Development of a sequence-specific PCR marker linked to the gene “pauper” conferring low-alkaloids in white lupin (Lupinus albus L.) for marker assisted selection

Seeds and plants of wild type Lupinus albus are bitter and contain high level of alkaloids. During domestication, at least three genes conferring low-alkaloid content were identified and incorporated into commercial varieties. Australian lupin breeders exclusively utilize one of these sweetness genes, “pauper”, in all varieties to prevent possible bitterness contamination via out-crossing. A cross was made between a sweet variety Kiev Mutant (containing pauper gene) and a bitter type landrace P27174, and the population was advanced into F₈ recombinant inbred lines (RILs). Twenty-four plants representing sweetness and bitterness were subjected to DNA fingerprinting by the microsatellite-anchored fragment length polymorphism (MFLP) technique. A dominant polymorphism was discovered in an MFLP fingerprint. The MFLP marker was converted into a co-dominant, sequence-specific, simple PCR-based marker. Linkage analysis by the software program MapManager with marker score data and alkaloid phenotyping data from a segregating population containing 190 F₈ RILs indicated that the marker is linked to the pauper gene at the genetic distance of 1.4 centiMorgans (cM). This marker, which is designated as “PauperM1”, is capable of distinguishing the pauper gene from the other two low-alkaloid genes exiguus and nutricius. Validation on germplasm from the Australian lupin breeding program showed that the banding pattern of the marker PauperM1 is consistent with the alkaloid genotyping on a wide range of domesticated varieties and breeding lines. The PauperM1 marker is now being implemented for marker assisted selection in the Australian albus lupin breeding program.     

Alkaloid profile of leaves and seeds of Lupinus hintonii C. P. Smith

L. hintonii C. P. Smith grows in the Central Highland forests of Mexico at altitudes between 2800 m to 3200 m above see level. Members of the genus Lupinus produce quinolizidine alkaloids as main chemical defensive compounds against herbivores. Surprisingly alkaloid profiles are rather constant within this species, while substantial variation was found when compared to morphologically closely related other taxa. As part of a phytochemical project on Mexican wild lupins, we report on the alkaloid profiles of seeds and leaves of L. hintonii. 19 alkaloids could be identified by capillary GLC-MS. Six major alkaloids occurred in leaves and seeds: 13-hydroxylupanine (28% and 45% respectively), tetrahydrorhombifoline (31% and 23% respectively), angustifoline (2% and 4% respectively), lupanine (7% and 5% respectively), 13alpha-tigloyloxylupanine (19% and 5% respectively) and 4alpha-angeloyl-3beta-hydroxylupanine (9% and 2%). This chemical pattern resembles that of the North American lupin L. floribundus.     

Equilibrium between basic nitrogen compounds in lupin seeds with differentiated alkaloid content

The results of studies on the content of the nitrogen basic compounds, viz. quinolizidine alkaloids, biogenic polyamines and basic amino acids in lupin seeds are presented. The investigations concerned three lupin species (Lupinus angustifolius L., Lupinus albus L. and Lupinus luteus L.) and 10 bitter and sweet cultivated varieties. Content of quinolizidine alkaloids in L. angustifolus ranged from 11.4 to 19.6 microg mg(-1) dw (bitter cultivars), from 0.18 to 0.47 microg mg(-1) dw (sweet), in L. albus from 0.58 microg mg(-1) dw (sweet) to 29.6 microg mg(-1) dw (bitter) and in L. luteus from 0.59 (sweet) to 14.7 microg mg(-1) dw (bitter). Total biogenic polyamine content ranged in L. angustifolius from 2,773.9 to 3,180.2 pmol mg(-1) dw (bitter) and from 315.0 to 599.0 pmol mg(-1) dw (sweet), in L. albus from 432.6 pmol mg(-1) dw (sweet) to 1,832.0 pmol mg(-1) dw (bitter) and in L. luteus from 506.9 pmol mg(-1) dw (sweet) to 2,091.8 pmol mg(-1) dw (bitter). Total basic amino acids varied in L. angustifolus from 1,034.3 to 1,704.6 pmol mg(-1) dw (bitter) and from 1,761.9 to 2,101.9 pmol mg(-1) dw (sweet), in L. albus from 696.9 pmol mg(-1) dw (bitter) to 1,269.2 pmol mg(-1) dw (sweet) and in L. luteus from 927.6 pmol mg(-1) dw (bitter) to 1,598.3 pmol mg(-1) dw (sweet). We found a close dependence between alkaloid content and level of biogenic polyamines and basic amino acids in all three lupin species tested. All bitter lupin seeds also contain high level of biogenic polyamines but a low content of basic amino acids. The reverse relationship in sweet lupin seeds was found. The findings demonstrate that lupin nitrogen basic compounds are in steady equilibrium and that change of content in one compound leads to corresponding change in the content of another.     

Post-damage alkaloid concentration in sweet and bitter lupin varieties and its effect on subsequent herbivory

Abstract:  While most lupin species possess quinolizidine alkaloids, sweet (low alkaloid) varieties are more palatable but at the same time more susceptible to herbivory. Nevertheless, as they are not totally devoid of alkaloids, it may be possible that their alkaloid levels increase after damage. The aim of this study was to compare inductive responses to herbivory in sweet and bitter varieties of Lupinus albus (L.) and Lupinus angustifolius (L.), and to assess if these responses were effective to stop subsequent herbivory. Two experiments were carried out; in the first, Anticarsia gemmatalis (Hübner; Lep., Noctuidae) caterpillars were introduced in field-growing lupin plants and allowed to feed for 72 h, after which leaves were collected and analysed for alkaloid content and composition. The second experiment was a bioassay, in which leaves collected from experiment 1, from treated and control plants, were offered to another set of Anticarsia caterpillars, and consumption was recorded after 24 h. We found that both L. albus varieties (sweet and bitter) had an increase in their alkaloid concentration after damage, while none of the L. angustifolius varieties had. The sweet L. albus variety, Rumbo, had a greater inductive response than the bitter variety. When leaves were offered to caterpillars (bioassay), this variety showed the greatest difference between consumption of controls and previously eaten leaves, implying that alkaloid levels reached after damage were effective to deter subsequent herbivores as a result of plants probably overcoming a 'palatability threshold'.     

Ecotypic differentiation reveals seed colour‐related alkaloid content in a crop wild relative

• Crop wild relatives can be a useful source of genotypes that maximise crop survival and yield in specific habitats. Lupinus angustifolius is an annual forb with crop varieties derived from a narrow genetic basis but that are cultivated worldwide. Its seeds have high nutritional value, but they naturally contain alkaloids with anti‐nutritive factors. The study of its wild populations can be useful to find genotypes that contribute to higher climate resilience and greater yield under stressing environmental conditions.
• Using a common garden, we evaluated ecotypic differentiation in four natural populations from two contrasting latitudes in terms of plant biomass, seed mass and number, alkaloid content in seeds for the three main alkaloids present in the plant and seed colour, including its possible influence on post‐dispersal predation. Correlations among traits were also assessed.
• We found differences among populations for all traits except final biomass. Northern populations had lighter seeds and a tendency to yield more seeds when they produced white seeds, compared to southern populations and variegated seeds. Regardless of latitude, populations showed differences in alkaloid concentration, with all three alkaloids found generally in high or low concentrations in each population. Proportion of white seed morphs varied in each population. Seed colour did not influence predator preference. In addition, white seed colour was related to a low alkaloid content.
• Our results evidence the existence of natural ecotypic differentiation in L. angustifolius not only due to latitudinal range, but also to local environmental factors. White seed coat colour could be used as a visual clue for identification of low‐alkaloid genotypes, a priority trait in L. angustifolius breeding programmes.

     

Effect of a traditional processing method on the chemical composition of local white lupin (Lupinus albus L.) seed in North-Western Ethiopia

The effect of a traditional Ethiopian lupin processing method on the chemical composition of lupin seed samples was studied. Two sampling districts, namely Mecha and Sekela, representing the mid- and high-altitude areas of north-western Ethiopia, respectively, were randomly selected. Different types of traditionally processed and marketed lupin seed samples (raw, roasted, and finished) were collected in six replications from each district. Raw samples are unprocessed, and roasted samples are roasted using firewood. Finished samples are those ready for human consumption as snack. Thousand seed weight for raw and roasted samples within a study district was similar (P > 0.05), but it was lower (P < 0.01) for finished samples compared to raw and roasted samples. The crude fibre content of finished lupin seed sample from Mecha was lower (P < 0.01) than that of raw and roasted samples. However, the different lupin samples from Sekela had similar crude fibre content (P > 0.05). The crude protein and crude fat contents of finished samples within a study district were higher (P < 0.01) than those of raw and roasted samples, respectively. Roasting had no effect on the crude protein content of lupin seed samples. The crude ash content of raw and roasted lupin samples within a study district was higher (P < 0.01) than that of finished lupin samples of the respective study districts. The content of quinolizidine alkaloids of finished lupin samples was lower than that of raw and roasted samples. There was also an interaction effect between location and lupin sample type. The traditional processing method of lupin seeds in Ethiopia has a positive contribution improving the crude protein and crude fat content, and lowering the alkaloid content of the finished product. The study showed the possibility of adopting the traditional processing method to process bitter white lupin for the use as protein supplement in livestock feed in Ethiopia, but further work has to be done on the processing method and animal evaluation.     

The interaction of phosphorus and potassium with seed alkaloid concentrations, yield and mineral content in narrow-leafed lupin (Lupinus angustifoliusL.)

We tested the impact of P deficiency, K deficiency, and their interaction on seed alkaloid concentrations and profile, yield and mineral content in sweet (low-alkaloid) and bitter (high-alkaloid) varieties of narrow-leafed lupin (Lupinus angustifolius L.). P deficiency reduced seed alkaloid concentrations in sweet, but not in bitter, varieties. Under P deficiency, the alkaloid profile in harvested seed of sweet varieties mimicked that of the bitter variety Fest, with 13-hydroxylupanine dominating over lupanine. With adequate or abundant P, lupanine was the predominant alkaloid in sweet varieties. K deficiency was associated with an 8-fold increase of seed alkaloid concentrations in the sweet variety Danja (from 1000 to 8000 mg kg^–1 DM), mostly due to the stimulation of lupanine production. There was a significant interaction between P and K that affected seed alkaloid concentrations in two ways: (i) the inhibitory effect of P deficiency was only apparent under K deficiency and (ii) the lowest seed alkaloid concentrations occurred with abundant K (240 mg K kg^–1) and P (60 mg P kg^–1). Seed yield of all varieties increased asymptotically with increasing P and reached a maximum at adequate P (30 mg P kg^–1). There was no impact of K deficiency on seed yield. In sweet and bitter varieties P supply increased seed N, P and Zn concentrations, but not K. In contrast, seed K concentrations increased and P concentrations decreased with increasing K supply. These findings suggest that P fertiliser should be supplemented with K, to avoid high seed alkaloid concentrations stimulated by asymptomatic K deficiency at high P levels.     

Evaluation of correlation between the content of alkaloids and chemical elements in Washington lupin (Lupinus polyphyllus Lindl.) besed on statistical analysis and mathematical simulation

A considerable number of linear and nonlinear correlations between concentrations of quinolizidine alkaloids and concentrations of K, Ca, Mg, Ba, Zn, Cu, Ni, and V were found in lupin seeds and leaves by using methods of correlation and regression analysis. Mathematical models of regulation of alkaloid metabolism by these chemical elements were constructed. The models of regulation were considered separately for individual plant organs.     

Lupin alkaloids from sophora chrysophylla

A new lupin alkaloid, (?)-mamanine N-oxide, was isolated from Sophora chrysophylla together with 18 known alkaloids including some unusual lupin alkaloids such as kuraramine, lamprolobine, epilamprolobine, epilamprolobine N-oxide, (+)-mamanine and (?)-pohakuline. It was also shown that the alkaloid constituents of S. chrysophylla differed considerably in the leaves, stems and seeds.     

Inhibition of seed germination by quinolizidine alkaloids

Germination of Lactuca sativa L. was inhibited by mixtures of quinolizidine alkaloids. The alkaloid esters resulted in the strongest inhibition: 6 mM 13-tigloyloxylupanine inhibited germination by 100%, whereas the other lupin alkaloids, such as lupanine and sparteine, gave a 45 and 20% inhibition, respectively. Seedlings of Lupinus albus L., which are not affected by quinolizidine alkaloids, excrete lupanine and 13-tigloyloxylupanine into the surrounding medium by their roots. It is assumed that lupin alkaloids are potential compounds of plant-plant interaction (i.e. allelopathy) besides their role in plant-herbivore interrelations.     

Chemische Verteidigung der Lupinen: Zur biologischen Bedeutung der Chinolizidinalkaloide

Quinolizidine alkaloids deter the feeding of herbivores (mammals, insects, molluscs), inhibit the growth and the development of bacteria and fungi and inhibit the germination of grass seeds and lettuce. Half-maximal inhibitor concentrations fall in the range between 0.5 and 15mM (= 0.01–0.3%). The actual concentrations of lupin alkaloids in the plant are between 5–200mmol/kg, i.e. much higher than the inhibitory concentrations. Since alkaloid-free lupins have a higher incidence of herbivory and disease, we assume that the biological function of lupin alkaloids is chemical defense besides minor roles as nitrogen transport and nitrogen storage compounds.

Teil der Habilitationsschrift (Wink 1984c). — Vorgetragen auf der Tagung der Deutschen Botanischen Gesellschaft, Wien, September 1984.     

Modelling of quinolizidine alkaloid net flows in Lupinus albus and between L. albus and the parasite Cuscuta reflexa: new insights into the site of quinolizidine alkaloid synthesis

The present work reveals new and completely different conclusionsabout the alkaloid economy of symbiotically fed Lupinus albusand L. albus parasitized by Cuscuta reflexa in the study periodof 43–55 d after sowing of lupin. Net flows of alkaloidswithin lupin and between host and parasite were calculated usingthe molar ratio of alkaloid nitrogen: total nitrogen combinedwith known net flows of nitrogen in the transport fluids andanalysing alkaloid accumulation in plant organs by HRGC. Incontrast to previous studies, quinolizidine alkaloids were predictedto be synthesized mainly in the root of L. albus and to be predominantlytransported via xylem to the apical plant shoot organs. Parasitismby C. reflexa for 12 d induced a decline of alkaloid contentin the host L. albus up to 53% compared to control plants andalkaloid synthesis was halved—apparently due to a shortageof the precursor lysine. In spite of an additional decreasein nitrogen levels at the second harvest, the host-parasitesystem showed a1.3-fold higher alkaloid content than the controlplants, 63% of the total alkaloids being attracted by Cuscuta.This indicates (a) restriction of catabolic processes withininfected lupins, (b) a massive shift of nitrogen metabolismin the direction of alkaloids and (c) an enormous sink potentialof Cuscuta for nitrogenous compounds. Although xylem was foundto be the main translocation system for alkaloids, the modellingof alkaloid flows predicts Cuscuta to derive only 4.5% of itstotal alkaloid supply from the xylem and 95.5% from the phloem.By analogy with nitrogen flows, this finding requires xylemphloemtransfers which were assumed to occur within the stem axis oflupin. A similar proportion regarding the contribution of xylemand phloem to the supply of Cuscuta was obtained for the netflows of two selected alkaloids, lupanine and 13     

Two cage-type lupin alkaloids from Sophora franchetiana

Two new cage-type lupin alkaloids, (?)-tsukushinamine-B and tsukushinamine-C, have been isolated from the fresh epigeal parts of Sophora franchetiana, along with (?)-cytisine, (?)-N-formylcytisine, (?)-rhombifoline, (?)-anagyrine, (?)-baptifoline and (±)-ammodendrine, as well as (?)-tsukushinamine-A. The structures of these novel tsukushinamine-type lupin alkaloids were determined by spectroscopic data and partly by a chemical reaction. Variations of the alkaloid contents in the seeds, seedlings and various parts of S. franchetiana were also examined.     

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.     

Secreted acid phosphatase is expressed in cluster roots of lupin in response to phosphorus deficiency

The roots of white lupin (Lupinus albus L. cv. Kievskij mutant) secrete acid phosphatase, S-APase, when they grow under conditions of low available phosphorus (P). S-APases hydrolyze organic phosphate compounds in the rhizosphere and supply inorganic phosphate to the plants. Low phosphorus availability also induces vigorous growth of cluster roots. In this study, the function of cluster roots was investigated with reference to S-APase secretion. White lupins were grown in hydroponic culture in a greenhouse under P-deficient and P-sufficient conditions. S-APase in the excised roots after treatment was detected by staining with 4-methylumbelliferone phosphate (MUP). Gene expression of S-APase in cluster and normal roots was also investigated. Activity was greatest in the roots of plants grown under conditions of P -deficiency, particularly in cluster roots. S-APase gene expression was induced by a decrease in internal P concentrations, and was especially high in cluster roots formed under conditions of P -deficiency. It was suggested that decrease of internal P concentration stimulated both of the S-APase expression and cluster root formation.     

Potassium nutrition effects on seed alkaloid concentrations,yield and mineral content of lupins (Lupinus angustifolius)

To ensure that narrow-leafed lupin (Lupinus angustifolius L.) meets feed quality standards, the concentration of alkaloids must be kept under the maximum acceptable limit of 200 mg kg^–1 DM. One of the factors that may affect seed alkaloid concentration is soil nutrient deficiency. In this paper, we report the results of glasshouse and field experiments that tested the effect of potassium (K) deficiency on seed alkaloid concentrations. In the glasshouse, seed alkaloid concentrations increased by 385, 400 and 205% under severe K deficiency in sweet varieties (Danja, Gungurru and Yorrel, respectively) of L. angustifolius. The concentration of alkaloids in Fest, the bitter variety, was always high regardless of soil K status. At all levels of applied K (0–240 mg kg^–1 soil), lupanine was the predominant alkaloid in sweet varieties, whereas 13-hydroxylupanine prevailed in the bitter variety. Seed yield of all varieties increased exponentially with increasing amounts of applied K, reaching a maximum at 60 mg K kg^–1 soil. In the field, application of K to deficient soils decreased seed alkaloid concentration at Badgingarra, Western Australia (WA) but not at Nyabing, WA, in 1996. In both field trials, seed yield and mineral content were not affected by the amounts of K fertiliser applied. These findings highlighted the need for adequate K fertilisation of deficient soils in WA to avoid the risk of producing low quality lupin seed with high alkaloid concentrations. K deficiency is involved in stimulating alkaloid production in sweet varieties of L. angustifolius.     

Whole-genome assembly and resequencing reveal genomic imprint and key genes of rapid domestication in narrow-leafed lupin

Shifting from a livestock-based protein diet to a plant-based protein diet has been proposed as an essential requirement to maintain global food sustainability, which requires the increased production of protein-rich crops for direct human consumption. Meanwhile, the lack of sufficient genetic diversity in crop varieties is an increasing concern for sustainable food supplies. Countering this concern requires a clear understanding of the domestication process and dynamics. Narrow-leafed lupin (Lupinus angustifolius L.) has experienced rapid domestication and has become a new legume crop over the past century, with the potential to provide protein-rich seeds. Here, using long-read whole-genome sequencing, we assembled the third-generation reference genome for the narrow-leafed lupin cultivar Tanjil, comprising 20 chromosomes with a total genome size of 615.8 Mb and contig N50 = 5.65 Mb. We characterized the original mutation and putative biological pathway resulting in low seed alkaloid level that initiated the recent domestication of narrow-leafed lupin. We identified a 1133-bp insertion in the cis-regulatory region of a putative gene that may be associated with reduced pod shattering (lentus). A comparative analysis of genomic diversity in cultivars and wild types identified an apparent domestication bottleneck, as precisely predicted by the original model of the bottleneck effect on genetic variability in populations. Our results identify the key domestication genetic loci and provide direct genomic evidence for a domestication bottleneck, and open up the possibility of knowledge-driven de novo domestication of wild plants as an avenue to broaden crop plant diversity to enhance food security and sustainable low-carbon emission agriculture.     

The alkaloid content of sweet lupin seed used in feeding trials on pigs and rats

The alkaloid content of the seeds of several new cultivars of “sweet” lupins was determined. The alkaloid contents ranged from < 0.01% for L. angustifolius cv. Uniwhite to 0.09% for L. albus cv. Neuland (a). The major alkaloid component (70%) of Neuland (a) was identified as dl-lupanine. This was the sample of L. albus that markedly suppressed feed intake and growth of young pigs; alcohol extraction reduced the alkaloid content to 0.02% and the extracted lupin-seed meal supported growth and food consumption equivalent to Uniwhite.Rats given a diet containing 50% L. albus cv. Neuland (a) grew normally over 13 weeks. Rats given Neuland (a) as the sole source of protein at 10% of the diet, had a protein efficiency ratio of 0.13. Supplementation of the diet with methionine increased the p.e.r. to 2.45. The addition of increasing amounts of lupin alkaloid, up to maximum content of 0.15% of the diet, had no effect on p.e.r. The p.e.r. obtained from diets containing ethanol-extracted Neuland (a), with and without methionine, were 0.94 and 2.61 respectively, as compared to 0.36 and 2.60 for the unextracted Neuland (a) diets, suggesting that lupin alkaloids can reduce protein quality in methionine deficient diets.The conclusion is that, in marked contrast to pigs, rats are more resistant to dietary lupin alkaloids.     

Quinolizidine alkaloid status of Styphnolobium and Cladrastis (Leguminosae)

Reports of quinolizidine alkaloids in Styphnolobium Schott and Cladrastis Raf. (Leguminosae) conflict with their position in recent molecular phylogenies because they are not members of a major clade of quinolizidine alkaloid-accumulating taxa. The alkaloid status of these two genera was therefore re-investigated using gas chromatography–mass spectrometry. Quinolizidine alkaloids could not be detected in extracts of leaves, flowers or seeds of S. japonicum (L.) Schott, nor in leaves of S. affine (Torrey & A. Gray) Walp., C. delavayi (Franch.) Prain, C. kentukea (Dum.-Cours.) Rudd or C. platycarpa Mak. In contrast, Calia secundiflora (Ortega) Yakovlev, also currently placed outside the major clade of quinolizidine alkaloid-producing genera in molecular phylogenies, was confirmed to accumulate a range of quinolizidine alkaloids.     

Phytochemical differences between Calia secundiflora (Leguminosae) growing at two sites in Mexico

The ecology and quinolizidine alkaloid chemistry of Calia secundiflora (Ortega) Yakovlev growing at two sites in Mexico were compared. At one site (Hidalgo) the vegetation was dominated by Flourensia resinosa and C. secundiflora, at the other site (Queretaro) C. secundiflora and Dodanaea viscosa were dominant. The Hidalgo site had shallower soils with less organic matter, N, P, and CaCO3. Seeds of C. secundiflora from each site accumulated a similar range of quinolizidine alkaloids, but the profile of alkaloids in the leaves and roots were different. The leaves and roots of plants at Hidalgo accumulated a similar range of alkaloids to the seeds with cytisine and/or N-methylcytisine being most abundant, whereas at Queretaro the leaves and roots accumulated lupinine, with other alkaloids being relatively minor constituents. The latter profile has not been reported previously for C. secundiflora.