Mapping of repeated DNA sequences in plant chromosomes by PRINS and C-PRINS

 The primed in situ DNA labelling (PRINS) procedure was optimised for the rapid physical mapping of several types of repetitive DNA sequences on the mitotic chromosomes of Vicia faba, Pisum sativum and Secale cereale. A localization of the highly repeated FokI sequence on V. faba chromosomes was achieved after a 7-min total reaction time. In addition, we report a procedure for direct cycling-PRINS (C-PRINS), a variation of PRINS which involves a sequence of thermal cycles analogous to the polymerase chain reaction. Compared to PRINS, C-PRINS was more sensitive. Further work is needed to improve the sensitivity of the reaction to allow for the reliable detection of low-copy DNA sequences. Received: 17 September 1996 / Accepted: 18 October 1996     

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.     

Deploying strain specific hypersensitive resistance to diminish temporal virus spread

Spread of necrotic and non‐necrotic strains of Bean yellow mosaic virus (BYMV) was compared when aphid vectors moved both types from external or internal virus sources to plots of Lupinus spp. (lupin). Regardless of whether virus sources were internal or external, removed or left in place, and spread was within plots with homologous sources or across buffers to plots containing the opposite type of virus source, non‐necrotic BYMV always spread faster than necrotic BYMV in plots of L. angustifolius (narrow‐leafed lupin). When necrotic BYMV spread from external sources into plots sown with two L. angustifolius genotypes differing in their necrosis responses to different BYMV strain groups and one genotype of L. luteus (yellow lupin) giving only non‐necrotic responses, differing symptom reactions in the two L. angustifolius genotypes revealed presence of two distinct necrotic BYMV strain groups and overall virus spread was greater in this species than in L. luteus. Spread of non‐necrotic BYMV in L. angustifolius was always polycyclic in nature. However, when it came initially from external sources, spread of necrotic BYMV was largely monocyclic. This work demonstrates how temporal virus spread can be diminished when hypersensitive (necrotic) resistance is deployed and the limitations associated with employing hypersensitivity that is strain specific.     

A molecular marker linked to the mollis gene conferring soft-seediness for marker-assisted selection applicable to a wide range of crosses in lupin (Lupinus angustifolius L.) breeding

To broaden the gene pool of domesticated commercial cultivars of narrow-leafed lupin (Lupinus angustifolius L.), wild accessions are used as parents in crossing in lupin breeding. Among the progenies from wild × domesticated (W × D) crosses, the soft-seediness gene mollis is the most difficult domestication gene to be selected by conventional breeding methods, where molecular marker-assisted selection (MAS) is highly desirable. MAS in plant breeding requires markers to be cost-effective and high-throughput, and be applicable to a wide range of crosses in a breeding program. In this study, representative plants from an F8 recombinant inbred line (RIL) population derived from a W × D cross, together with four cultivars and four wild types, were used in DNA fingerprinting by microsatellite-anchored fragment length polymorphisms (MFLP). Two co-dominant MFLP polymorphisms were identified as candidate markers linked to the mollis gene, and one of the candidate markers was selected and converted into a co-dominant, sequence-specific PCR marker. This marker, designated MoLi, showed a perfect match with phenotypes of seed coat permeability on a segregating population consisting of 115 F8 RILs, confirming the close genetic linkage to the mollis gene. Validation tests showed that the banding pattern of marker MoLi is consistent with all the 25 historical and current commercial cultivars released in Australia, and is consistent with mollis genotypes in 119 of the 125 accessions in the Australian L. angustifolius core collection. Marker MoLi provides a cost-effective way to select the mollis gene in a wide range of W × D crosses in lupin breeding.     

Variation in the growth of lupin species and genotypes on alkaline soil

Commercial lupins grow poorly on alkaline and neutral fine-textured soils. Genotypic variation exists among lupins. The present study compared the growth of 13 lupin genotypes, including introduced cultivars and wild types, in an alkaline loamy soil and an acid loamy soil.Plants grown in the alkaline and acid soils did not show obvious symptoms of iron deficiency at any stage. There was however a large variation of shoot fresh weight among genotypes in response to the alkaline soil with L. atlanticus and L. pilosus being more tolerant than L. luteus, L. cosentinii, L. albus and L. angustifolius. Some variation also existed among genotypes of L. angustifolius. In addition, root growth was retarded on the alkaline soil except for L. atlanticus, L. pilosus P20955 and L. albus Kiev mutant. In the alkaline soil, root growth at week 2 correlated well with the shoot fresh weight at week 12. The results suggest that early root elongation may be useful for screening tolerant genotypes for alkaline soils.     

Lupinus species differ in their requirements for iron

The effect of iron supply on the growth and nodulation ofLupinus angustifolius L. (Gungurru),Lupinus luteus L. (R-1171) andLupinus pilosus Murr. (P20957) was studied in acid solutions. Plants of the three species were grown together in the same solution and inoculated withBradyrhizobium (Lupinus) WU 425. Plants were then grown with or without applied NH₄NO₃. The lupin species differed greatly in their sensitivity to low iron concentrations in solution withL. pilosus being most tolerant andL. luteus most sensitive.L. pilosus had the highest iron concentration in tissues and had a higher ratio of iron concentration in the youngest fully expanded leaf blades (YEB) to that in roots than the other two species.L. luteus had higher iron concentrations in roots but lower iron concentration in YEB and shoots than didL. angustifolius. The requirements of internal iron for the maximal chlorophyll synthesis in YEB were 65 μg g^-1 forL. angustifolius andL. luteus, and 52 μg g^-1 forL. pilosus. In contrast to effects on growth, the three species had similar external iron requirements for nodule formation in roots and for maximal nitrogen concentrations in shoots. The results indicate that iron tolerant lupin species require lower internal and external iron supply and have a greater ability than sensitive species to translocate iron from roots to shoots.     

The symbiotic performance of lupin bacteria under glasshouse and field conditions

Summary Bacteria from the nodules ofLupinus angustifolius L.,L. digitatus Forsk., andL. luteus L. have been isolated, and the symbiotic relationships of nine of these strains and the three lupin species investigated in a glasshouse experiment. All symbioses were effective and no interaction or specificity was detected among the symbionts.Soil factors modified expression of the strains' symbiotic abilities in the field, but several strains have shown successful symbiosis with all three lupin species.One lupin species,L. digitatus, nodulates freely under natural conditions in Western Australia, whereasL. luteus andL. angustifolius do not. This lupin is more readily infected by native strains of rhizobia than the others.     

The first gene-based map of Lupinus angustifolius L.-location of domestication genes and conserved synteny with Medicago truncatula

We report the first gene-based linkage map of Lupinus angustifolius (narrow-leafed lupin) and its comparison to the partially sequenced genome of Medicago truncatula. The map comprises 382 loci in 20 major linkage groups, two triplets, three pairs and 11 unlinked loci and is 1,846 cM in length. The map was generated from the segregation of 163 RFLP markers, 135 gene-based PCR markers, 75 AFLP and 4 AFLP-derived SCAR markers in a mapping population of 93 recombinant inbred lines, derived from a cross between domesticated and wild-type parents. This enabled the mapping of five major genes controlling key domestication traits in L. angustifolius. Using marker sequence data, the L. angustifolius genetic map was compared to the partially completed M. truncatula genome sequence. We found evidence of conserved synteny in some regions of the genome despite the wide evolutionary distance between these legume species. We also found new evidence of widespread duplication within the L. angustifolius genome.Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorised users.     

Physiological responses of lupin roots to high pH

High pH seems to be a major constraint limiting the production of narrow-leafed lupin (Lupinus angustifolius L.) on alkaline soils. Whereas there has been much interest in soil acidity, relatively little is known about the effect of high pH on the growth of roots of higher plants.Elongation of roots of L. angustifolius was particularly sensitive to pH6.0 compared with other species. The effect of high pH in decreasing root elongation in L. angustifolius occurred within one hour. It was via an effect on cell elongation and not cell division and the effect was readily reversible. The mechanisms of the adverse effect of high pH are unknown. The permeability ratio of K⁺ to Na⁺ in the plasma-membrane of the root cortical cells was similar in solutions of both low pH and high pH. Reduced cell growth at high pH was not associated with an inefficiency of proton extrusion to the bulk solution by roots of this species. Nevertheless, increasing buffer concentration in the external solution decreased root elongation more in L. angustifolius than in Lupinus pilosus and Pisum sativum.     

Species-specific EcoRI repetitive elements of at least 16 kb in length are present in Lupinus luteus

Summary A genomic DNA library of Lupinus luteus cv. Ventus was constructed in the phage vector EMBL3 using Mb oI-digested DNA. Screening with a 1070 bp labelled repetitive unit from L. luteus yielded several DNA clones. The repetitive family is composed of elements whose length is at least 16 kb. The average copy number of the cloned fragments is 5.0 × 10⁴ per haploid genome and constitutes approximately 3% of the total L. luteus genome. The homologous repeats were found in all ten cultivars of L. luteus tested but were not detected in two cultivars each of the closely related species Lupinus albus and Lupinus angustifolius. The EcoRI family fragments could thus be considered as species-specific DNA elements. These fragments may be useful as molecular markers in the genetic manipulation of L. luteus.     

Electronically monitored cowpea aphid feeding behavior on resistant and susceptible lupins

The feeding behavior of cowpea aphid, Aphis craccivora Koch (Homoptera: Aphididae) was examined on seedlings of narrow leafed lupin, Lupinus angustifolius L., and yellow lupin, L. luteus L., using electronic monitoring of insect feeding behavior (EMIF). Aphid feeding behavior was first compared between resistant (cv. Kalya) and susceptible (cv. Tallerack) varieties of narrow-leafed lupin. Aphids spent significantly more time in non- penetration and stylet pathway activities, and significantly less time in the sieve element phase on Kalya than on Tallerack, suggesting that feeding deterrence is an important component of aphid resistance in Kalya. Aphid feeding on a susceptible yellow lupin variety (cv. Wodjil) was then compared with that on two resistant lines, one (Teo) with high and the other (94D024-1) with low seed alkaloid content. There were no consistent differences in aphid feeding behavior between Wodjil and Teo. Total, mean and percentage sieve element phase times were significantly lower, and total and percentage times in non-phloem phase were greater on 94D024-1 than on Wodjil, suggesting the possibility of phloem-based deterrence in 94D024-1.     

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.     

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.     

Cluster-root production and organic anion exudation in a group of old-world lupins and a new-world lupin

We examined the capacity of several Old-World lupin species (Lupinus luteus L., L. hispanicus Boiss. et Reuter and L. angustifolius L.) and one species of a New-World lupin (L. mutabilis Sweet) to form cluster roots under a range of conditions in solution culture. The effect of the synthetic auxin, IBA (indole-3-butyric acid), on cluster-root development in L. luteus and L. albus L. provided with an adequate phosphorus (P) supply was also investigated. In addition, the effect of a high nitrate-N (NO₃-N) supply on the efflux of citrate and malate from roots of L. angustifolius was examined to determine if specific regions of the root system exuded these organic anions. When P-deficient, L. hispanicus, L. luteus and L. mutabilis formed cluster roots that secreted organic anions. Citrate was generally the dominant organic anion exuded, although succinate was also exuded in large quantities from L. luteus. Citrate efflux by L. hispanicus and L. luteus was at least comparable to that reported for P-deficient L. albus[up to 1.092 nmol g^–1 fresh weight (FW) s^–1], but was over an order of magnitude lower in L. mutabilis (0.036 nmol g^–1 FW s^–1). Citrate and malate were not detected in significant amounts from either the lateral roots or the root tips of any species grown under P-sufficient or -deficient conditions. Citrate efflux from cluster roots of L. luteus showed a diurnal pattern, similar to that reported for L. albus, with maximum efflux during the day, and declining to a minimum before dawn. IBA added to the nutrient solution induced cluster-root formation on both L. albus and L. luteus at concentrations of P that would normally suppress the production of these roots. However, the IBA-induced cluster roots did not exude significant amounts of citrate. Although L. angustifolius did not produce cluster roots when P-deficient, it produced cluster-like root structures that exuded citrate (0.053 nmol g^–1 FW s^–1) when grown at a high nitrate-N (NO₃-N) supply. L. angustifolius did not exude significant citrate or malate from lateral roots or root tips when grown at either high or low NO₃-N supply. Our findings for L. hispanicus and L. luteus are the first reports of cluster-root formation in response to P deficiency for these Old-World species, and for L. mutabilis, it is the first report of cluster roots for a New-World lupin species. These reports indicate that evolutionary and biogeographical aspects of cluster-root formation in the genus Lupinus need to be revised. Furthermore, investigation is warranted to determine the capacity of species of the large group of New-World lupins to form cluster roots in soils of their native habitats.     

Primedin situ labeling (PRINS)

PRimedIn Situ labeling (PRINS) is a fast and sensitive alternative to fluorescencein situ hybridization (FISH) for identification of chromosome aberrations. In this article, we present the detailed protocols for detection of repeat sequences using oligonucleotides or fragments of cloned probes as primers for PRINS. We describe a multicolor PRINS procedure for simultaneous visualization of more probes in different colors on a metaphase preparation, and a PRINS-painting procedure, which combines PRINS and chromosome painting. Finally, a protocol for detection of single-copy genes is presented.     

Mapping of rRNA genes and telomeric sequences in Danube salmon (<Emphasis Type="Italic">Hucho hucho</Emphasis>) chromosomes using primed in situ labeling technique (PRINS)

In the current paper we described the application of primed in situ (PRINS) labeling approach for the chromosomal mapping of repetitive DNA sequences in Danube salmon (Hucho hucho) (2n = 82, NF = 112). PRINS was successfully performed with primers enabling amplification of 5S rRNA genes (minor rDNAs), NOR building DNA sequences (major rDNAs), and telomeric sequences. Two loci of 5S rRNA were observed on distinct chromosome pairs; the minor arrays were located interstitially on the long (q) arms of two large metacentrics (chromosomes No. 3) and the large clusters of 5S rDNAs were assigned to the short (p) arms of two subtelocentric chromosomes No. 18. Major rDNA clusters were observed on the p-arms of two submeta-subtelocentric chromosomes No. 10. These chromosomal areas were built with GC-rich chromatin what was proved in the course of chromomycin A(3) (CMA(3)) staining performed sequentially. Major and minor rDNA families were not co-localized in the Danube salmon chromosomes.The distinct hybridization signals at the ends of all the chromosomes were provided in the course of PRINS with (CCCTAA)( n ) primer. The chromosomal localization of rRNA genes and telomeric DNA sequences was discussed in the context of Salmonidae karyotype evolution.     

Feeding preferences of the weevils Sitona gressorius and Sitona griseus on different lupin genotypes and the role of alkaloids

The weevils Sitona gressorius and Sitona griseus are specialist herbivores on lupins in Europe. The adult weevils feed on the leaves, and the larvae on the root nodules of the plants. This causes severe damage to lupin crops. In the present study, the feeding preferences of lupin weevil adults on different lupin genotypes were examined with respect to a possible effect of lupin alkaloids on host selection. A total of 12 genotypes from the species Lupinus albus, L. angustifolius, L. luteus, and L. nanus were grown in a field experiment and the feeding damage on the leaves caused by naturally occurring lupin weevil adults was estimated. Additionally, a feeding choice test with S. gressorius adults was performed to examine feeding preferences under laboratory conditions. A gas chromatographic analysis provided information on the alkaloid content and profiles in the leaves of the tested lupin genotypes. In the field experiment, significant differences in the extent of the feeding damage within the 12 lupin genotypes were observed. The dual-choice feeding bioassay did not show discrimination of lupin species, but two L. angustifolius genotypes were significantly less affected than the standard L. luteus “Bornal”. The alkaloid analysis revealed large contrasts in alkaloid concentrations and profiles in the leaves of the tested genotypes. Correlation analysis with the results from the field and laboratory did not indicate a significant influence of the total foliar alkaloid content on the extent of weevil feeding.     

<Emphasis Type="Italic">In vitro</Emphasis> micropropagation of four lupin species

The complete protocols for long-term micropropagation of some cultivars of four lupin species: Lupinus luteus, L. albus, L. angustifolius and L. mutabilis were elaborated. The shoots were regenerated in vitro via induction of axillary buds development. Plantlets were multiplicated on lowered salts MS-derived media containing BAP in diverse and generally low concentrations. Significant differences in regeneration capacity between species and cultivars were observed. The highest multiplication ratio revealed L. mutabilis and L. luteus. Regenerated shoots were rooted in vitro on low-salts MS-derived media with B5 vitamins. Media were supplemented with different auxins that affected roots formation of particular species and cultivars. Rooting ability of regenerated shoots decreased rapidly through in vitro culture. For that reason, grafting was applied as an alternative method of transfer of shoots to in vivo conditions. This method turned out to be successful for the majority of studied species and cultivars. Complete rooted or grafted plantlets were cultivated in pots with perlit in greenhouse. An erratum to this article is available at .