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

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  Development of a sequence-specific PCR marker linked to the  Ku  gene which removesthe vernalization requirement in narrow-leafed lupin J. G. Boersma 1,2,3 B. J. Buirchell 1 ,  K. Sivasithamparam 2 and  H. Yang 11 Department of Agriculture and Food, Western Australia, 3 Baron-Hay Court, South Perth, WA 6151, Australia; 2 The University of Western Australia, 35 Stirling Highway, Nedlands, WA 6009, Australia  3 Corresponding author, E-mail: With 2 figures and 2 tablesReceived June 30, 2006/Accepted November 1, 2006Communicated by W. E. Weber Abstract Wild types of   Lupinus angustifolius  require vernalization to promoteflowering. Modern domesticated cultivars carry the early-floweringgene  Ku  which removes this requirement. A microsatellite-anchoredfragment length polymorphism marker was identified as co-segregatingwith the  Ku  gene in a recombinant inbred line (RIL) populationderived from a domesticated  ·  wild-type cross. DNA sequencingshowed that the marker contained a 7 bp insertion/deletion poly-morphism, as well as a single nucleotide polymorphism. A pair of sequence-specific primers was designed and successfully converted thesize polymorphism into a simple polymerase chain reaction basedco-dominant marker. This marker is closely linked to the  Ku  gene, as itco-segregates with the  Ku  phenotyping in a population consisting of 106 RILs. Key words:  Lupinus angustifolius  — early flowering — simplesequence repeat–PCR markerOne of the climatic requirements of many wild narrow-leafedlupin ( Lupinus angustifolius  L.) including those srcinating inthe Mediterranean region is a period of vernalization topromote flowering (Gladstones 1970). Previous genetic studies(Gladstones and Hill 1969) have shown that a single dominantgene  Ku , srcinally selected from a natural mutant of theSwedish cultivar   Borre  , had a major effect on the initiation of flowering by removal of the need for vernalization andadvanced flowering by 2–5 weeks. This gene greatly increasesthe flexibility of lupins as a crop in that it not only enablesthem to be grown in warmer areas with insufficient cold tosatisfy the vernalization requirements, but also allows them tobe sown in areas with a short growing season as experienced inlarge areas of the Western Australian wheat-belt (Gladstonesand Hill 1969). Most modern cultivars of   L. angustifolius  bredin Western Australia and Europe since 1971 carry this gene(Cowling 1999).As a crop,  L. angustifolius  has a short history of only80 years. Because the gene pool in domesticated cultivars isvery narrow, the inclusion of wild germplasm into the lupinbreeding programme will give greater diversity and scope forincreased genetic gains by the inclusion of further desirabletraits. However, when a wild type is crossed with a domesti-cated line, it involves the risk of losing desirable domesticationgenes if they are not actively selected for. Field-based selectionfor the  Ku  gene is time-consuming because a large number of progeny has to be maintained until flowering prior to selection.Furthermore, it is impossible to directly select plants with thehomozygous early-flowering genotype ( KuKu ) from heterozy-gous early-flowering individuals ( Kuku ) as both genotypes willexhibit the early-flowering phenotype in the field. The devel-opment of a molecular marker linked to the  Ku  gene wouldgreatly facilitate the selection of this important domesticationgene in lupin breeding programmes by enabling selection at theF 2  stage and concomitant 75% reduction in plant populationsin the same generation.For marker-assisted selection (MAS) to be viable in apractical plant breeding programme, a molecular marker mustpossess the following characteristics (Eagles et al. 2001, Yanget al. 2004). 1  The marker is closely linked to a gene of interest. 2  The operation of the marker must be more cost-effective thanconventional glasshouse or field-based selection. 3  The marker must be amenable to large numbers of samples. The microsatellite-anchored fragment length polymorphism(MFLP) method is capable of producing DNA polymorphismswith high efficiency (Yang et al. 2001). Recently, MFLPmarkers were used to construct a molecular map of   L. angust-ifolius  (Boersma et al. 2005). Several domestication genes werealsomapped. However,MFLPmarkerscannotbeuseddirectlyforMASbecausetheyaretootime-consumingandexpensivetoimplement. The aim of this research is to develop a sequence-specific, simple polymerase chain reaction (PCR)-based markerlinked to the  Ku  gene desirable for MAS in lupin breeding. Materials and Methods Plant materials and phenotyping of   Ku  gene:  An F 8  recombinantinbred line (RIL) of a domestic  ·  wild type (D  ·  W) population of  L. angustifolius  was previously developed by the Department of Agriculture and Food, Western Australia using as parents lines P27255(wild) and 83A : 476 (domesticated). The resulting population fromthis cross has been used to produce linkage maps for  L. angustifolius (Boersma et al. 2005, Nelson et al. 2006).The parents and 106 RILs of this population were sown in thescreen-house at the beginning of winter as randomized plots eachconsisting of three plants. There were three replicates. Anthesis wasdetermined as the first floret on the main stem of any one plant havingopened in a particular plot (Gladstones and Hill 1969, Rahman andGladstones 1972). The mean date for the three replicate plots was thenused to place the RILs and the parent lines into groups designated aseither   kuku   (late flowering) or   KuKu   (early flowering). www.blackwell-synergy.comPlant Breeding  126 , 306—309 (2007) doi:10.1111/j.1439-0523.2007.01347.xJournal compilation    2007 Blackwell Verlag, BerlinNo claim to srcinal US government works  Marker development: The two parents and 89 RILs were subjected toMFLP tests involving 10 simple sequence repeat (SSR)-anchor primerseach in combination with 16  Mse I-primers (Boersma et al. 2005).MFLP markers having the best correlation to the  Ku  phenotyping datawere selected. DNA fragments of candidate MFLP markers wereisolated from MFLP gels, re-amplified by PCR, ligated into plasmids  pGEM-T Easy Vector   (Promega, Annandale, Australia) and clonedinto  Escherichia coli   according to the manufacturer’s instructions.Plasmid DNA with MFLP fragment inserts were isolated from  E. coli  ,and sequenced using the BigDye Terminator system (Applied Biosys-tems, Melbourne Australia) as described by Yang et al. (2002, 2004).A pair of sequence-specific primers was designed flanking the insertion/deletion site (Yang et al. 2002, 2004, You et al. 2005). Primers weredesigned so that the annealing temperature was approximately 54  Cbased on the calculations using the nearest-neighbour model ( DNA amplification and sequencing gel electrophoresis:  Marker DNAfragments were amplified in a 10  l l PCR consisting of 1.5  l l of thetemplate DNA and with one of the sequence-specific primers labelledwith  c ) 33 P as previously described by Yang et al. (2001, 2002). Theselective PCR products were separated on a 5% polyacrylamidedenaturing sequencing gel (7  M  urea) using a 38  ·  50 cm (0.4 mm inthickness) Sequi-Gen GT sequencing cell (Bio-Rad, Hercules, CA,USA). After electrophoresis at 55 W for about 3 h, the gel was driedunder vacuum on a gel drier (Model 583, Bio-Rad). Marker bandswere detected by autoradiography (Yang et al. 2002, You et al. 2005)with overnight exposure of the X-ray film on the dried gel.The converted sequence-specific marker was tested on 106 F 8  RILsof the population derived from the D  ·  W cross. The marker scoredata and the  Ku  phenotyping data on the 106 F 8  RILs were mergedand analysed by the program MapManager (Manly et al. 2001) todetermine the genetic linkage between the marker and the  Ku  gene. Results Phenotyping of   Ku  gene In 2005, the domesticated parent (83A : 476) being earlyflowering ( Ku ), reached anthesis 72 days after sowing. Thewild parent (P27255;  ku ) reached anthesis 97 days aftersowing. Field-testing of 106 RILs of the marker populationshowed that anthesis dates could be clustered into a number of smaller groups contiguous with the parental values (Table 1).There was a 5-day interval between these two groups duringwhich no RILs initiated flowering. Anthesis dates ranged from69 to 84 days after sowing ( KuKu ) and 90 to 110 days aftersowing ( kuku ) with the majority of RILs flowering within1 week of either parent. Combining these groups resulted in 53RILs being designated as   early flowering   ( Ku ) and another 53RILs being designated as   late flowering   ( ku ) based onthe method described by Rahman and Gladstones (1972).The segregation of plants with the  KuKu  allele to plants withthe  kuku  allele fits perfectly with the expected 1 : 1 ratio for theF 8  progeny. DNA sequencing of the candidate MFLP marker A dominant MFLP marker designated   DAWA428.290  ,srcinally produced during a mapping study (Boersma et al.2005), co-segregated with the  Ku  phenotyping scores of theRILs tested. This marker was selected as a candidate fordevelopment of a sequence-specific marker tagging the  Ku gene. Marker DAWA428.290 was generated by SSR-anchorprimer MF42 (5 ¢ -GTCTAACAACAACAACAAC-3 ¢ ) in com-bination with primer  Mse I-CAC (5 ¢ -GATGAGTCCTGA-GTAACAC-3 ¢ ).The DNA sequencing showed that the dominant markerDAWA428.290 was a 318 bp fragment with sequences of SSR-anchor MF42 and  Mse I-CAC at either end as expected(Table 2). A sequence-specific primer   KuHMS1   (3 ¢ -AGA-CATACCT TGTATGCGG-5 ¢ ) was designed to replace pri-mer  Mse I-CAC. PCR amplification of this marker by primerKuHMS1 in combination with the SSR-anchor primer MF42and using as template, the DNA from the two parents and 10randomly selected RILs revealed a pair of co-dominant bands.Apart from the band produced by the 298 bp fragment of plants with the  KuKu  allele, plants with the  kuku  alleleproduced a longer DNA band fragment that migrated moreslowly (Fig. 1). DNA fragments from the  ku  allele weresequenced and aligned with the sequence from the  Ku  allele(Table 2). It was revealed that the  ku  allele fragmentscontained a 7 bp insertion, as well as a single nucleotidepolymorphism of an adenosine substituting the guanine in the Ku  allele (Table 2). Marker   KuHM1  Because the insertion/deletion site is within the SSR- Mse Ifragment, a second sequence-specific primer, KuHSR1(3 ¢ -CAAAAACAATAATAACGACAAC-5 ¢ ) was designed,flanking the mutation site on the side opposite to KuHMS1.The pair of primers successfully converted the MFLP markerinto a co-dominant, sequence-specific, simple PCR-basedmarker. We designate this marker as   KuHM1  . MarkerKuHM1 shows a 280 bp band for the  Ku  allele, and a287 bp band for the  ku  allele (Fig. 2).Marker KuHM1 was tested on the parents and 106 RILsof the D  ·  W population. The score of marker and plantphenotypes showed perfect correlation on all the RILs. All53 RILs with the  Ku  allele developed the shorter band(280 bp), and the 53 RILs carrying the  ku  allele developedthe longer band (287 bp). Linkage analysis by computerprogram MapManager suggested that the genetic distancebetween the marker and the gene is <0.5 cM if we assumeone crossover in that interval in a population of 107 ormore. Table 1: Phenotyping of the early-flowering gene  Ku  on an F 8  derived RIL population from a cross 83A : 476 ( KuKu )  ·  P27255 ( kuku ) in Lupinus angustifolius  LFlowering date (DAS) 65–69 70–74 1 75–79 80–84 85–89 90–94 95–99 2 100–104 105–110Numbers of RILS 1 7 33 12 0 14 24 12 3Total 53  KuKu  53  kuku 1 The domesticated parent 83A : 476 flowered on day 72. 2 The wild parent P27255 flowered on day 97. There is a discontinuity between days 84 and 90 showing the boundary between the two majorgroups based on the  Ku  gene.DAS, days after sowing; RIL, recombinant inbred line.Vernalization requirement in narrow-leafed lupin 307  Discussion Anthesis dates for this marker population were clusteredaround the parental values, but showed signs of segregationthat could be indicative of two or more genes being involved.Rahman and Gladstones (1972) found that flowering beha-viour of non- Ku L. angustifolius  cultivars was dominated bytheir high vernalization requirement and, that anthesis wasalso accelerated by increased photoperiod. Higher growingtemperatures, although a factor in several species did notappear to significantly alter the time to anthesis in  L. angust-ifolius  (Rahman and Gladstones 1972). It is therefore possiblethat in this population, photoperiod is also a factor influencinganthesis. Nevertheless, as vernalization has such a major effect(Gladstones and Hill 1969, Rahman and Gladstones 1972) wecould separate the lines into two distinct groups, classifyingRILs as either  KuKu  or  kuku  with confidence.DAWA428.290 was srcinally a dominant marker in MFLPfingerprinting. However, after sequencing we were able todirectly convert this MFLP candidate marker into co-domin-ant marker KuHM1. Because the two alleles gave excellentDNA amplification when SSR-anchor primer MF42 is used incombination with primer KuHMS1 (Fig. 1), it suggests thatthere is no difference in DNA sequences between the twoalleles at the annealing site for primer SSR-anchor primerMF42. It is possible that a mutation exists on the cutting sitefor restriction enzyme  Mse I on the  ku  allele which preventedligation of the  Mse I-adaptor (Yang et al. 2001), causing theMFLP to develop only a dominant band for the  Ku  allele.Marker KuHM1 reported in this paper meets the require-ments for a sequence-specific, size-based simple PCR markerof low-cost, making it potentially suitable for routine MAS inlupin breeding (You et al. 2005). The marker bands can also be Table 2: DNA sequence of the MFLP marker DAWA428.290 showing primers giving rise to sequence-specific markers KuHM1 and KuHSNP.The underlined sequences correspond to the sequences/annealing sites of the following:The 7 bp insertion/deletion mutation site, and the SNP mutation site (G/A) are shown in bold italic. 1 Primer  Mse I-CAC (5 ¢ -GATGAGTCCTGAGTAACAC-3 ¢ ). 2 Primer KuHMS1 (3 ¢ -AGACATACCTTGTATGCGG-5 ¢ ). 3 Primer KuHMS2 (3 ¢ -GAACAGTATTCATTATTCC-5 ¢ ). 4 Annealing site of primer KuHSR1 (3 ¢ -CAAAAACAATAATAACGACAAC-5 ¢ ). 5 Annealing site of SSR-anchor primer MF42 (5 ¢ -GTCTAACAACAACAACAAC-3 ¢ ).G/A, guanine/adenosine; MFLP, microsatellite-anchored fragment length polymorphism; SNP, single nucleotide polymorphism.Fig. 1: Polymerase chain reaction amplification products of 12recombinant inbred line plants of   Lupinus angustifolius  withsequence-specific primer KuHMS1 (3 ¢ -AGACATACCT TGTAT-GCGG-5 ¢ , labelled with  c ) 33 P) in combination with simple sequencerepeat-anchor primer MF42 (5 ¢ -GTCTAACAACAACAACAAC-3 ¢ ).Five plants having  KuKu  allele (298 bp band) are the domesticatedparent 83A : 476 (lane 1), A13 (lane 5), A14 (lane 6), A15 (lane 7), A17(lane 9). The seven other plants including the wild parent P27253 (lane2), A10 (lane 3), A12 (lane 4), A16 (lane 8), A18 (lane 10), A19 (lane11) and A21 (lane 12) carry the  kuku  allele (305 bp band)Fig. 2: Screening molecular marker KuHM1 on the parents and 28 F 8  derived recombinant inbred lines (RILs) of   Lupinus angustifolius  usingpolymerase chain reaction with sequence-specific primer pair KuHMS1 (3 ¢ -AGACATACCTTGTATGCGG-5 ¢ ) and KuHSR1 (3 ¢ -CA-AAAACAATAATAACGACAAC-5 ¢ ). The parent plant 83A : 476 (lane 1) and 15 RILs: A64 (lane 6), A65 (lane 7), A69 (lane 10), A70 (lane 11),A73 (lane 13), A74 (lane 14), A75 (lane 15), A76 (lane 16), A77 (lane 17), A78 (lane 18), A83 (lane 23), A84 (lane 24), A86 (lane 26), A87 (lane 27)and A93 (lane 30) carrying the  KuKu  allele produced a 280 bp band. The wild-type parent P27253 (lane 2) and 13 RILs: A61 (lane 3), A62 (lane4), A63 (lane 5), A66 (lane 8), A67 (lane 9), A71 (lane 12), A79 (lane 19), A80 (lane 20), A81 (lane 21), A82 (lane 22), A85 (lane 25), A88 (lane 28)and A92 (lane 29) carrying the  kuku  allele produced a 287 bp band 308 Boersma, Buirchell, Sivasithamparam  and  Yang  amplified by PCR, using the one sequence-specific primerKuHMS1 with the SSR-anchor primer MF42. However,replacement of the SSR-anchor primer with the specific primerKuHSR1 increases the specificity and makes the PCR ampli-fication more robust giving a significant reduction in back-ground signal (Fig. 2).The tight linkage of marker KuHM1 (derived fromDAWA428.290) to the  Ku  gene is indicated by the perfectcorrelation between plant phenotype and the marker score onthe available RIL population, giving a distance estimate of <0.5 cM from the gene. However, there were only 106 RILsavailable in this instance. A more precise estimation of thegenetic distance between the marker and the gene wouldrequire further testing on a larger population. The usefulnessof this marker for practical lupin breeding needs to be furtherevaluated by testing on a wide range of wild accessions,breeder’s lines and cultivars.The co-dominant resolution of sequence-specific markerKuHM1 makes it amenable to a simple PCR-based assay forthe presence of the  Ku  gene in lupin, able to distinguishhomozygous lines from those heterozygous for the two alleles.This feature should make it particularly useful for MAS in ahigh-throughput situation (Kumar 1999). Furthermore, thismarker could be used to probe a Bacterial Artifical Chromo-some (BAC) library of this species to walk the chromosome tothe actual gene. Successfully locating and sequencing the  Ku gene will enable development of a   perfect   marker that targetsthe active gene sequence (e.g. Bradbury et al. 2005). Acknowledgement The help of Mr D. Renshaw and Mr C. Smith (Department of Agriculture and Food, Western Australia) in data collection isgratefully acknowledged. References Boersma, J. G., M. Pallotta, C. Li, B. J. Buirchell, K. Sivasithampa-ram, and H. Yang, 2005: Construction of a genetic linkage mapusing MFLP and identification of molecular markers linked todomestication genes in narrow-leafed lupin ( Lupinus angustifolius L.). Cell. Mol. Biol. Lett.  10,  331—344.Bradbury, L. M. T., R. J. Henry, Q. Jin, R. J. Renke, and D. L. E.Waters, 2005: A perfect marker for fragrance genotyping in rice.Mol. Breed.  16,  279—283.Cowling, W. A., 1999: Pedigrees and characteristics of narrow-leafedlupin cultivars released in Australia from 1967–1998. AgricultureWestern Australia Bulletin 4365.Eagles, H. A., H. S. Bariana, F. C. Ogbonnaya, G. J. Rebetzke, G. J.Hollamby, R. J. Henry, P. H. Henschke, and M. Carter, 2001:Implementation of markers in Australian wheat breeding. Aust.J. Agric. Res.  52,  1349—1356.Gladstones, J. S., 1970: Lupins as crop plants. Field Crops Abstr.  23, 123—148.Gladstones, J. S., and G. D. Hill, 1969: Selection for economiccharacters in  Lupinus angustifolius  and  L. digitatus . 2. Time of flowering. Aust. J. Exp. Agric. Anim. Husb.  9,  213—220.Manly, K. F., R. H. Cudmore Jr, and J. M. Meer, 2001: MapManagerQTX, cross-platform software for genetic mapping. Mamm. Gen. 12,  930—932.Nelson, M. N., H. T. T. Phan, S. R. Ellwood, P. M. Moolhuijzen,M. Bellgard, J. Hane, A. Williams, C. E. O’Lone, J. Fosu-Nyarko,M. Scobie, M. Cakir, M. G. K. Jones,M. Bellgard, M. Ksi a˛  _ zkiewicz,B. Wolko, S. J. Barker, R. P. Oliver, and W. A. Cowling, 2006: Thefirst gene-based map of   Lupinus angustifolius  L. – location of domestication genes and conserved synteny with  Medicago trunca-tula . Theor. Appl. Genet.  113,  225—238.Pusch, W., J. H. Wurmbach, H. Thiele, and M. Kostrzewa, 2002:MALDI-TOF mass spectrometry-based SNP genotyping. Pharmac-ogenomics  3,  537—548.Rahman, M. S., and J. S. Gladstones, 1972: Control of lupin flowerinitiation by vernalization, photoperiod and temperature undercontrolled environment. Aust. J. Exp. Agric. Anim. Husb.  12, 638—645.Yang, H., M. W. Sweetingham, W. A. Cowling, and P. M. C. Smith,2001: DNA fingerprinting based on micro-satellite anchored frag-ment length polymorphisms, and isolation of sequence-specific PCRmarkers in lupin ( Lupinus angustifolius  L.). Mol. Breed.  7,  203—209.Yang, H., M. Shankar, B. J. Buirchell, M. W. Sweetingham,C. Caminero, and P. M. C. Smith, 2002: Development of molecularmarkers using MFLP linked to a gene conferring resistance to Diaporthe toxica  in narrow-leafed lupin ( Lupinus angustifolius  L.).Theor. Appl. Genet.  105,  265—270.Yang, H., J. G. Boersma, M. You, B. J. Buirchell, and M. W.Sweetingham, 2004: Development and implementation of asequence-specific PCR marker linked to a gene conferring resistanceto anthracnose disease in narrow-leafed lupin ( Lupinus angustifolius L.). Mol. Breed.  14,  145—151.You, M., J. G. Boersma, B. J. Buirchell, M. W. Sweetingham, K. H.M. Siddique, and H. Yang, 2005: A PCR-based molecular markerapplicable for marker-assisted selection for anthracnose diseaseresistance in lupin breeding. Cell. Mol. Biol. Lett.  10,  123—134.Vernalization requirement in narrow-leafed lupin 309
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