CONSTRUCTION OF A GENETIC LINKAGE MAP USING MFLP AND ...

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alkaloid), Ku (early flowering) and moustache pattern on seed coats; were included. Three to 7 molecular markers were identified within 5cM of each of.
CELLULAR & MOLECULAR BIOLOGY LETTERS

Volume 10, (2005) pp 331 – 344 http://www.cmbl.org.pl Received 2 February 2005 Accepted 27 April 2005

CONSTRUCTION OF A GENETIC LINKAGE MAP USING MFLP AND IDENTIFICATION OF MOLECULAR MARKERS LINKED TO DOMESTICATION GENES IN NARROW-LEAFED LUPIN (Lupinus angustifolius L.) JEFFREY G. BOERSMA1,2*, MARGARET PALLOTTA3, CHENGDAO LI1, BEVAN J. BUIRCHELL1, KRISHNAPILLAI SIVASITHAMPARAM2 and HUAAN YANG1 1 Department of Agriculture, Western Australia, 3 Baron-Hay Court, South Perth, W.A. 6151, Australia, 2School of Earth and Geographical Sciences, The University of Western Australia, Nedlands, W.A. 6009, Australia, 3 Australian Centre for Plant Functional Genomics, Hartley Grove, Waite Campus, Urrbrae, S.A. 5064, Australia

Abstract: A mapping population of F8 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 5cM of each of these domestication genes. The anthracnose resistance gene Lanr1 was also mapped. Linkage groups were constructed using MapManager version QTXb20, resulting in 21linkage groups consisting of 7 or more markers. The total map length was 1543cM, with an average distance of 3.4cM 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. * Corresponding author, e-mail: [email protected] Abbreviations used: AFLP - amplified fragment length polymorphisms; cM centiMorgan; LG - linkage group; MAS - marker assisted selection; MFLP microsatellite-anchored fragment length polymorphism; PCR - polymerase chain reaction; RAPD - random amplified polymorphic DNA; RILs - recombinant inbred lines; SSR - simple sequence repeat.

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Key Words: Lupinus angustifolius, Linkage Map, MFLP, Molecular Marker INTRODUCTION The narrow-leafed lupin (Lupinus angustifolius L.) was grown as a crop in classical Greek and Roman times [1], being used for both stock feed and human consumption. Plant breeding in L. angustifolius started in Germany early last century, followed by extensive work in Australia in the 1950’s [2]. Key domestication genes incorporated into modern cultivars of L. angustifolius include the low-alkaloid gene iucundis (iuc), seed coat permeability gene mollis (moll), white flower gene leucospermum (leuc), nil vernalisation requirement gene Ku and, reduced pod shattering genes tardus (ta) and lentus (le) [1, 3, 4]. Domesticated cultivars of L. angustifolius are currently grown in Europe, Africa and North America [2, 4], but Australia remains the world's largest producer of this species with approximately 1.5 million hectares planted annually in Western Australia alone [5]. The objectives of modern lupin breeding programs include improved yield, higher protein content, environmental adaptation and disease and pest resistance, particularly resistance to anthracnose which is the most devastating known disease of lupins [6]. Recently two molecular markers linked to the anthracnose resistance gene Lanr1, have been developed and used in marker-assisted selection for anthracnose resistance in breeding of lupins in Australia [7, 8]. L. angustifolius is a diploid species containing (2n) 40 chromosomes [1]. Molecular genetic mapping in L. angustifolius was first attempted in Poland [9, 10], with 60 RAPD markers and 4 isozyme markers mapped into 17 groups. Brien et al. [5] generated 705 polymorphic AFLP markers, which mapped into 52 linkage groups covering approximately 1000cM. To date, no comprehensive genetic map of L. angustifolius has been published. In recent years, the AFLP technique [11] has been widely used in plant genetic mapping because of its high efficiency in generating large numbers of molecular markers [12-14]. The use of simple sequence repeat (SSR) markers also known as microsatellite markers, is increasing in popularity because they occur at high frequencies in plant genomes [15], and exhibit high mutation rates [16]. The MFLP technique was derived from a combination of the AFLP concept with SSR-anchor primers [17] and is capable of producing DNA markers with high efficiency, with each of the detected DNA polymorphisms including an SSR motif [7, 17, 18]. The objectives of this study are (1) to employ the MFLP technique to generate large numbers of DNA markers for construction of a genetic map of Lupinus angustifolius and (2) to identify molecular markers linked to the domestication genes and anthracnose resistance gene Lanr1. MATERIALS AND METHODS Plant material An F8 RIL mapping population (DxW) was developed from a cross between a domesticated line 83A:476 (maternal) and a wild type P27255 from Morocco

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(paternal) by single seed descent. This DxW population segregates for a number of traits and domestication genes including: permeable (soft) seed coat gene mollis [19]; non-shattering genes lentus and tardus [20]; sweetness (low alkaloid) gene iucundis [21]; early flowering gene Ku [22] and, white flower/seed colour gene leucospermus [23]. In addition, it is known that the domesticated parent carries the gene for resistance to anthracnose, Lanr1 [7]. A total of 89 sibs were used in this study. Phenotyping Mollis (moll). This gene is recessive (moll) and allows rapid imbibition and germination of viable seeds when soaked in water [19, 24]. Leucospermus (leuc). This gene controls flower colour, which is either blue (Leuc) or white (leuc). Both flower colour and the production of a white seed coat have previously been found to be associated with this gene [1, 23]. Lentus (le ). This gene is one of two complementary reduced pod-shatter genes Lentus and Tardus [20]. It reduces but does not eliminate pod shattering after ripening. When a plant possesses both reduced shattering genes (genotype lele tata), pod shatter is eliminated. Iucundis (iuc). This gene controls alkaloid levels. Plants with the Iuc allele are bitter, and plants with the iuciuc genotype are sweet [25]. Ku. This gene controls the need for vernalisation. Plants with the Ku genotype do not require vernalisation, resulting in their flowering 2 - 5 weeks earlier under Western Australian (W.A.) conditions than those not possessing this allele [22]. Lanr1 (Anthracnose disease resistance gene). Seedlings were vernalised for three weeks at 4°C (to encourage uniform flower initiation) before being planted in 25cm diameter pots filled with a mixture of river sand and potting mix. Five single plants of different RILs were selected at random and planted per pot. All treatments were replicated 4 times. Plants were inoculated with conidial spore suspension of the strain “VCG-2” of Colletotrichum lupini [6] (previously classified as Colletotrichum gloeosporioides) [26]. Details of disease inoculation and scoring were the same as in the other studies [7]. Moustache (mou) patterning on seed coat. The moustache pattern, also known as eyebrow or arrowhead (C. Smith, pers. comm.) is of no known agronomic value but has been used by Australian plant breeders in lupin seed descriptions. DNA extraction Leaflets from 10 to 15 plants were used for each RIL. The two F7 lines used were each tested as a single plant. Total genomic DNA was extracted from 0.5g of fresh leaf material for each RIL using the CTAB extraction method as described by Rogers and Bendich [27]. Extracted DNA was re-suspended in TE 0.1 buffer (10 mM TrisHCl, 0.1 mM EDTA). MFLP protocol DNA from each RIL line and the two parents was digested by the restriction enzyme Tru9I (Roche Diagnostics, Australia), an isoschizomer of MseI. The

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MseI-adaptor [11] was ligated onto the restriction fragments using T4 DNAligase (Roche Diagnostics Australia). To reduce and optimise the number of DNA bands on the sequencing gel, the template DNA was further digested with a frequent cutter HpaII (recognition site 5’-C/CGG-3’, GeneWorks Pty Ltd, Australia). Detailed methodology for MFLPs have been described elsewhere [17]. Pre-selective MFLP reactions were set up using 10 SSR-anchor primers (Tab. 1), each in combination with an MseI-primer with one selective nucleotide C at the 3’ end (5’-GAT GAGTCCTGAGTAAC-3’) [11]. A total of 153 selective-MFLP reactions were run using the 10 SSR-anchor primers in combination with 16 MseI primers having two additional selective nucleotides (MseI-Cxx, [11]). The SSR-anchor primers of the selective-MFLP reactions were labelled with γ-[33P] ([17, 18]). The PCR amplification products of the MFLP were resolved on 5% denaturing sequencing gels and, DNA polymorphisms were detected by autoradiography [7, 17, 18]. A 100 bp DNA ladder (100 bp to 1000 bp in 100 bp increments; catalogue number "DMW-100L", GeneWorks Pty Ltd, Australia) was run with the samples on each gel. Tab. 1. DNA sequences of SSR-anchor primers used in MFLP

Primer Name MF128 MF129 MF51 MF42 MF43 MF62 MF52 MF11 MF151 MF152 a

Sequences (5’ - 3’) DVDTCTCTCTCTCTCTCa HVHTGTGTGTGTGTGTGb GGGAACAACAACAAC GTCTAACAACAACAACAAC CCTCAAGAAGAAGAAGAAG CCCAAACAACAACAAC GGGAAGAAGAAGAAG GGACCTCTCTCTCTCT CACGTCTCTCTCTCTCT GATGCTCTCTCTCTCTC

D=A+G+T, V=A+G+C; b H=A+C+T

Data analysis and map construction The majority of MFLP markers were scored as dominant and the remainder as co-dominant markers. Ambiguous genotypes were treated as missing data. Marker scoring was checked at least twice. Added to the data were phenotype scores given each RIL for the traits and anthracnose resistance gene described above under 'plant material'. Linkage group construction was accomplished with aid of the computer program MapManager, version QTXb20 [28]. Linkage groups were determined to consist of markers linked with a minimum LOD score of 2.5. The order of the markers in each linkage group was initially determined by MapManager's 'find linkage groups' tool (Linkage evaluation: Self RI; Search Link criterion P = 0.001; Map Function: Kosambi) followed by a critical visual evaluation of all data. The map was drawn using the computer program MapChart [29].

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Genome length estimates The recombination length of the L. angustifolius genome was estimated using two methods, Hulbert et al. [30]'s method-of-moment estimator and Chakravarti et al. [31]’s modification of this method which corrects for upward bias related to chromosome ends. The confidence intervals (p = 0.05) for the latter method were calculated as per Gerber & Rodolphe [32]. RESULTS Phenotyping Phenotyping of parental lines confirmed that the domesticated parent (83A:476) bore all the domestication gene alleles (moll, leuc, iuc, le, and Ku), whereas the wild parent (P27255) carried the wild type alleles (Moll, Leuc, Iuc, Le and ku). Parent 83A:476 is resistant to anthracnose, whereas P27255 is susceptible. Testing of all RILs for the domestication traits and for anthracnose resistance found most of the phenotypes segregated in a 1:1 ratio ( Tab. 2). These results support earlier findings that those traits are controlled by single major genes [19, 20, 22, 23]. Exceptions were the phenotypes for low alkaloids (iuc) and anthracnose resistance (lanr1), both of which have previously also been shown to be controlled by single genes [7, 21]. Tab. 2. Chi-square test on the segregation ratios of domestication traits and anthracnose disease resistance among the 89 RILs derived from cross 83A:476 x P27255 of Lupinus angustifolius Expected segregation 43.5:43.5

χ2

P

Seed coat permeability

Observed segregation (M:P) a 36:51 (moll:Moll)b, c

2.586

0.108

Flower colour

43:42 (leuc:Leuc)d, c

42.5:42.5

0.012

0.914

44.5:44.5

7.022

0.008**

Traits

e

Plant alkaloid

32:57 (Iuc: iuc)

Early flowering

44:38 (Ku:ku)f, c

41:41

0.439

0.508

Reduced pod shatter 1

45:44 (le:Le) g

44.5:44.5

0.011

0.916

43.5:43.5

0.287

0.592

40:40

4.050

0.044*

Seed Moustache Anthracnose disease

41:46 (Mou :mou) 31:49 (R: S)j,c

h,i,c

a

M = maternal, P = paternal numbers; bMoll = hard-seeded coat, moll = soft-seeded seed coat; cTotal plant numbers do not equal 89 as a result of either inconclusive phenotyping or early plant mortality;.dLeuc = blue flower colour, leuc = white flower colour; eIuc = bitter, iuc = sweet; fKu = early flowering, ku = late flowering; gLe = shattering, le = reduced shattering; hMou = broad moustache , mou = narrow moustache; iMou - parental dominance not determined; jR = resistant to anthracnose, S = susceptible to anthracnose. * = significant at the 5% level, ** = significant at the 1% level.

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Marker polymorphism and segregation MFLP polymorphisms were screened from 153 primer combinations ( Tab. 3). A total number of 1083 polymorphic markers were scored. Marker fragment lengths ranged from ~50 bp to 800bp, with the majority in the range of 100 - 450 bp. The number of polymorphic markers detected for individual primer pairs ranged from nil to 16 with the average number being 6.7 per primer combination. There were 522 unique marker loci. It was calculated that 21% of markers were co-dominant and 79% dominant. Tab. 3. Primer combinations in MFLP giving rise to DNA polymorphic markers for genetic linkage mapping in Lupinus angustifolius1. SSRMseI-primers anchor -CAA -CAG -CAC -CAT -CGA -CGG -CGC -CGT -CCA -CCG -CCC -CCT -CTA -CTG -CTC -CTT primers

MF128 142

64

75

87

MF129 178 188 191 198 204 211

25

32

39

55

nil

216 221 227 234 240 246 254 262 269

96

106 115 127 133 144 155 164

MF51

281 289 299 312 320 324 325 347 351 355 359 367 376 385 396 410

MF42

413 421 425 436 443 444 451 461 464 474 478 493 503 507 512 520

MF43

527 532 546 558 564 566 571 575 579 593 599 601 603 613 620 632

MF62

641 644 649 657 661 670 678 686 692 701 709 723 733 738 742 747

MF52

748 758 770 776 787 789 792 796 798 808 815 823 829 837 844 853

MF11

869 873 885 895 902 912 920 928 935 945 951 961 973 976 988 995

MF151 1002 nil 1008 1012 1018 1021 1025 1027 1032 1038 1040 1047 1056 1058 1061 1068 MF152 1072 1085 1089 1091 nil

nil 1094 nil 1095

1

The prefix WADA applies to all markers listed in the table; 2Numbers within each cell denote the first scored polymorphic marker for that particular primer combination; for example, primer combination of MF128 with MseI-CAA gave rise to 11 markers from “WADA14” to “WADA24”.

Chi-squared analysis revealed that almost 91% of the mapped loci segregated in the expected 1:1 ratio, whereas 41 (9.0%) showed distortion at P < 0.05 (13 or 2.9% at P < 0.01). The majority of these markers were clustered in Linkage Groups (LG) 2 (9), LG 9 (13), LG 10 (7) and LG 15 (5) (Fig 1, Tab. 4). Some of the distortion in LG2 was in the region of the gene for anthracnose resistance (Lanr1) and in LG 9 the distortion was in the region of the iucundis gene (iuc). Of the other 7 distorted markers, in at least 2 cases the distortion may be due to missing data (markers (DAWA)1046.125c; 948.215 in LGs 3, 20 respectively). Overall, the ratio of maternal to paternal phenotypes within the distorted sectors was 7:10. Map construction A framework linkage map was constructed for Lupinus angustifolius (Fig 1). Markers and trait phenotypes were placed into 21 linkage groups (LGs)

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consisting of 7 or more markers. Linkage groups have been numbered in order of decreasing marker numbers and length. The map distance covered by the 21 LGs was 1543cM. Linkage distance spanned by the individual LGs ranged from 19.9cM (LG 21) to 137.3cM (LG 1). Linkage groups had a mean length of 73.5 and a standard deviation of 34.5cM. The average distance between adjacent markers was 3.4cM. The longest distance between neighbouring markers (28cM) is located on LG 1. Some clustering of markers was noted in most LGs. There were a further 68 unlinked markers that we were unable to place. Mapping of domestication and the anthracnose resistance genes Except for the reduced pod-shatter gene ta for which the phenotype data is not yet complete, all domestication genes were integrated into the molecular genetic map (Fig. 1, Tab. 4). The low alkaloid gene iuc was found in LG 9. Seven Tab. 4. Characteristics of 21 LGs of L. angustifolius constructed with 454 MFLP markers based on 89 RILs. LG No. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 Total / mean a

LG No. of length markers (cM) 137.3 45 124.7 33 123.9 106.2 95.5 92.4 89.1 88.0 85.5 74.6 73.4 72.7 71.5 87.4 65.8 36.9 33.0 31.7 27.1 26.4 19.9

39 30 26 22 15 19 29 27 24 22 17 20 17 17 14 9 11 11 7

1543

454

Density No. of Morphological traits; (marker gapsa Marker numbersb,c /cM) 3.0 1 3.8 0 Lanr1; AntjM2b1, 224.170, 1019.350c 3.2 1 3.5 0 3.7 0 4.2 0 5.9 1 4.6 1 moll; 561.180, 783.180 2.9 0 iuc; 632.550, 604.335 2.8 0 mou; 479.425, 198.450c 3.1 0 leuc; 520.350, 1045.150 3.0 0 4.2 0 le; 323.15, 964.275 4.4 0 3.9 0 2.2 0 2.4 0 Ku; 428.290, 306.255c 3.5 0 2.5 0 2.4 0 2.8 0 3.4

4

Distortion regions marker intervalb, (totals) 758.470 (1) P d 563.130-824.400 (5) P; 207.325-1019.350c (4) P 972.100c -1046.125c (3)P

305.278-679.430 (14) P 93.090-41.400 (7) P

350.050-742.850 (4)M 229.250, 193.240 (2)P 341.230 (1)M 948.215c (1)M

(42)

A distance of more than 20cM between two adjacent markers is termed a 'gap', b The pre-fix DAWA has been omitted from marker names for ease of reading, b1 The prefix DAWA does not apply here, c Only the two most closely associated markers are indicated for any given trait, d The letter P represents distortion in favour of the paternal parent. M denotes distortion in favour of the maternal parent.

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Fig.1. Genetic linkage map of Lupinus angustifolius constructed from 89 F8 derived recombinant inbred lines. Loci names, on the right side of the bars have a first number denoting the numerical order of markers (Tab. 3) from MFLP gels, and another number after the decimal denoting the size of the fragments in bp. The suffix 'c' on the end denotes that the marker is co-dominant. Genetic distances on the left side of the bar are in cM (Kosambi function). Genes of interest are located in the following Linkage groups: LG 2 - Anthracnose resistance gene Lanr1; LG 8 - Mollis; LG 9 - iucundis; LG 10 - moustache; LG 11 - leucospermus; LG 13 - lentus; LG 17 - Ku. Distorted loci are marked with * ( P < 0.05) or ∗∗ (P < 0.01).

molecular markers flank the iuc gene on either side at distances of less than 5cM, among which the closest two markers flanking the gene are DAWA632.550 (1.1 cM to iuc) and DAWA604.335 (1.2 cM to iuc). The gene for early flowering (Ku) in LG 17 co-segregated with marker DAWA428.290 and is flanked by a further 5 markers within 5cM. The gene for soft-seeds (moll) was located in LG 8. This gene was found to co-segregate with marker DAWA561.180. Two more markers, DAWA783.180 and DAWA664.290 are positioned at 3.8 and 5.6cM from moll respectively. The le gene, one of two reduced shattering genes, was located in LG13 flanked by markers DAWA964.275 (0.6cM) and DAWA323.150 (1.2cM). The gene leuc was located on LG 11. It is flanked on one side by marker DAWA1045.15 at a distance of 0.7cM, and on the other by marker DAWA520.350 at a distance of 1.1cM. The anthracnose resistance gene Lanr1 and marker AntjM2 [8] were both located in LG2 separated by a distance of 2.6cM. The next nearest marker

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to Lanr1 is DAWA224.170 at a distance of 4.1cM and on the far side of Lanr1 to AntjM2. The anthracnose resistance marker AntjM1 [7] was also tried but found to be monomorphic in this population. Estimation of genome size of L. angustifolius According to the method of Hulbert et al. [30], the genome size of L. angustifolius is expected to be in the range 1596cM (LOD 7.0) to 1896cM (LOD 3.0) with an average value of 1746cM. Alternatively, by using the methods of Chakravarti et al. [31] and Gerber & Rodolphe [32], the expected genome length was calculated as being between1407cM (LOD 7.0) and 1673cM (LOD 3.0) with an average length of 1540cM. DISCUSSION We have reported the first comprehensive linkage map for Lupinus angustifolius, with an average distance of 3.4cM between adjacent markers. We found 522 unique marker loci of which 454 (87%) could be connected into 21 linkage groups covering 1543cM. We have identified a weak linkage between LGs 17 and 20 as well as between some of the other linkage groups and residual fragments (data not presented). However, it was considered that these linkages were not sufficiently strong for mapping purposes. Additional markers are needed to strengthen and confirm the linkages to these fragments, enabling their incorporation into the map. Some clustering of markers was observed in most linkage groups. Clustering of markers is not an uncommon occurrence [33-36] and has been suggested to occur frequently although not exclusively in centromeric regions, possibly as a result of reduced recombination in such regions [34]. Clustering in our linkage groups has mostly been consistent with this theory in that several tend to be approximately central. Exceptions including LGs 9, 10, 11 and 12 are probably the result of incomplete linkage groups as suggested by the residual fragments. Distorted sib segregation of markers in RIL populations have been reported by others as being potentially indicative of non-random sib selection or the presence of genes linked to either the maternal or paternal parent [14], or other distorting factors [37]. In our instance the distortions appear to strongly favour the wild parent as shown by the observed segregations in Tab. 2 and indicated in Tab. 4. The reason for this observation is unknown and may be coincidental as the level of distortion at 9.2% (P < 0.05 ; 2.9% at P < 0.01) is low and within the range reported as normal by other workers [e.g. 14]. When compared to the expected genomic map length as calculated using the formulae of Chakravarti et al. [31] and Gerber & Rodolphe [32] and the limits set by Hulbert et al. [30]'s formula, it could be suggested that much of the genome has been mapped. The close proximity of markers mapped and linked to each of the domestication genes at a distance of 2cM or less is indicative of a good coverage of the genome within the mapped regions. This is supported by our data for the observed distance between Lanr1 and marker AntjM2 of 2.6cM

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c.f. the 2.3cM measured by You et al. [8] for 184 RILS of a cross between the cultivars Tanjil (resistant) and Unicrop (susceptible). However, the presence of as yet un-linked fragments and several larger gaps in the LGs, indicates that the map is not yet quite complete. Using MFLPs to construct a linkage map is advantageous as many MFLP polymorphisms can easily be converted into simple PCR based markers desirable for routine marker implementation in MAS [7, 17, 18]. The relatively high proportion of co-dominant markers in MFLP is an advantage when converted into sequence-specific, simple PCR based markers for MAS, as they are able to identify homozygous and heterozygous individuals. We are currently in the process of developing sequence-specific PCR primers for several of the markers associated with the domestication genes we have mapped. With the development of PCR primers for these markers it will be possible to make a much greater use of wild plant accessions without the need for lengthy phenotypic screening procedures to ensure retention of these important domestication traits. The marker AntjM2 for the anthracnose resistance gene Lanr1 is already being used [8]. With further development of this map and the identification of QTLs for yield and other quality traits it is anticipated that the breeding of lupins using MAS will be greatly enhanced, resulting in the release of superior cultivars over a reduced time scale. Acknowledgements. The help of Dr M. You (Department of Agriculture; Western Australia) in carrying out DNA extractions and anthracnose disease screening is gratefully acknowledged. Mr C. Smith (DAWA) is thanked for his advice and contribution in sourcing and sorting references and for scanning the linkage map. REFERENCES 1. Gladstones, J.S. Lupins as crop plants. Fld. Crop Abstr. 23 (1970) 123-148. 2. Gladstones, J.S. Distribution, origin, taxonomy, history and importance. in Lupins as Crop Plants: Biology, Production and Utilization (Gladstones, J.S., Atkins, C. and Hamblin, J. Eds) CAB International, Oxon, U.K., 1998, 1-39. 3. Pate, J.S., Williams, W and Farrington, P. Lupin (Lupinus spp.). in Grain Legume Crops (Summerfield, R.J. and Roberts, E.H. Eds.) Collins, London, 1985, 37-72. 4. Swiecicki, W. and Swiecicki, W.K. Domestication and breeding of narrowleafed lupin (Lupinus angustifolius L.), J. Appl. Genet. 36 (1995) 155-167. 5. Brien, S.J., Cowling, W.A., Potter, R.H., O'Brien, P.A., Jones, R.A.C. and Jones, M.G.K. A molecular marker for early maturity (Ku) and markerassisted breeding of Lupinus angustifolius. in Proc. 11th Aust. Plant Br. Conf. (Adelaide) 2 (1999) 204-205.

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