Diploid/Polyploid Syntenic Shuttle Mapping and Haplotype ... · high polyploidy in the successive steps of map-based cloning approaches, including diploid/polyploid syn-tenic shuttle

Copyright � 2008 by the Genetics Society of AmericaDOI: 10.1534/genetics.108.091355

Diploid/Polyploid Syntenic Shuttle Mapping and Haplotype-SpecificChromosome Walking Toward a Rust Resistance Gene (Bru1) in

Highly Polyploid Sugarcane (2n � 12x � 115)

Loıc Le Cunff,*,1 Olivier Garsmeur,*,1 Louis Marie Raboin,† Jerome Pauquet,*,2

Hugues Telismart,† Athiappan Selvi,*,‡ Laurent Grivet,*,3 Romain Philippe,*Dilara Begum,§,4 Monique Deu,* Laurent Costet,† Rod Wing,§,5

Jean Christophe Glaszmann* and Angelique D’Hont*,6

*CIRAD, UMR DAP, 34398 Montpellier, Cedex 5, France, †CIRAD, UMR PVBMT, Pole de Protection des Plantes,97410 Saint-Pierre, Reunion, France ‡Sugarcane Breeding Institute, Coimbatore 641007, India and §Clemson

University Genomics Institute, Clemson, South Carolina 29634-5727

Manuscript received May 13, 2008Accepted for publication July 10, 2008

ABSTRACT

The genome of modern sugarcane cultivars is highly polyploid (�12x), aneuploid, of interspecific origin,and contains 10 Gb of DNA. Its size and complexity represent a major challenge for the isolation ofagronomically important genes. Here we report on the first attempt to isolate a gene from sugarcane bymap-based cloning, targeting a durable major rust resistance gene (Bru1). We describe the genomicstrategies that we have developed to overcome constraints associated with high polyploidy in the successivesteps of map-based cloning approaches, including diploid/polyploid syntenic shuttle mapping with twomodel diploid species (sorghum and rice) and haplotype-specific chromosome walking. Their applicationsallowed us (i) to develop a high-resolution map including markers at 0.28 and 0.14 cM on both sides and 13markers cosegregating with Bru1 and (ii) to develop a physical map of the target haplotype that still includestwo gaps at this stage due to the discovery of an insertion specific to this haplotype. These approaches willpave the way for the development of future map-based cloning approaches for sugarcane and othercomplex polyploid species.

SUGARCANE (Saccharum spp.) is an important tropi-cal grass crop that accounts for 70% of the raw sugar

produced worldwide. It is able to partition carbon tosucrose in the stem, a vegetative organ, in contrast withother cultivated grasses that usually accumulate theirreserve products within seeds. This unique feature wasselected by man who first used its soft watery culm forchewing. Sugarcane is a C4 photosynthetic plant which,combinedwithitsperennialnature,hasmadeitoneof themost productive cultivated plants. Recently, it has gainedincreased attention because it represents an importantsource of renewable biofuel. However, sugarcane prob-ably has the most complex of all crop genomes studied todate, mainly due to its very high degree of polyploidy

(�12x) and interspecific origin (D’Hont 2005). It thusrepresents a major challenge for genetic studies (Grivet

and Arruda 2002; D’Hont et al. 2008).Modern sugarcane cultivars are derived from the

combination of the polyploid species Saccharum offici-narum, the domesticated sugar-producing species with2n ¼ 8x ¼ 80, and S. spontaneum, a vigorous wild specieswith 2n¼ 5x¼ 40 to 2n¼ 16x¼ 128 and many aneuploidforms (Sreenivasan and Ahloowalia 1987; D’Hont

et al. 1998). Both species are thought to have an auto-polyploid origin (Sreenivasan and Ahloowalia 1987;Grivet et al. 1996). Prompted by disease outbreaks,breeders combined both genomes a century ago. Thehybrids were backcrossed to S. officinarum to recover thethick sugar-containing stalks of this species. This processwas accelerated through the selection of hybrids derivedfrom 2n transmission of S. officinarum chromosomes(Bremer 1961). Modern cultivars are derived fromthese interspecific crosses. They are highly polyploid(�12x) and aneuploid, with �120 chromosomes (re-view by Sreenivasan and Ahloowalia 1987). Molec-ular cytogenetics (D’Hont et al. 1996; Piperidis andD’Hont 2001; Cuadrado et al. 2004) and genetic map-ping studies (Grivet et al. 1996; Hoarau et al. 2001)showed that modern cultivars typically display 70–80%

1These authors contributed equally to this work.2Present address: Biogemma Mondonville, Domaine de Sandreau, 31700

Mondoville, France.3Present address: Syngenta Seeds S.A.S., F-31790 Saint-Sauveur, France.4Present address: Epicentre, Madison, WI 53713.5Present address: AGI, University of Arizona, Plant Sciences Department,

P.O. Tucson, AZ 85721-0036.6Corresponding author: CIRAD, UMR DAP, TA A-96/03 Ave. Agropolis,

34398 Montpellier, Cedex 5, France. E-mail: [email protected]

Genetics 180: 649–660 (September 2008)

of chromosomes entirely derived from S. officinarum,10–20% from S. spontaneum, and a few chromosomesderived from interspecific recombinations (Figure 1;D’Hont 2005).

Brown rust of sugarcane is a fungal disease caused byPuccinia melanocephala and is present in most sugarcanegrowing areas. We previously identified a major gene(Bru1) conferring resistance to brown rust in the mod-ern cultivar R570 (Daugrois et al. 1996). This was thefirst well-characterized Mendelian trait described in thecomplex genomic context of sugarcane. This source ofresistance is of particular interest since it is durable.Indeed, Bru1 resistance breakdown has never been ob-served despite intensive cultivation of R570 for .20 yearsin various regions of the world. Moreover, tests undercontrolled conditions demonstrated that this gene pro-vides resistance against diverse rust isolates collected inAfrica and America (Asnaghi et al. 2001). This gene iscurrently the focus of a map-based cloning approach.

Map-based cloning is becoming increasingly efficientin model crops such as rice (Sun et al. 2004; Ueda et al.2005; Xu et al. 2006) thanks to their simple diploidstructure, their small genome size, and tremendous mo-lecular resources. However, it is still a major challenge in

more complex crops with a large genome such as breadwheat (34 Gb/2C) or sugarcane (10 Gb/2C; D’Hont

2005). Two mechanisms are responsible for the increase ingenome size and thus complexity: an increase in mono-ploid genome size related mainly to transposable elementamplification (Bennetzen 2005; Piegu et al. 2006) andduplications of the whole genome, i.e., polyploidization.The large sugarcane genome size is due to a very highdegree of polyploidy (�12x). The size of its monoploidgenome (basic set of chromosome, Figure 1) is ~900 Mb(D’Hont and Glaszmann 2001; D’Hont et al. 2008),which is similar to that of sorghum (760 Mb) and onlytwofold that of the rice genome (390 Mb). As comparedto bread wheat, the sugarcane genome is around three-fold smaller, but its redundancy level is much higher,with 12 hom(oe)ologous highly heterozygous haplo-types on average at each locus as compared to threesets of highly homozygous pairs of haplotypes in breadwheat (2n ¼ 6x ¼ 42, AABBDD). This very high level ofgenetic redundancy in sugarcane makes it difficult tomonitor specific loci in genetic and physical mappingapproaches. In addition, in modern sugarcane cultivars,chromosomes form mainly bivalents at meiosis (Price

1963; Burner and Legendre 1994), but pairing amongthe hom(oe)ologs appears close to random with onlyoccasional preferential pairing (Grivet et al. 1996;Hoarau et al. 2001; Jannoo et al. 2004).

In bread wheat, map-based cloning approaches havebeen successfully developed especially through the use ofdiploid donors (or close diploid relatives) of bread-wheatsubgenomes and the abundant genomic resources fromthis well-studied plant (Keller et al. 2005). In sugarcane,there are no close diploid relatives; only polyploids areknown in the Saccharum genus. In addition, due to itsgenetic complexity, this species has received very littleresearch investment despite its economic importance,and molecular resources have just recently begun to bedeveloped (Grivet and Arruda 2002).

Despite this challenging complexity, we implementeda map-based cloning approach to isolate the rust resis-tance gene Bru1. Bru1 was originally linked to a singlemarker (CDSR29) on a map built using selfed cv R570progeny (Daugrois et al. 1996; Grivet et al. 1996).Asnaghi et al. (2000) refined the genetic map aroundBru1 on the basis of existing rice, maize, and sorghumgenetic maps. This approach revealed that the targetedregion is orthologous to one end of sorghum consensuslinkage group 4 (LG4), the end of the short arm of ricechromosome 2, and part of maize LG4 and LG5. It alsoenabled localization of Bru1 at the end of one cosegre-gation group of the R570 homology group VII. How-ever, it did not enable marker saturation of the regiondue to the distal position of the gene and the poordensity of markers in the distal orthologous map area ofrice and sorghum at that stage. Later, Asnaghi et al.(2004) identified AFLP markers flanking Bru1 at 2 cMon both sides using a BSA approach.

Figure 1.—Schematic of a typical modern sugarcane culti-var genome. Each bar represents a chromosome; open boxesrepresent regions originating from S. officinarum and shadedboxes regions from S. spontaneum. Chromosomes aligned inthe same row are hom(oe)ologous and represent a homologygroup (HG). Chromosomes assembled in the dotted verticalrectangle correspond to a monoploid genome (MG) of S. of-ficinarum. The key characteristics of this genome are the highlevel of ploidy, the aneuploidy, the bispecific origin of thechromosomes, the existence of structural differences betweenchromosomes of the two origins, and the presence of inter-specific chromosome recombinants.

650 L. Le Cunff et al.

In this article, we describe (i) strategies that we de-veloped to overcome various constraints associated withhigh polyploidy in the successive steps of map-basedcloning approaches, including diploid/polyploid syn-tenic shuttle mapping with two model diploid species,sorghum and rice, and haplotype-specific chromosomewalking and (ii) their successful application in progress-ing toward isolation of the rust resistance gene Bru1 insugarcane.

MATERIALS AND METHODS

Sorghum genetic mapping: A population of 110 recombi-nant inbred lines, derived from the Sorghum bicolor ssp. bicolorintraspecific cross IS2807 3 379, was genotyped by RFLP,according to the protocol of Dufour et al. (1996). The markerswere then integrated to the composite map of Boivin et al.(1999) using Mapmaker 3.0 software (Lander et al. 1987).

Sugarcane genetic mapping: Three subpopulations ob-tained by selfing the sugarcane cultivar R570 were used.R570 is a typical modern sugarcane cultivar obtained by theCentre d’Essai de Recherche et de Formation in Reunion. Afirst population of 88 individuals was used originally to dem-onstrate the existence of a major resistance gene to brown rustin R570 (Daugrois et al. 1996) and to construct an RFLP map(Grivet et al. 1996; Asnaghi et al. 2000). A second populationof 695 individuals was developed to initiate high-resolutionmapping of the gene Bru1 (Asnaghi et al. 2004). A total of 26locally recombinant individuals that displayed recombinationin a 10-cM region surrounding Bru1 were identified in thesetwo populations and described by Asnaghi et al. (2004). Athird population of 1600 individuals was developed later toincrease the resolution of the genetic map around Bru1.

Rust resistance was scored according to the presence/absence of sporulations. Individual-bearing sporulating pus-tules were classified as susceptible or otherwise as resistant.These field evaluations were performed in Reunion usingnatural infections as described in Asnaghi et al. (2004) andTai et al. (1981).

RFLP probes were first tested on DNA bulks of five resistantindividuals and five susceptible individuals (5 3 2 mg¼ 10 mg/lane) digested with HindIII, DraI, SstI, or EcorV. When a poly-morphic marker was identified between bulks, a subset of 28individuals (resistant and susceptible, 10 mg/lane) was used tovalidate cosegregation of the marker with Bru1. Then themarker was analyzed on the 26 locally recombinant individualsfrom the above populations and mapped using Morgan’smapping function. Genomic DNA extractions and RFLP wereperformed as described in Grivet et al. (1994).

AFLP: Four ALFP markers (actctt, aaccac6, actctg9R, andacgctt17) surrounding Bru1 (Asnaghi et al. 2004) were used toidentify new ‘‘local recombinants.’’ The AFLP procedure wasperformed as described by Asnaghi et al. (2004).

AFLP markers cloning: AFLP bands were cut from thepolyacrylamide gel and transferred into 10 ml of sterile waterovernight to allow the DNA to diffuse out of the gel. PCRamplifications were performed in an MJ Research PTC 100thermal cycler in 20-ml reaction mixtures containing 1 ml ofthe AFLP fragment, 50 mm KCl, 10 mm TRIS–HCl (pH 8.3),each primer at 0.2 mm (Invitrogen AFLP primers that revealedthe AFLP markers EcoRI-aac/MseI-cac for aaccac6 and EcoRI-att/MseI-cag for attcag), 125 mm of each dNTP (already mixedwith the Invitrogen MseI primer), and 1 unit of Taq poly-merase. The samples were denatured at 94� for 5 min andsubjected to 35 cycles at 94� for 30 sec, 52� for 45 sec, and 72�for 1 min, followed by an extension step for 8 min at 72�. The

PCR products were cloned in a pGEM-T vector (Promega) andthen transformed with DH5 a thermo-competent cells. Thecloned fragments were used as RFLP probes on the R570 map-ping population to confirm their cosegregation with the cor-responding AFLP marker.

BAC libraries: Four BAC libraries were used in this study,including three constructed at Clemson University GenomicsInstitute (CUGI; http://www.genome.clemson.edu):

The sorghum BAC library (SB_BBc) constructed with bicolorBtx623 genotype contains 110,592 clones with an averageinsert size of 120 kb, covering 17-fold the haploid genome.

The rice BAC library (OSJNBa) constructed with Nipponbaregenotype contains a total of 36,864 clones with an averageinsert size of 130 kb, covering 10-fold the haploid genome.

The sugarcane BAC library (CUGI, SHCRBa) constructed withthe R570 cultivar contains 103,296 clones with an averageinsert size of 130 kb representing 1.2 total genome equiv-alents (Tomkins et al. 1999; Grivet and Arruda 2002).

The sorghum and rice BAC libraries have been partiallyordered by fingerprinting at CUGI with HindIII usingFPCV6 software (Soderlund et al. 2000) and a cut-off of1e-13. Rice and sorghum BAC contigs are available at http://www.genome.arizona.edu/fpc/WebAGCoL/rice.WebFPCand http://www.stardaddy.uga.edu/fpc/WebAGCoL/bicolor/WebFPC, respectively.

We constructed a second sugarcane BAC library (CIR)during this study following the protocol described by CUGI(http://www.genome.clemson.edu/protocols). This librarywas built with the DNA of four selfed R570 individuals carryingtwo copies of Bru1. A total of 110,592 clones were producedwith an average insert size of 130 kb. This library was organizedin pools of six BACs and thus fits on one nylon membrane.Screenings of these BAC libraries were performed by hybrid-ization on high-density filters using a standard protocol(http://www.genome.clemson.edu/protocols). For the CIRlibrary, when a pool was identified, a new screening step wascarried out to identify the positive BAC in the positive pool.

BAC-ends isolation: Isolation of the BAC ends (terminalsequences) was performed by two different techniques. Thefirst one is based on an adapter-anchor PCR method describedby Devic et al. (1997) and adapted for BAC-ends isolation by E.Bourgeois (personal communication). This technique relieson the use of a blunt-end restriction enzyme, a specific adapter,and two steps of nested PCR amplification using primersspecific to the adapter and the BAC vector. Twenty-five nano-grams of BAC DNA was digested separately with four differentenzymes (DraI, EcorV, StuI, and HpaI). The adapter was pre-pared by annealing the complementarity oligonucleotides,Adema1 (59-CACTGAATCTTGCTGACTAGGTCTGGGGAGGT-39) and Adema2 (59-P-ACCTCCCCAGAC-NH2-39). PCR1was performed using a specific adaptor primer, MA1 (59-CTGAATCTTGCTGACT-39), and two specific BAC vector borderprimers, Lac283 (59-ACGACGTTGTAAAACGACGGCCAGTGAAT-39) to amplify the left BAC terminal sequence or Lac439(59-AGCTATGACCATGATTACGCCAAGCTATT-39) to amplifythe right BAC terminal sequence. PCR1 amplifications wereperformed in a MJ Research thermo-cycler with the followingconditions: 3 min at 94� followed by 14 cycles of 94� for 30 sec,65�–58� for 45 sec (the first cycle was 65�, and subsequentcycles were reduced by 0.5�) and then 20 cycles of 94� for 30sec, 58� for 45 sec, and 72� for 2 min and 30 sec. PCR2 was per-formed using a nested specific adaptor primer, MA2 (59-ATCTTGCTGACTAGGT-39), and two nested BAC vector primers,FBAC for the left BAC terminal sequence (59-AGTCGACCTGCAGGCATG-39) or RBAC for the right BAC terminal sequence(59-CGCCAAGCTATTTAGGTGA-39). PCR2 amplification was

Map-Based Cloning in a Highly Polyploid Genome 651

performed with 1/50 dilution of PCR1 product using the sameconditions as PCR1, except for the extension time at 72�,which was 1 min and 30 sec. The second technique is based ondirect sequencing of the BAC extremities using the specificBAC vector border primers, F-BAC and R-BAC. Analysis of thesequences obtained allows the definition of specific primerpairs, which were used to amplify the BAC ends by PCR.

PCR products that were obtained by the two differenttechniques were finally loaded on a 1% low-melting-pointagarose gel and, after staining with ethidium bromide, theamplification products were cut directly from the gel to beused as probes.

BAC subclones: BAC DNA (200 ng) was digested with tworestriction enzymes (HindIII and EcorV). DNA fragmentsgenerated were cloned into pBluescript2SK1 vector. Thesubclone sizes were determined on a 1% agarose gel afterPCR amplification with M13 universal primers and clones withthe same sizes were considered as identical. To eliminatesubclones corresponding to repeat sequences or bacterialDNA, a Southern blot was made and hybridized with R570 totalDNA and Escherichia coli DNA. The remaining BAC subcloneswere isolated from a 1% low-melting-point agarose gel to beused as probes.

BAC fingerprinting filters: Digested BAC DNA (500 ng) wasseparated in a 0.7% agarose gel at 70 V for 24 hr and trans-ferred onto nylon filters.

RESULTS

Diploid/polyploid syntenic shuttle high-resolutionmapping using sorghum as a model species: To refinethe location of the sorghum region orthologous to theR570 target region, we used our sorghum mapping pop-ulation and, in a second step, information from othersorghum genetic maps. The two closest AFLP markers(aaccac6 and attcag) distally flanking Bru1 (Figure 2, step1), from Asnaghi et al. (2004), were cloned and analyzedbyRFLPonoursorghummappingpopulation.Theaaccac6locus was mapped on LG4 (previously LG D; Boivin et al.1999) at 9.4 cM proximally from CDSR29. The attcag proberevealed no polymorphism in our sorghum mappingpopulation. However, its hybridization on the sorghumBAC library (Sb_BBc) identified several BACs, includingBAC 67N07. One end of this BAC, F67N07D, was map-ped by RFLP on LG4 at 8.8 cM proximally from CDSR29.These results allowed us to delimit the sorghum regionorthologous to the target sugarcane region to an 8.8-cMinterval between CDSR29 and F67N07D (Figure 2).They also revealed a local inversion on the R570 geneticmap as compared to the sorghum map. Indeed, al-though LGVII of the R570 sugarcane genetic map issyntenic and mostly colinear to sorghum LG4 (Asnaghi

et al. 2000), the two AFLP markers located distally fromCDSR29 in CG VII.1 of R570 were mapped proximallyfrom CDSR29 on sorghum LG4.

We aligned our sorghum LG4 (previously LG D;Boivin et al. 1999; our unpublished data) with LG4 (pre-viously LG F) of the high-density RFLP map of Bowers

et al. (2003). Two common RFLP loci (CDSR29 andM738) allowed us to delimit the target region on themap of Bowers et al. (2003) (Figure 2, step 1). Nine

RFLP loci located in the target sorghum region over thetwo maps at that stage were analyzed on the R570progeny (Figure 2, step 1). Four RFLP probes (pSB1445,cMWG652, pSB0084, and pPAP05H10) revealed a poly-morphic marker between the resistant and susceptiblebulks. The four markers were mapped in the target areawith the two closest to Bru1 being revealed by probespSB0084 and pPAP05H10 and mapped at 0.6 cM proxi-mally and 0.3 cM distally from Bru1, respectively (Figure2, step 1). These results reduced the target region inR570 to a 0.9-cM interval. These two loci are separatedby 3.1 cM on the sorghum maps of Bowers et al. (2003).The position of the mapped markers confirmed theinversion between sugarcane R570 CGVII.1 and sorghumand was in agreement with colinearity inside the inversion.

To further refine the genetic map of the Bru1 region,we built an orthologous sorghum BAC physical map andthen derived new probes from this physical map (Figure2, step 2). Four loci (pSB0084, pSB1565, pSB0927, andpPAP05H10) located in the 3.1 cM orthologous targetregion in sorghum were used to screen a partiallycontiged sorghum BAC library (Sb_BBc, http://www.genome.arizona.edu/genome/sorghum.html).Theiden-tified BACs were searched for on the sorghum phys-ical map (http://www.stardaddy.uga.edu/fpc/bicolor/WebAGCoL/WebFPC) to find the corresponding con-tig. Together, these four probes allowed us to identifyfour contigs (named at that stage Ctg1123, Ctg458,Ctg1363, and Ctg1455). To test if the four identifiedcontigs overlapped, (i) we used the ‘‘contig-end bestmatch’’ option, available on FPCV6, on CUGI BACfingerprint data; (ii) we compared the HindIII finger-prints of some key BACs from the four sorghum contigs;and (iii) we subcloned BAC ends from the contigextremities and hybridized them on the sorghum BAClibrary. Altogether, the results obtained demonstratedthat the four contigs overlapped and thus could bemerged into a unique contig of 91 BACs representing aregion of �350–400 kb (Figure 2, step 2).

Fifty-seven BAC ends spread over the sorghum contigwere cloned and analyzed. Among them, 14% did nothybridize well with sugarcane DNA and�56% appearedto correspond to repeated sequences in sugarcane.Among the remaining 30% (17 clones), 4 revealed a poly-morphic marker between resistant and susceptible bulksfrom the self-progeny of R570. They were all mapped inthe target area in R570. Two markers, revealed by theBAC ends R24P17eV and F57P2eV, mapped at 0.3 cMproximally to Bru1; one marker, revealed by the BAC-endF123K21D, cosegregated with Bru1 and one marker,revealed by the BAC end R195K15H, mapped at 0.3 cMdistally to Bru1 (Figure 2, step 2). The sorghum regionorthologous to the sugarcane target region (betweenF57P2eV and R195K15H) was reduced to �225 kb.Again, the position of the markers confirmed the in-version between sorghum and R570 CG VII.1 and was inagreement with colinearity inside the inversion.


Diploid/polyploid syntenic shuttle high-resolutionmapping using rice as a model species: To identify therice region orthologous to the R570 target region,probes revealing a marker surrounding Bru1 were tested

for hybridization with rice DNA and used to screen theOSJNBa rice BAC library (Figure 2, step 3). The riceprobe C673 proximal to Bru1 hybridized to a BAC contigthat, according to the rice physical map (http://www.

Figure 2.—Saturation of the sugarcane target genetic region from various resources. (Step 1) Three markers originally sur-rounding Bru1 in sugarcane (orange) were mapped on our sorghum genetic maps (green) (Boivin et al. 1999, and our unpub-lished data) to identify the corresponding sorghum orthologous region in this map and, by comparison, to the map of Bowers

et al. (2003). Double green arrows link common markers between these two sorghum maps. Several sorghum RFLP loci (under-lined) from these two sorghum maps were analyzed on R570 progeny, allowing for mapping of new markers (indicated by greenarrows) on the sugarcane map. (Step 2) Four RFLP probes (in italic) from the sorghum genetic map were used to construct a localsorghum orthologous physical map. Green arrows point to sorghum BAC contigs obtained through BAC library screening and byconsulting the online sorghum physical map. Double black arrows indicate links between contigs (see text for details). Most sor-ghum BAC ends were analyzed on R570 progeny, allowing four BAC ends (indicated by green arrows) to be genetically mapped inthe sugarcane target region. (Step 3) Rice RFLP loci C673 originally mapped proximally from Bru1 in sugarcane and the RFLP locipPAP5H10 loci (derived from the sorghum genetic map) mapped proximally from Bru1 were used to screen a rice BAC libraryand to identify the corresponding rice orthologous physical map (from Clemson University, http://www.genome.arizona.edu, rep-resented in red). Orange arrows point to the rice BACs identified. Sugarcane cDNAs with homology to the rice orthologous se-quence were analyzed on R570 progeny, allowing mapping of new markers (indicated by red arrows) on the sugarcane map.Genetic distances are indicated in centimorgans. For the sugarcane map, distances indicated on right are based on 312 individualsand those indicated on left and in parentheses are based on 712 individuals. Markers used to build the sugarcane physical map areindicated in boldface type.


genome.arizona.edu), corresponded to rice chromo-some 2. The probe pPAP05H10 distal to Bru1 identifiedBAC clones belonging to the same contig. Their posi-tions revealed that the orthologous target region in ricecorresponds to �600 kb on the short arm of chromo-some 2 (Figure 2, step 3). According to the orientationof the rice physical map, the target region in the R570sugarcane cultivar is inversed as compared to rice. Thisinversion, which we also noted between the target regionin R570 and sorghum, must have arisen in sugarcaneafter it diverged from sorghum.

The sequence of five BAC clones covering most of therice region orthologous to the target sugarcane regionwas available at this stage (AP005304, AP005311, AP004885,AP004078, and AP004121). We compared these sequen-ces with the SUCEST sugarcane EST database (238,000sugarcane ESTs assembled in 43,000 clusters; Vettore

et al. 2003). Ninety EST clusters displaying at least 80%homology on at least 100 bp with the rice BACsequences were identified. cDNAs corresponding to 34of the sugarcane EST clusters were used as probes onbulks of DNA from susceptible and resistant R570progeny, and 7 revealed a polymorphic marker. Theywere all mapped within the sugarcane target area, withthe closest to Bru1 being cBR32 and cBR33 thatcosegregated with Bru1 and cBR23 and cBR37 thatsurrounded Bru1 at 0.3 cM on both sides (Figure 2, step3). These latter two belonged to BAC AP004885 andwere separated by �100 kb. The marker positionsconfirmed the inversion between rice and R570 CGVII.1 and were in agreement with colinearity inside theinversion. BLAST of the 7 mapped EST clusters did notreveal significant homology to any cloned resistancegene or resistant gene analog.

High-resolution genetic mapping in sugarcane: Anew population of 1600 individuals was developed toincrease the resolution of the genetic map around Bru1.The 400 susceptible individuals identified in this pop-ulation were analyzed with four AFLP markers surround-ing the gene (actctt, aaccac6, actctg9R, and acgctt17mapped at 3.5 and 2.5 cM distally and at 2.2 and 3.1 cMproximally from Bru1, respectively) to identify individ-uals locally recombinant in the target area. We used onlysusceptible individuals because the susceptible pheno-type is easier to establish with certainty since susceptibleindividuals represent a quarter of the population butencompass half of the detectable recombinant individ-uals in the region. Among the identified locally recom-binant individuals, 35 displayed recombination betweenAFLP markers aaccac6 and actctg9R, in accordance withthe previously observed genetic distance between thesemarkers (4.7 cM). The nine markers closest to Bru1 onthese individuals were analyzed. Two new recombina-tions were identified in the 0.6-cM target area but theydid not allow separation of previous cosegregatingmarkers. The new high-resolution map of the target regionincluded three markers mapped at 0.28 cM on both

sides of Bru1 (F57P2eV, cBR37, and R24P17eV proxi-mally; pPAP05H10, R195K15H, and cBR23 distally) andthree markers cosegregating with Bru1 (cBR32, cBR33,and F123K21D) (Figure 2).

Physical mapping of the target locus in sugarcane:We screened the whole R570 BAC library with nineprobes mapped in a 0.70-cM region around Bru1 (be-tween loci pPAP05H10 and PSB0084). We also used 14probes that were not mapped but corresponded to sixsorghum BAC ends from the sorghum orthologous BACcontig, and eight sugarcane cDNAs displaying homol-ogy to the orthologous rice sequence. To build thephysical map, we used BACs that were detected by atleast 2 probes, including 1 probe that revealed a markerlinked to Bru1 and 1 probe associated in another BAC toa probe that revealed a marker linked to Bru1. We werethus able to build a physical map of 32 BACs coveringthe Bru1 region (Figure 3). The physical map at thatstage contained one gap (between loci cBR32 andR195K15H). To complete this map, 10 BAC ends fromsome of the 32 sugarcane BAC clones were isolated andanalyzed on bulks of DNA of susceptible and resistantR570 progeny. One BAC end (R15N23S) revealed apolymorphic marker between the resistant and suscep-tible bulks and was mapped at 0.14 cM on the distal sideof Bru1. This allowed us to reduce the target area betweenprobe cBR37 (0.28 cM proximally) and R15N23S (0.14 cMdistally) and to eliminate the gap from the target area.The BAC end R15N23S was used to screen the R570BAC library and allowed us to identify an additionalBAC (164H22) that partially covered the target area.

Finally, at this stage, the entire target region (betweenprobe/locus cBR37 and R15N23) was contained in threeR570 BAC clones (15N23, 25N07, and 253G12, presentinga size of 120, 90, and 135 kb, respectively) and seven R570BAC clones contained part of the target area (Figure 3).

Identification of the target haplotype: As sugarcane ispolyploid and heterozygous, the obtained physical mapdid not correspond to a unique BAC contig but toseveral hom(oe)ologous BAC contigs corresponding tohom(oe)ologous chromosomes. To differentiate thehaplotypes, we compared the restriction profiles ofmost of the BAC clones of the studied region, includingall those belonging to the target region (betweenprobes/loci cBR37 and R15N23S). Four hom(oe)olo-gous haplotypes (Figure 3, a–d) were identified forBACs located on the proximal side of the target area.Five hom(oe)ologous haplotypes (Figure 3, a–e) wereidentified for BACs located on the distal side of the targetarea. Seven hom(oe)ologous haplotypes (Figure 3, 1–7)were identified among the 10 BAC clones covering thetarget area.

To identify the haplotype bearing Bru1 (target hap-lotype), we had to determine which BAC clones werebearing the markers (alleles) linked to Bru1. We thusanalyzed the BAC clones by RFLP with the probe/enzyme combinations that revealed the markers linked


Figure 3.—Physical map of the Bru1 region. BAC clones are represented by vertical lines: orange for the target haplotype,brown for the hom(oe)ologous haplotypes, and green for sorghum. Dotted lines and open circles indicate the localization ofprobes used on the sorghum or/and sugarcane physical map and/or on the genetic map of the Bru1 region in sugarcane cvR570. Boxes assemble BAC clones for the same haplotype. Probes in green represent those from the sorghum genetic or physicalmap, those in red are from the sugarcane cDNA library, and those in orange are BAC ends or subclones of sugarcane BACs.


to Bru1 and compared their RFLP profiles with thatof R570 (Figure 4). The RFLP patterns of BAC clonescorresponding to the haplotype bearing Bru1 shouldinclude the bands/markers linked to Bru1. Using thisprocedure, we were able to identify only one BAC clonecorresponding to the target haplotype, BAC 164H22.This BAC clone hybridized only with marker R15N23Sand thus only partially covered the target (Figure 3).

Haplotype-specific chromosome walking using aBru1-enriched BAC library: To complete the physicalmap of the target haplotype, a new BAC library of110,592 clones with an average insert size of 130 kb wasbuilt. We used a mix of DNA from four resistant selfedR570 progeny that contained two copies of Bru1 with theaim of increasing the proportion of the target haplotypein this library. These four individuals were selectedaccording to the band intensity observed on the RFLPprofiles for four markers surrounding Bru1 (F57P2eV,F123K21D, pPAP05H10, and R195K15H), the bandintensity reflecting to the number of marker doses. Thenew library, which covers 2.8-fold the target haplotypeand 1.4-fold the total genome, was screened with fiveprobes (cBR37, F123K21D, cBR33, cBR32, and R15N23)mapped in a 0.42-cM region around Bru1. Only BACsbelonging to the target haplotype, identified as ex-plained above, were selected. Two new BACs were recov-ered, i.e., BAC CIR9O20 (90 kb), which hybridized withprobes cBR37, F123K21D, cBR33, and BAC CIR12E03(125 kb), which hybridized with probe cBR32. Wedesigned primers at the extremities of these two BACsand tested them against each other and BAC 164H22.This revealed that two gaps still remained in the physicalmap, i.e., between BAC CIR9O20 and CIR12E03 andbetween CIR12E03 and 164H22 (Figure 3).

BAC ends and BAC subclones from the three BACsfrom the target haplotype (CIR9O20, CIR12E03, and164H22) were isolated and a chromosome walk wasundertaken to fill the two gaps remaining on the targethaplotype. Due to polyploidy, only mapped probes canbe used for this chromosome walking step to ensure thatthis walking is actually on the target chromosome. Wethus first had to map the subclones and select the onesrevealing a marker linked to Bru1. Nine such subclones(R-CIR9O20, R-CIR12E03, CIR9O20-D10, CIR9O20-F4,CIR12E03-A5, CIR12E03-B6, CIR12E03-D7, CIR12E03-D10, and CIR12E03-H6) were obtained, and they allcosegregated with Bru1. They were used to screen theBAC libraries and revealed three new BAC clones fromthe target haplotype [CIR43P06, 22M06 (85 kb), andCIR12H16]. Analysis of these BAC clones showed thatCIR12H16 was included in BAC CIR12E03 and thatCIR43P06 and 22M06 partially overlapped with BACCIR12E03 but not with CIR9O20 or 164H22 (Figure 3).BAC-end sequences of CIR43P06 and 22M06 corre-sponded to repeated sequences. Both BAC ends ofCIR12H16 and one subclone of 22M06 (22M06-H5)were mapped and cosegregated with Bru1. However,they did not reveal any new BAC of the target haplotypewhen used to screen the BAC libraries.

Finally, after all these chromosome walking steps, thephysical map of the target haplotype contained six BACsbut two gaps remained. The genetic map included 15markers that cosegregated with Bru1 (Figure 3).

All subclones that revealed a marker linked to Bru1were sequenced. Interestingly, subclone CIR9O20-F4showed homologies with the X and XI domains of Rpg1(Brueggeman et al. 2002), a barley rust resistance proteinthat corresponds to a serine (S)/threonine (T) kinase,and subclone CIR12E03-A5 displayed homology with theVIII, IX, and X domains of a rice S/T kinase (9639.t0149on The Institute for Genomic Research database) andRpg1.

Identification of an insertion—impact on recombi-nation: During the BAC library screening steps with theBAC subclones, we noted that nine of the subclones (R-CIR9O20, R-CIR12E03, CIR12E03-D10, R-CIR12H16,CIR12E03-A5, 22M06-H5, CIR12E03-B6, CIR12E03-H6,and CIR12E03-D7) that revealed a marker cosegregatingwith Bru1, and thus belonging to the target haplotype,did not hybridize with the other hom(oe)ologous BACclones (Figure 3). This highlighted the presence of aninsertion specific to the target haplotype. This insertedsegment corresponds to part of BAC CIR9O20 and BACCIR12E03 and thus comprises one of the two gaps re-maining on the target haplotype contig.

To assess the impact of this insertion on recombina-tion, we attempted to map a few probes/loci surroundingthe insertion on the other hom(oe)ologous cosegrega-tion groups. Five probes were analyzed, including threeprobes mapped distally from Bru1 (cBR8, cBR20, andR15N23S), one mapped proximally from Bru1 (cBR56),

Figure 4.—MethodforidentifyingtheBACcorrespondingtothe target haplotype vs. hom(oe)ologous BACs. (A) Schematicof hom(oe)ologous chromosome segments (haplotypes) bear-ing four allelic RFLP markers with probeA/HindIII (a1–a4).The target haplotype bearing allele a1 is in black. (B) RFLP pro-files of R570 and six BACs analyzed with probe A (R15N23) incombination with HindIII. Asterisks indicate BACs selected withprobe A. BAC 164H22 contains marker a1 (indicated by an ar-row) and thus belongs to the target haplotype.


and one that genetically cosegregated with Bru1(F123K21D) but physically mapped proximally from theinsertion (Figure 5). For the mapping, we used a subsetof 112 individuals from the selfed R570 progeny. Fourmarkers (revealed by probes cBR8, cBR20, R15N23S,and F123K21D) were mapped on CG VII-b, two markers(revealed by probes cBR20 and F123K21D) were map-ped on CG VII-c, and two markers (revealed by probescBR8 and cBR56) were mapped on VII-d. The geneticdistance between a given pair of markers varied betweenthe hom(oe)ologous cosegregation groups. For all theintervals composing the insertion, the distance waslower for the target cosegregation group (CG VII-1) ascompared to the other hom(oe)ologous cosegregationgroup (Figure 5). This showed that the insertion in-duced a reduction of recombination on the targethaplotype.

DISCUSSION

In this article, we report on the first map-basedcloning attempt in highly polyploid sugarcane.

Diploid/polyploid syntenic shuttle mapping facili-tated the development of a high-density genetic maparound Bru1: Modern sugarcane cultivars have a largegenome with an estimated complete genetic map size of17,000 cM (Hoarau et al. 2001) and up to 12 alleles onaverage that can coexist at a given locus. This impliesthat only single-dose markers [i.e., markers present ononly one of the hom(oe)ologous haplotypes] can beused for high-resolution mapping. Our strategy forhelping to rapidly saturate a given target area was touse bulk segregant analysis while also benefiting fromthe good syntenic relationships between sugarcane and

two model diploid Poaceae species, sorghum and rice(Grivet et al. 1994; Dufour et al. 1997; Glaszmann et al.1997; Guimaraes et al. 1997; Ming et al. 1998; Jannoo

et al. 2007), which are estimated to have diverged fromsugarcane �8 and 50 MYA, respectively (Wolfe et al.1989; Jannoo et al. 2007). Genetic maps, physical maps,and/or genome sequence data from these two specieswere tapped to select loci potentially present in thetarget area. The derived RFLP probes were analyzed onbulks of DNA from resistant and susceptible selfed R570progeny. This allowed us in one step to select single-dosemarkers that were linked to Bru1 and thus to eliminatemultiple dose markers and markers not linked to Bru1.

We fully tapped the various genomic resources of thethree species to develop a high-resolution genetic maparound Bru1. The origin and type of probes tested duringthese steps gave contrasting results. Around 44% of theprobes derived from sorghum genetic maps revealed amarker linked to Bru1, as compared to only 7% of theBAC ends tested from the sorghum orthologous physicalmap and to 20% of the sugarcane cDNAs selected fortheir homology to the rice orthologous sequence. Thiswas due to the fact that the probes derived from thesorghum genetic map were already selected as corre-sponding to single- or low-copy loci in sorghum. Most ofthem were from Gramineae species other than sorghumand thus were already selected for cross-hybridizationwith other species. Sugarcane cDNAs selected for theirhomology to the rice orthologous sequence also generallycorresponded to conserved single- or low-copy loci, butconservation of synteny between rice and sugarcane islower than between sorghum and sugarcane (Glaszmann

et al. 1997). By contrast, we estimated that �56% of thesorghum BAC ends tested corresponded to high or

Figure 5.—Impact of the inser-tion on recombination geneticdistances are indicated in centi-morgans and are based on 112 in-dividuals. Shaded areas indicatehaplotype segments composingthe insertion.


moderate repeated sequences in sugarcane. This percent-age is approximate because, due to polyploidy, the RFLPprofiles of single-copy loci in sugarcane are typicallymultiply banded and cannot be clearly separated frommoderately repeated sequences. In addition, 14% of thesorghum BAC ends did not hybridize well with sugarcaneDNA.

The combined use of synteny relationships betweensugarcane, sorghum, and rice and BSA was very success-ful since it allowed us to reduce the size of the geneticinterval containing the Bru1 gene from 4 to 0.56 cM andto identify nine markers within this genetic interval.These nine markers allowed us to initiate the sugarcanephysical map. These results illustrate the value of ex-ploiting model diploid species for mapping complexgenomes. The efficiency of this study, in contrast to thatof Asnaghi et al. (2000), could be explained mainly bythe fact that we were able to refine the location of theorthologous sorghum and rice region and access newlyavailable molecular resources, in particular high-densitysorghum RFLP genetic maps, a partially ordered sor-ghum BAC library, and the rice genome sequence. Theavailability of the complete sorghum genome sequence(http://www.phytozome.net/sorghum) should greatlyaccelerate the high-resolution mapping steps in futurestudies of this type in sugarcane.

Haplotype-specific chromosome walking: To developa physical map again since sugarcane is polyploid andhighly heterozygous, we had to develop specific method-ologies. To identify among hom(oe)ologous BAC clonesthe ones corresponding to the haplotype bearing Bru1,we had to determine which ones encompass the markers(alleles) linked to Bru1. This meant that, to develop thephysical map, and for further steps of chromosomewalking toward the gene, only probes revealing a markerslinked to Bru1 could be used. Therefore, physical andgenetic mapping had to be tightly associated through-out the physical mapping process.

Another difficulty, due to the large size of the sug-arcane genome, was that, to obtain a BAC library withreasonable genome coverage, a very large number of BACclones was required. For example, although the R570BAC library (Tomkins et al. 1999) that we first usedincludes as many as 103,296 BAC clones with an averagesize of 130 kb, this corresponded to a coverage of only1.3-fold the total genome and thus to a probability ofonly 73.9% of finding any particular DNA segment. Bycomparison, the sorghum library used in this studyincludes a similar number of BAC clones (110,592) witha similar average size but, since sorghum is diploid andhomozygous, represents a coverage of 17-fold the genomeand a 99.9% probability of finding a given segment. Oneway to overcome this problem is to build a much largerlibrary, which then becomes very tedious to manipulate.We thus developed an alternative strategy that involvedbuilding a library with a genotype comprising morecopies of the target haplotype. This material allowed us

to specifically double the proportion of the targethaplotype in the new BAC library (as compared to theR750 library). Genotypes with even more copies of thetarget haplotype could be used to continue increasingthe proportion of the target haplotype. For example, itcould be possible to again self the selfed R570 progenyand select individuals with four copies of the targethaplotype. To accelerate screening of the new library,BAC clones were picked and pooled by six in each well.The new BAC library represents 1.4 genome equivalentsbut statistically contains 2.8-fold the target area. Addedto the 1.3 genome coverage of the R570 BAC library ofTomkins et al. (1999), we now have BAC libraries cover-ing 4-fold the target area overall, with a 98.5% proba-bility of finding the target segment.

Using these strategies, we were able to build a physicalmap that encompasses seven hom(oe)ologous BAC con-tigs, including three hom(oe)ologous BAC clones thatentirely cover the target region. In addition, we reducedthe target interval to 0.42 cM and identified 12 addi-tional markers cosegregating with Bru1.

Structure of the target haplotype: We showed thatpart of the target haplotype corresponded to an inser-ted chromosome segment with no homology to thehom(oe)ologous haplotypes. This type of discovery hasalready complicated several map-based cloning projectsin diploid species (Stirling et al. 2001; Barker et al.2005). This is due to the fact that these insertions gen-erally induce severe repression of recombination (Wei

et al. 1999; Stirling et al. 2001; Neu et al. 2002; Barker

et al. 2005), as we observed in the Bru1 region. Theseinsertions may result from genomic rearrangement or,as frequently observed for resistance genes, to intro-gression of resistance from an alien source. Hoarau

et al. (2001) suggested that the chromosome carryingBru1 originated from S. officinarum, a species that repre-sents the main component of modern cultivar genomes.In the inserted segment, we identified two subclones(12E3-D07 and 22M06-H05) that revealed an atypicalsugarcane RFLP profile with only one band (thatcosegregating with Bru1). This suggested that the in-serted chromosome segment might originate fromS. spontaneum, the minority part of the modern sugar-cane cultivar genome. This would be in line with the factthat the interspecific crosses from which all moderncultivars are derived were performed 100 years ago toovercome disease outbreaks in S. officinarum by utilizingresistance from the wild S. spontaneum species. Recently,Raboin et al. (2006) identified a second major brownrust resistance gene that is also suspected to originatefrom S. spontaneum and mapped on a linkage groupnonorthologous to the R570 linkage bearing Bru1(Raboin et al. 2006).

Two BAC subclones from the target haplotype showedhomology with the S/T kinase barley rust resistance geneRpg1. This suggests the possible presence of an S/Tkinase cluster in the target region. These clones are being


further investigated as they represent potential candidategenes for Bru1. In sorghum, rust resistance loci have beenlocalized (Tao et al. 1998), including a major rust re-sistance QTL, genetically associated with a homolog ofthe maize rust resistance gene Rp1-D (McIntyre et al.2004). In addition, McIntyre et al. (2004) showed thatsome RGAs can be found in a syntenic position in sug-arcane and sorghum. According to available databasesand reports, no resistance genes or QTL have been iden-tified in the Bru1 orthologous rice and sorghum region.However, the colinearity between sorghum, rice, andsugarcane in the target region has been upset by theinsertion identified in the target haplotype.

We are currently sequencing 10 sugarcane BAC clonesrepresenting the different haplotypes that are hom(oe)ol-ogous or cover the Bru1 haplotype. These sequences willbe mined to complete the cloning of Bru1, especially (i) tocharacterize the two S/T kinase candidate sequencesidentified in this study and identify additional candidatesgenes, (ii) to localize the sorghum region orthologous tothe sugarcane target haplotype-inserted chromosomesegments, and (iii) to develop new probes to completethe physical map of the target haplotype.

In addition, we are currently characterizing a pop-ulation of sugarcane cultivars with different rust resis-tance phenotypes with the markers cosegregating withBru1 to identify recombinant haplotypes to reduce thesize of the target region. Linkage disequilibrium wasevaluated in sugarcane cultivars and varies from 0 to 30cM with a sharp decrease after 5 cM ( Jannoo et al. 1999;Raboin et al. 2008).

We thank P. Arruda for providing access to the SUCEST sugarcaneEST and cDNA resources and N. Yahiaoui and Rob Miller for criticalreading of the manuscript. We also thank the International Consor-tium for Sugarcane Biotechnology for financial support for this work.

LITERATURE CITED

Asnaghi, C., F. Paulet, C. Kaye, L. Grivet, J. C. Glaszmann et al.,2000 Application of synteny across the Poaceae to determinethe map location of a rust resistance gene of sugarcane. Theor.Appl. Genet. 10: 962–969.

Asnaghi, C., A. D’Hont, J. C. Glaszmann and P. Rott,2001 Resistance of sugarcane cultivar R570 to Puccinia melano-cephala isolates from different geographic locations. Plant Dis.85: 282–286.

Asnaghi, C., D. Roques, S. Ruffel, C. Kaye, J. Y. Hoarau et al.,2004 Targeted mapping of a sugarcane rust resistance geneBru1 using bulked segregant analysis and AFLP markers. Theor.Appl. Genet. 108: 759–764.

Barker, C. L., T. Donald, J. Pauquet, M. B. Ratnaparkhe, A. Bouquet

et al., 2005 Genetic and physical mapping of the grapevine pow-dery mildew resistance gene, Run1, using a bacterial artificialchromosome library. Theor. Appl. Genet. 111: 370–377.

Bennetzen, J. L., 2005 Transposable elements, gene creation andgenome rearrangement in flowering plants. Curr. Opin. Genet.Dev. 15: 621–627.

Boivin, K., M. Deu, J. F. Rami, G. Trouche and P. Hamon,1999 Towards a saturated sorghum map using RFLP and AFLPmarkers. Theor. Appl. Genet. 98: 320–328.

Bowers, J. E., C. Abbey, S. Anderson, C. Chang, X. Draye et al.,2003 A high-density genetic recombination map of sequence-tagged sites for sorghum, as a framework for comparative struc-

tural and evolutionary genomics of tropical grains and grasses.Genetics 165: 367–386.

Bremer, G., 1961 Problems in breeding and cytology of sugarcane.I. A short history of sugarcane breeding: the original forms of Sac-charum. Euphytica 10: 59–78.

Brueggeman, R., N. Rostoks, D. Kudrna, A. Kilian, F. Han et al.,2002 The barley stem rust-resistance gene Rpg1 is a novel dis-ease-resistance gene with homology to receptor kinases. Proc.Natl. Acad. Sci. USA 99: 9328–9333.

Burner, D. M., and B. L. Legendre, 1994 Cytogenetic and fertilitycharacteristics of elite sugarcane clones. Sugar Cane 1: 6–10.

Cuadrado, A., R. Acevedo, S. Moreno Diaz de la Espina, N. Jouve

and C. de la Torre, 2004 Genome remodelling in three mod-ern S. officinarum 3 S. spontaneum sugarcane cultivars. J. Exp. Bot.55: 847–854.

Daugrois, J. H., L. Grivet, D. Roques, J. Y. Hoarau, H. Lombard

et al., 1996 A putative major gene for rust resistance linked withan RFLP marker in sugarcane cultivar R570. Theor. Appl. Genet.92: 1059–1064.

Devic, M., S. Albert, M. Delseny and T. J. Roscoe, 1997 EfficientPCR walking on plant genomic DNA. Plant Physiol. Biochem. 35:331–339.

D’Hont, A., 2005 Unraveling the genome structure of polyploidsusing FISH and GISH: examples of sugarcane and banana. Cyto-genet. Genome Res. 109: 27–33.

D’Hont, A., and J. C. Glaszmann, 2001 Sugarcane genome analysiswith molecular markers: a first decade of research. Proc. Int. Soc.Technol. 24: 556–559.

D’Hont, A., L. Grivet, P. Feldmann, S. Rao, N. Berding et al.,1996 Characterisation of the double genome structure of mod-ern sugarcane cultivars Saccharum spp by molecular cytogenetics.Mol. Gen. Genet. 250: 405–413.

D’Hont, A., D. Ison, K. Alix, C. Roux and J. C. Glaszmann,1998 Determination of basic chromosome numbers in the ge-nus Saccharum by physical mapping of ribosomal RNA genes.Genome 41: 221–225.

D’Hont, A., G. M. Souza, M. Menossi, M. Vincentz, A-M. Marie-Anne

Van-Sluys et al., 2008 Sugarcane: a major source of sweetness, al-cohol, and bio-energy, pp. 483–513 in Tropical Crop Plant Genomics,edited by P. H. Moore and R. Ming. Springer-Verlag, New York.

Dufour, P., L. Grivet, A. D’Hont, M. Deu, G. Trouche et al.,1996 Comparative genetic mapping between duplicated seg-ments on maize chromosomes 3 and 8 and homeologous regionsin sorghum and sugarcane. Theor. Appl. Genet. 92: 1024–1030.

Dufour, P., M. Deu, L. Grivet, A. D’Hont, F. Paulet et al.,1997 Construction of a composite sorghum genome map andcomparison with sugarcane, a related complex polyploidy. Theor.Appl. Genet. 94: 409–418.

Glaszmann, J. C., P. Dufour, L. Grivet, A. D’Hont, M. Deu et al.,1997 Comparative genome analysis between several tropicalgrasses. Euphytica 96: 13–21.

Grivet, L., and P. Arruda, 2002 Sugarcane genomics: depicting thecomplex genome of an important tropical crop. Curr. Opin.Plant Biol. 5: 122–127.

Grivet, L., A. D’Hont, P. Dufour, P. Hamon, D. Roques et al.,1994 Comparative genome mapping of sugarcane with otherspecies within the Andropogoneae tribe. Heredity 73: 500–508.

Grivet, L., A. D’Hont, D. Roques, P. Feldmann, C. Lanaud et al.,1996 RFLP mapping in cultivated sugarcane Saccharum spp.: ge-nome organization in a highly polyploid and aneuploid interspe-cific hybrid. Genetics 142: 987–1000.

Guimaraes, C. T., G. R. Sillis and B. W. Sobral, 1997 ComparativemappingofAndropogoneae:SaccharumL.sugarcaneanditsrelationto sorghum and maize. Proc. Natl. Acad. Sci. USA 94: 14261–14266.

Hoarau, J. Y., B. Offmann, A. D’Hont, A. M. Risterucci, D. Roques

et al., 2001 Genetic dissection of a modern sugarcane cultivarSaccharum spp I genome mapping with AFLP markers. Theor.Appl. Genet. 103: 84–97.

Jannoo, N., L. Grivet, A. Dookun, A. D’Hont and J. C. Glaszmann,1999 Linkage disequilibrium among modern sugarcane culti-vars. Theor. Appl. Genet. 99: 1053–1060.

Jannoo, N., L. Grivet, J. David, A. D’Hont and J. C. Glaszmann,2004 Differential chromosome pairing affinities at meiosis inpolyploidy sugarcane revealed by molecular markers. Heredity93: 460–467.


Jannoo, N., L. Grivet, N. Chantret, O. Garsmeur, J. C. Glaszmann

et al., 2007 Orthologous comparison in a gene-rich regionamong grasses reveals stability in the sugarcane polyploid genome.Plant J. 50: 574–585.

Keller, B., C. Feuillet and N. Yahiaoui, 2005 Map-based isolationof disease resistance genes from bread wheat: cloning in a super-size genome. Genet. Res. 85: 93–100.

Lander, E. S., P. Green, J. Abrahamson, A. Barlow, M. J. Daly et al.,1987 MAPMAKER, an interactive computer package for con-structing primary genetic linkage maps of experimental and nat-ural populations. Genomics 1: 174–181.

McIntyre, C. L., S. M. Hermann, R. E. Casu, D. Knight, J. Drenth

et al., 2004 Homologues of the maize rust resistance gene Rp1-D are genetically associated with a major rust resistance QTL insorghum. Theor. Appl. Genet. 109: 875–883.

Ming, R., S. C. Liu, Y. R. Lin, J. Da Silva, W. Wilson et al.,1998 Detailed alignment of Saccharum and Sorghum chromo-somes: comparative organization of closely related diploid andpolyploid genomes. Genetics 150: 1663–1682.

Neu, C., N. Stein and B. Keller, 2002 Genetic mapping of theLr20-Pm1 resistance locus reveals suppressed recombinationon chromosome arm 7AL in hexaploid wheat. Genome 45:737–744.

Piegu, B., R. Guyot, N. Picault, A. Roulin, A. Saniyal et al.,2006 Doubling genome size without polyploidization: dynamicsof retrotransposition-driven genomic expansions in Oryza aus-traliensis, a wild relative of rice. Genome Res 16: 1262–1269.

Piperidis, G., and A. D’Hont, 2001 Chromosome compositionanalysis of various Saccharum interspecific hybrids by genomicin situ hybridization GISH. Proc. Int. Soc. Technol. 24: 565–566.

Price, S., 1963 Cytogenetics of modern sugar canes. Econ. Bot. 17:97–105.

Raboin, L. M., K. M. Oliveira, L. Le Cunff, H. Telismart, D.Roques et al., 2006 Genetic mapping in sugarcane, a high poly-ploid, using bi-parental progeny: identification of a gene control-ling stalk colour and a new rust resistance gene. Theor. Appl.Genet. 112: 1382–1391.

Raboin, L. M., J. Pauquet, M. Butterfield, A. D’Hont and J. C.Glaszmann, 2008 Analysis of genome-wide linkage disequilib-rium in the highly polyploid sugarcane. Theor. Appl. Genet116(5): 701–714.

Soderlund, C., S. Humphray, A. Dunham and L. French,2000 Contigs built with fingerprints, markers, and FPC V47. Ge-nome Res. 10: 1772–1787.

Sreenivasan, T. V., and B. S. Ahloowalia, 1987 Cytogenetics, pp.211–253 in Sugarcane Improvement Through Breeding, edited by D. J.Heinz. Elsevier Science, Amsterdam.

Stirling, B., G. Newcombe, J. Vrebalov, I. Bosdet and H. D. Bradshaw,2001 Suppressed recombination around the MXC3 locus, a ma-jor gene for resistance to poplar leaf rust. Theor. Appl. Genet103: 1129–1137.

Sun, X., Y. Cao, Z. Yang, C. Xu, X. Li et al., 2004 Xa26, a gene con-ferring resistance to Xanthomonas oryzae pv. oryzae in rice,encodes an LRR receptor kinase-like protein. Plant J. 37: 517–527.

Tai, P. Y. P., J. D. Miller and J. L. Dean, 1981 Inheritance of resis-tance to rust in sugarcane. Field Crops Res. 4: 261–268.

Tao, Y. Z., D. R. Jordan, R. G. Henzell and C. L. McIntyre,1998 Identification of genomic regions for rust resistance insorghum. Euphytica 103: 287–292.

Tomkins, J. P., Y. Yu, H. Miller-Smith, D. A. Frisch, S. S. Woo et al.,1999 A bacterial artificial chromosome library for sugarcane.Theor. Appl. Genet. 99: 419–424.

Ueda, T., T. Sato, J. Hidema, T. Hirouchi, K. Yamamoto et al.,2005 qUVR-10, a major quantitative trait locus for ultraviolet-B resistance in rice, encodes cyclobutane pyrimidine dimer pho-tolyase. Genetics 171: 1941–1950.

Vettore, A. L., F. R. da Silva, E. L. Kemper, G. M. Souza, A. M. da

Silva et al., 2003 Analysis and functional annotation of an ex-pressed sequence tag collection for tropical crop sugarcane. Ge-nome Res. 13: 2725–2735.

Wei, F., K. Gobelman-Werner, S. M. Morroll, J. Kurth, L. Mao

et al., 1999 The Mla powdery mildew resistance cluster is asso-ciated with three NBS-LRR gene families and suppressed recom-bination within a 240-kb DNA interval on chromosome 5S 1HS ofbarley. Genetics 153: 1929–1948.

Wolfe, K. H., M. Gouy, Y. W. Yang, P. M. Sharp and W. H. Li,1989 Date of the monocot-dicot divergence estimated fromchloroplast DNA sequence data. Proc. Natl. Acad. Sci. USA 86:6201–6205.

Xu, K., X. Xu, T. Fukao, P. Canlas, R. Maghirang-Rodriguez et al.,2006 Sub1A is an ethylene-response-factor-like gene that con-fers submergence tolerance to rice. Nature 10: 705–708.

Communicating editor: J. A. Birchler


Documents

Diploid/Polyploid Syntenic Shuttle Mapping and Haplotype ... · high polyploidy in the successive steps of map-based cloning approaches, including diploid/polyploid syn-tenic shuttle