these aphid species and of the turnip aphid (Lipaphis erysimi) in transmitting CMV from lupins to lupins were examined in glasshouse tests, short acquisition access times favoured transmission. With 5–10 min acquisition access times, overall transmission efficiencies were 10.8%, 9.4%, 6.1% and 3.9% for the green peach, cowpea, blue-green and turnip aphids respectively.     

Construction of a genetic linkage map using MFLP and identification of molecular markers linked to domestication genes in narrow-leafed lupin (Lupinus angustifolius L.)

A mapping population of F(8)derived recombinant inbred lines (RILs) was established from a cross between a domesticated breeding line 83A:476 and a wild type P27255 in narrow-leaf lupin (Lupinus angustifolius L.). The parents together with the 89 RILs were subjected to DNA fingerprinting using microsatellite-anchored fragment length polymorphism (MFLP) to rapidly generate DNA markers to construct a linkage map. Five hundred and twenty two unique markers of which 21% were co-dominant, were generated and mapped. Phenotypic data for the domestication traits: mollis (soft seeds), leucospermus (white flower and seed colour); Lentus (reduced pod-shattering), iucundis (low alkaloid), Ku (early flowering) and moustache pattern on seed coats; were included. Three to 7 molecular markers were identified within 5 cM of each of these domestication genes. The anthracnose resistance gene Lanr1 was also mapped. Linkage groups were constructed using MapManager version QTXb20, resulting in 21 linkage groups consisting of 7 or more markers. The total map length was 1543 cM, with an average distance of 3.4 cM between adjacent markers. This is the first published map for a lupin species. The map can be exploited for marker assisted selection for genetic improvement in lupin breeding programs.