genetic mapsof saccharum officinarum l. and saccharum …

Genetics and Molecular Biology, 22, 1, 125-132 (1999)

GENETIC MAPS OF Saccharum officinarum L. AND Saccharum robustumBRANDES & JEW. EX GRASSL *

Claudia T Guimarães', Rhonda J. Honeycutr, Gavin R. Sills' and Bruno Ws. Sobrai'

ABSTRACT

Genetic analysis was performed in a population composed 01 100 F, individuais derived lrom a cross between a cultivatedsugarcane (S. officinarum 'LA Purple') and its proposed progenitor species (S. robustum 'Moi 5829'). Various types (arbitrarilyprimed-PCR, RFLPs, and AFLPs) 01 single-dose DNA markers (SDMs) were used to construct genetic linkage maps lor bothspecies. The LA Purple map was composed 01 341 SDMs, spanning 74 linkage groups and 1,881 eM, while the Moi 5829 mapcontained 301 SDMs, spanning 65 linkage groups and 1,189 eM. Transmission genetics in these two species showed incom-plete polysomy based on the detection 01 15% 01 SDMs linked in repulsion in LA Purple and 13% 01 these in Moi 5829.Because 01 this incomplete polysomy, multiple-dose markers could not be mapped for lack 01 a genetic model lor their segre-gation. Due to inclusion 01 RFLP ancho r probes, conserved in related species, the resulting maps will serve as uselul tools lorbreeding, ecology, evolution, and molecular biology studies within the Andropogoneae.

INTRODUCTION

Saccharum L. is part of a polyploid complex withinthe Andropogoneae tribe. Cultivated forms of Saccharum(sugarcane) are most notably used for sugar and alcoholproduction worldwide, especially in the tropics. Sugarcaneis the most genetically complex crop for which genomemapping has been achieved (AI-Janabi et al., 1993; daSilvaet al., 1995). Polyploidy in Saccharum is widespread andis largely responsible for its genetic and taxonornic com-plexity. Studies using DNA markers and molecular cyto-genetics revealed polysornic inheritance and octoploidy(x = 8) within S. spontaneum (2n = 64, from India) (AI-Janabi et al., 1993;daSilva et al., 1995; D'Hont et al.,1996). The basic chromosome number and levei of ploidyhave not been conclusively determined for other Saccha-rum species.

Due to its genetic peculiarities, molecular geneticmarkers cannot be applied to sugarcane as they are to mostplants. Use of DNA markers has recently allowed geneticmapping in polyploids (daSilva and Sobral, 1996). A novelgenetic approach to direct mapping of polyploid plants wasproposed by Wu et al. (1992). This approach is based onsingle-dose markers (SDMs). SDMs are present in oneparent, absent in the other parent, and segregatel: 1 in the

'Universidade Federal de Viçosa - BIOAGRO, 36571-000 Viçosa, MG,Brasil. Send correspondence to c.T.G. Ennail: [email protected] Sidney Kimmel Cancer Center; 3099 Science Park Rd #200, San Diego,CA 9212/, USA.3 Department of Crop and Soil Sciences, Washington State University, Pull-I1UII1, WA 99164, USA.4 National Centerfor Genome Resources, 1800 Old Pecos Trail, Santa Fe,87505 NM, USA.* Pari of a thesis presented by c.T.G. to lhe Department of Genetics andBreeding, Universidade Federal de Viçosa, 1999, in partial [uljillment oflhe requirements for lhe Ph.D. degree.

progeny. More recently, daSilva (1993) and Ripol (1994)presented a methodology for mapping multiple dose mark-ers in polysornic polyploids, which greatly improved theaccuracy of identification of homology groups (daSilva etal., 1993, 1995).

Restriction fragment length polymorphisms(RFLPs) were the first DNA markers used to constructgenetic maps of higher organisms (Botstein et al., 1980).DNA fingerprinting methods, based on amplification ofrandom genornic DNA fragments by arbitrarily selectedprimers (Welsh and McClelland, 1990; Williams et al.,1990), have also been used for genetic mapping (Al-Janabiet al., 1993) among other applications (Welsh et al., 1991).More recently, amplified fragment length polymorphisms(AFLPs), a technique based on selective PCR amplifica-tion of genomic restriction fragments, have provided an-other very powerful tool for genornic research (Vos et al.,1995). When mapping with single-dose polymorphisms,all bands are scored as dominant markers, therefore thetypical advantage of RFLPs, namely codominance ofmarkers, is lacking. Thus, PCR-generated markers withan inherently higher data output per unit labor are goodchoices for generating and saturating linkage maps(Sobral and Honeycutt, 1993; Vos et al., 1995). How-ever, RFLPs remain the most informative marker to de-termine homologous relationships among chromosomeswithin Saccharum and among grasses, including maizeand sorghum.

S. officinarum is a domesticated species, which isthought to have been derived primarily from S. robustum,a wild species in Papua New Guinea (Brandes, 1929). Weherein report the development of SDM linkage maps foreach of these species using RFLP- and PCR-based mark-ers for progeny of an interspecific cross. These maps havealso been used in comparative studies among sugarcane,sorghum and maize, and in the analysis of quantitative traitsin these two species (Guimarães et al., 1997).

126

scoring of amplified products were performed accordingto Al-Janabi et ai. (1993). Arbitrarily primed PCR prod-ucts amplified with (X32P-dCTPwere resolved in 5% poly-acrylamide-50% urea gels in lx Tris-borate-EDTA, andvisualized by autoradiography using BioMax film (Kodak)at room temperature for 1-3 days. Over 400 ten-mers ofarbitrary sequence (Operon Technologies, Inc.) and fourRY-repeat twelve-mers (CG6 - 5-'TCGCTGCGGCGG-3',CG7 - 5'-CTGCGGTCGCGG-3', CG8 - 5'-CAGCCGTAGCGG-3' , andCG9 - 5' -CCGCGACTGCGG-3') werescreenedagainst the mapping parents.

Guimarães et aI.

MATERIAL AND METHODS

Plant materiais

Plant materials were kindly provided by the Ha-waiian Sugar Planters' Association (Aiea, HI). The popu-lation consisted of 100 FI individuals produced by cross-ing S. offieinarum 'LA Purple' as female with 5. robustum'MoI 5829'. Cytological evaluation of the populationshowed that parents and progeny displayed strict bivalentpairing at meiosis and had 2n = 80 chromosomes, as de-scribed previously by Al-Janabi et al. (1994a).

DNA markers

RFLPs

Genomic DNA was extracted according to themethod of Honeycutt et ai. (1992). Fifteen ug of genomicDNA from parents and 100 progeny was restricted indi-vidually with DraI, EeoRI, HindID, and XbaI, and resolvedin agarose gels. The gels were blotted and Southem hybrid-ization was performed according to daSilva (1993). Afterhybridization, blots were exposed to BioMax film (Kodak)at -80°C for 3 to 7 days depending on signal intensity. Onehundred and ninety probes were surveyed against parentalDNA blots digested individually with the four enzymes toidentify scorable polymorphisms. Subsequently,probes werehybridized to genomic DNA blots of the FI population thathad been digested with the appropriate enzyme.

Heterologous maize genomic clones (UMC - Uni-versity of Missouri-Columbia, and BNL - Brookhaven Natl.Laboratory) and maize cDNAs (ISU - Iowa State Univer-sity) previously mapped in maize and sorghum were usedas RFLP probes. Sugarcane genomic DNA (SG) clonesand cDNA clones from buds (CSB), cell culture (CSC),and roots (CSR), which were previously mapped in Sac-eharum spontaneum 'SES 208' (daSilva et al., 1993, 1995),were also used as RFLP probes. Cloned genes from su-crose metabolism and transport pathways, including smp-1, a sugarcane membrane protein and putati ve glucosetransporter (Bugos and Thom, 1993), sps-L, sucrose phos-phate synthase from maize (Worrell et al., 1991), 55-1,maize sucrose synthase (McCarty et al., 1986), and HBr-1, a maize phosphoglucomutase-encoding probe (kindlyprovided by S. Briggs, Pioneer Hi-Bred International,Johnston IA), were also used as RFLP probes.

Arbitrarily primed PCR

Genomic DNAs from parents and progeny (25 ng)served as templates for thermal cycling in a System Cycler9600 (Perkin Elmer), using the protocol described bySobral and Honeycutt (1993). Arbitrarily primed PCRproducts were resolved on either agarose or polyacryla-mide gels. Agarose gel electrophoresis and recording and

Selective restrietion fragment amplifieation

AFLPs (Voset al., 1995)were generated using AFLPAnalysis System I (Gibco-BRL). Two hundred and fifty ngof genomic DNA from parents and progeny was simulta-neously digested to comp\etion with EeoRI and MseI. Re-stricted genomic DNA fragments were ligated to EeoRI andMseI adapters, diluted 1:10, and pre-amplified using AFLPcore primers, each having one selective nucleotide. Pre-amplification products were then diluted to 1:50 and usedas a template for selective amplification using the combina-tions of MseI- and EeoRI-specific primers, each containingthree selective nucleotides. EcoRI-se\ective primers werelabeled with 'f2P-ATP before amplification. The thermalprofile for both steps of amplification, primer labeling, andselective primer combinations were performed as recom-mended by the manufacturer. The selective amplified prod-ucts were resolved by electrophoresis in denaturing poly-acrylamide gel, as described for arbitrarily primed PCR.

Marker identification

RFLPs were named by using the original probes'identification (ume, bnl, isu, esb, esc, esr or sg), followedby the first letter of the restriction enzyme used (d, e, h, orx for DraI, EeoRI, HindID, or XbaI, respectively), followedby a period and the molecular size (in base pairs). Sizewas a single-gel estimate calculated by linear regressionand standardization against a l-kb ladder (Gibco, BRL)for each blot. Arbitrarily primed-PCR polymorphisms werenamed using the Operon denomination (from A to Z andfrom 1 to 20), or the RY-repeat primer designation (CG6-CG9), followed by a period and the molecular size (in basepairs). The arbitrarily primed PCR polymorphisms that arefollowed by the letter p were resolved in denaturing poly-acrylamide gels, while the others were resolved in agarosegels. AFLPs were coded by the EeoRI (E) and MseI (M)selective primer combination and the respective molecu-lar size (in base pairs).

Linkage analysis

Polymorphisms were scored for presence (1) andabsence (O), and analyzed for dosage among FI progeny

Sugarcane genetic maps

using chi-square tests (P < 0.05), as described by Wu et ai.(1992) and daSilva et ai. (1995). Because of the double-pseudo-testcross mating strategy used (reviewed in daSilvaand Sobral, 1996), SDMs are identified in each of the par-ents, resulting in two maps: one for the male parent andone for the female parent. Linkage relationships amongSDMs were deterrnined using MapMaker v 2.0 for theMacintosh (Lander et ai., 1987) by coding the data as hap-loid (as the population is resultant from a double pseudo-testcross mating strategy). SDMs were grouped using aminimum LOD of7.0 and a maximum recombination frac-tion (r) of r < 0.25 (Wu et ai., 1992). Linkage groups werethen ordered using multi-point analyses. Markers at r < 3cM could not be ordered accurately because of the rel a-tively small sample size; however, the best possible orderwas always accepted, even if the LOD score supportingthe order was not large. Map distance in centimorgans wascalculated using the Kosambi mapping function. Linkagesin repulsion phase were determined as described by AI-Janabi et ai. (1993).

RESULTS AND DISCUSSION

Linkage maps

A total of 341 single-dose DNA markers weremapped in the LA Purple genome, yielding 74 linkagegroups (Figure 1) with 58 unlinked SDMs. 1n MoI 5829,linkage analysis of301 SDMs generated 65linkage groups(Figure 2), while 93 SDMs remained unlinked under thechosen criteria. In LA Purple the linked markers spanned1,881 centimorgans (cM) for an average of 6.65 cM permarker. 1n MoI 5829, linked markers covered 1,189 cM,an average of 5.74 cM per marker.

Marker distribution and mapping output

The total number of polymorphisms between thetwo genomes analyzed was significantly different with X2

= 4.49; P < 0.05 (503 for LA Purple and 438 for MoI 5829)

127

(Table I). S. officinarum showed a higher level of poly-morphism for all marker types. Sixty-eight percent of thepolymorphisms generated were single-dose. This percent-age is similar tõ the results of daSilva (1993), that found73% ofpolymorphisms in S. spontaneum to be single dose.

Markers generated by different methods were notuniforrn1y distributed across the linkage groups. In LAPurple, 26% of the linkage groups had all three markertypes, and in MoI 5829, just 9% of the linkage groups werecovered by all types of markers. Lack of uniforrn distribu-tion may be accounted for simply by the different num-bers of each type of marker mapped on each genome (TableI) and the incomplete saturation of both genomes withmarkers. daSilva et aI. (1995) mapped 208 AP-PCR mark-ers and 234 RFLPs in S. spontaneum 'SES 208', and theydid not find significant deviation from a random distribu-tion of the markers among linkage groups.

Of the 190 maize probes surveyed against the pa-rental sugarcane DNA, 131 probes produced a good hy-bridization pattem. The signal produced with maize genomicand cDNA probes suggests a high degree ofDNA sequencesimilarity among these species, despite at least 25 millionyears of evolution since they shared a common ancestor (AI-Janabi et aI., 1994b; Sobral et aI., 1994). A similar resultwas reported by daSilva et aI. (1993), in which 78% of maizeprobes surveyed produced a strong signal in S. spontaneum.

Chromosome assortment in S. officinarum andS. robustum

Both repulsion and coupling phase linkages wereobserved in S. officinarum and S. robustum genomes. Fif-teen percent of LA Purple markers were detected in repul-sion phase and were assigned to 17 linkage groups havingat least one repulsion phase SDMs. Similarly, 13% of MoI5829 were in repulsion phase and were assigned to l l link-age groups.

If complete preferential pairing of homologouschromosomes (as in diploids and disomic polyploids) wereobserved in these species, then linkages in both repulsion

Table ISummary of marker data.

RFLP AP-PCR ARP Ali markers

LAP Moi Total LAP Moi Total LAP Moi Total LAP Moi Total

Number of experiments" 166 64 12Number of polymorphisms 271 268 539 126 78 204 135 107 242 532 453 985Number of SDMsb 172 173 345 73 54 127 96 74 170 341 301 642SDMs Iinked 151 129 280 55 31 86 77 47 124 283 207 490Total polymorphisms/exp. 3.2 3.2 20.2SDMs/exp. 2.1 2.0 14.2

'Each experiment consists of: RFLP, probe/restriction enzyme combination; AP-PCR, primer reaction; AFLP, Eco RI and MseIseletive primer combination. "Single-dose markers (SDMs) determined using a X' test (P < 0.05), as described by Wu et ai. (1992).LAP, Saccharum officinarum LA Purple; Moi, Saccharum robustum Moi 5829.

128 Guimarães et al.

Figure 1 - Genetic linkage map of Saccharum officinarum 'LA Purple'. Seventy-four linkage groups were detected with an LOD = 7 and r = 0.25 for two-point analysis. The numbers to the left of the linkage groups represent the genetic distance in centimorgans as calculated by using the Kosambi function. Themarkers are shown on the right of the linkage groups; markers followed by an asterisk (*) are linked in repulsion phase.

Linkage Group 1 Linkage Group 2 Linkage Group 3 Linkage Group 4 Linkage Group 5

H8r-le.4240 smp-Ih.2730· Vlp.430 712p.340 urnc81x.2420·

16.6- 15.5- 13.3-20.3-22.0.-

EacaMcat.210 .•EaggMcac.260 * EacaMcal.640 .•

EacaMcac.4606.4- 8.8- sg26d.5900 16.2-EacaMcal.200 • 110.600 • 6.7-

umc1Ox.S73011.0- EacaMcat.40016.3- 16.7-0.0 umc29d. 1610" 14.8- 10..1-

sg2fid.3540 1.0 bn116.06dJ150 •EacaMctg.l10 .• umc113d.2060

umc 149x. 4480 * umc1 03e.556012.8- 9.4- 12.8-

csr15h5030 21.3- csrI5h.2/40 NZ5802.2- 05.430

15.5- 11.0-16.1- 0.0 isUI03h.8030umc45e.6630 ísu94e.8860 PI/.3403.2

umc 103x.21 00umc97h.4040 • 2.2 EaggMcla.125 13.3-6.0- EaacMc/g.51Ocsr 15h.7560 • ~1f EacaMcta.260 ísu64h.80S08.8-

EacaMcac.335N/B.770· 12.6

smp-/h.38oo

Linkage Group 6 Linkage Group 7 Unkage Group 8 Linkage Group 9 Unkage Group 10

isu66h.4240 Ul1p.5TO V/TB60 isu/10M.5110 3.4- EactMcal300D3.330

17.4- 16.2-20.4- 11.1-

26.4- umc 1326.2760EacaMcta.280 • K6.l30·

csb69d. 1860 • umcll4e.5310 10.3-12.2- umcesx. 4450 •

10.8- D7.510EaacMclg.290EactMcta330 .• umc66x4420 •

EaggMclg.3004.1-

AZO.4IXJ 12.5-umc114e.7100 .• 4.7-11.2-isu45e.56oo EacaMcac. 185 10.0- csb22h..50 70

umc13ge.6010 •• umcll4e.2490 13.0- csc37e.9890umc43e.31fJO 15.8-umc51h.6720 umc65x4180 15.8-20.7- 7.8- EaacMctg.540bn/8.45h.6100 umc66x.4120 RI5p.520

bn/15.21h.9620· VI5A40J5.41OEactMcat.130


E.acMcag.200 EaggMcta.290 • ísu66h.7280 EactMcat. 170 G4p.370·

12.8- 9.6- 10.3-umcI47x.6200·15.2 17.3- RI5p.870

sg26d.8580 2.0umc95d. 7050 •

7.7 -0.0 umcI4x.3150·

csr 1336. 3560 isu42h.63103.5 bnl6.25h.36/0 •6.2-19.0- 10..8- al- H19.520 07.780 •

csbI5h.6540 • X17.5006.0-

3.0-isu95d.2860 11.4-

:16- EaggMcta.150 OO/5.62e.7910 8.7-EactMcag.580 EaacMctg.450 sg426h.3270· umc27h.2310

13.0- 15.4- 9.5- 7.610.2-

EacaMcac.365· umc83h4300umc36h.4480 EaacMctg.275 EaacMcag.570

linkage Group 16 Linkage Group 17 Unkage Group 18 Linkage Group 19 Linkage Group 20umcI68h.102BOXI7.180 • csb 10e.4190 umc47e.9530 bn/5.62e. 11910

12.7- a9- bn/5.09x.3180 11.4-20.0- EacaMcat.520 umc94d.4330 813.670

1.1 umc32h.319011.1- EactMcta.350

umc121e.1930 EaacMctg.340 12.3-EacaMcat260 EaacMclg.455 19.4- EacaMcat.22022.3- 9.9-

15.4-isuJ8d. 1500 72- Ea.caMcac.320

4.0-umc85e.9530 EacaMcat.225

AZO.6oo Q2.710


2.3- J4.800 csr120h.651O • CG6p.440· umc 135h.9030 EaggMcac.3400.0.

csrI2h..2390 7.2-bn/7.49X.5030 EacaMcac.3603.01:.umc 16x.2980 18.4- 20.0- 20.3-

5.0 T- umc34d.42S0 15.6-5.0

umc44h. 11880 2.1 sg54x.3350 umc 131ki.6660 EactMcag.27015.6- sg54x.1100 umc31 e. 750010.0- 10.5- 10.0- 7.2-

umc34d.3230 • EacaMcac.370 EaggMcta.135 112.460 isu5ge,1850

Continued on next page

Sugarcane genetic maps 129

Figure J Continued


bn/15.208.6200 csr12h.2120 i'''~ ·l"'~ i-=11.1- 11.0- 10.3- 11.0-14.9-

2.2- urnc39x.5530 2.8_ /su43x.2740 EacaMctg.21O 4.2_ 811.3602.0 uma 1179.2520 EacaMcat. 140 smp-lh.4410 CSt918.66308.1..r cs1968.3430

5.5-EaacMeag.280 12.1- 12.7- 9.3-

5.2- umc117e.2690 • 7.1- smp-lh.2510· 2.5- EaggMcta.64O 016.380isu38d.3770 • RI5p.650 EaggMcta.l90

Linkage Group 31 Linkage Group 32 Linkage Group 33 Linkage Group 34 Linkage Group 35l-'~t-"ro l"M" ,"~E __ "~ l~~-7.1- 5.6- EaaeMctg.53010.5-bn/8.45h.8570 12.2- 3.4- CI3.650

4.5- umc51h.l0270 20.5-umcl02xA290 2.0 ume1378.7910 ume49d.4580

10.5-1.3 umc88h.4690 10.9-

12.8- 10.4 T E19.3602.2_ cst91x.8390 •cst918.461O csrI2h.3540

urneI148.4870 cst91s.7940· EacaMcat.150

Linkage Group 36 Linkage Group 37 Linkage Group 38 Linkage Group 39 Linkage Group 40t~- l-"~.; t.: t-'&'~10.5-

212- ume 1078.8090 19.8- 19.8- 18.4-

9.7-

sg426x.38oo • W15.870 Kl0.980 cst918.9530 M9.550


lE~- t-&'~ l_h~ tw", !-~9.5- 8.2- 11.0-

umcl04d.4080 17.1- tscasa.zeoo 14.7-

5.1- csr8h.49605.7- EaetMeag.4702.4_ EaggMcta.350 5.9- 1.0 csr969.531O

3.1- isu42h.822O EacaMcat.300 EaggMcac.390 EacaMcac. 150csc568.6630

Linkage Group 46 Linkage Group 47 Linkage Group 48 Linkage Group 49 Linkage Group 50t"~ t~- t=,m,m tE'-= =tE-.'"14.7- 13.4- 11.8- 10.8-

14.5-csr748.5650 1.0 umc36h.5030

EacaMera. 115 EaggMcac.290 EacaMcac.295 csb7s.5730

Linkage Group 51 Linkage Group 52 Linkage Group 53 Linkage Group 54 Linkage Group 55t~- t'."",tterc t-oo

'" t~"~ '7~r~11.6- 11.4- 11.3- 11.3- 1.0 sg99h.2070

EaggMcta250 isu120x46B0 EaeaMcac.3802.0 sg99s.5530

EaetMcag.2303.5r csr15h.6oo0

H6.55O

Linkage Group 56 Linkage Group 57 Linkage Group 58 Linkage Group 59 Linkage Group 60t-'-- t=""'*3" t~'~' t"'''~.,- t->x-10.8- 10.8- 10.3- 10.1- 10.0-

bn/5. 1Od.3080 EaetMcat.270 EaetMcat.260 /16.530 /11.420

Linkage' Group 61 Linkage Group 62 Linkage Group 63 Linkage Group 64 Linkage Group 65

t"'- 1.1 tisu87d.5190 t umc85h.l0280 il= csb588.4900 i=W.4809.8- 8.2- isu87x6oo0 6.4- 7.3- 6.4-

EacaMcat.3.50 EaacMctg.3.202.8- 015.3.20 NI5.670 CG6p.230

EaggMctg. 135


:ij=CSr/33s.2460 :§::811.860 5.1_tX1.470 3.3 _::§::umc85h.7460 3.3_::§:: cst968.104206.0- 5.1-

isul05d.6660 EaacMctg.450 EacaMcac.24O isul05d.4030 isu77x.2800

Linkage Group 71 Linkage Group 72 Linkage Group 73 Linkage Group 74

3.0_::§:: G4p.630 1.2~ csb23h.3470 1.0~isU759.48oo 0.0~sg293X.13t50EactMcag.280 X17A30 isu699.5360 isu140x4150

130 Guimarães et al.

Figure 2 - Genetic linkage map of Saccharum robustum 'Moi 5829'. Sixty-five linkage groups were detected with an LOD = 7 and r = 0.25 for two-pointanalysis. The numbers to the left of the linkage groups represent the genetic distance in centimorgans as calculated by using the Kosambi function. Markersare shown on the right of the linkage groups; markers followed by an asterisk (*) are linked in repulsion phase.


EacaMcta. 130 EactMcag.470 N15.670 sg54x.7030 EactMcta.220 '"

13.2- 13.0-8,4-

19.3- 4.5- EactMcat.230EaacMctg.270 G15.330 212-

EaacMcag.420

umcll4e.7640 11.8-15.1-19.5- isu75a.3440

11.5- umc36h.9150

C 13.250 2.0 umc34d.53104.9- isu38d.4210 22.4- ~~y.: umc6d.2030

EactMcag.21O24.7- umc44h.5410

9.5- 13.3- 6.6,f umc6d.3780 •4.1- EaggMcta 180 0.5 csrI338.2360 csblOe.6010 111.810 •

isu48d.5340 EacaMcat. 130 0.5 csr133d.l067011.9- 1.1 csr133e.5010

isull0Ah.541O csr133d 1570


2.0 isu32h.3460 umc14x.6700 4.1- umc55h.10050 .• 03.440 umc 104d.46209.1 isu32h. 1770 •

1.0 umc55x. 13750 9.1-14.5-

sg54x.2120 • 17.9- 112- umc55h.7730 isu35a.461O •9.1 umc49d. 8100 7.1-

isu64x.5270 umc54d.7590 .•.sg426x.3350 Dl.680·

7.1-11.3- EaggMcta.270· 19.7- 12.6-

19.9-016.720 • 4.7-

EacaMcat.240 .•13.3-

7.8- TI2p.460· N17.210 EacaMcac.365EactMcag.300 • umc84h.4090 .•


EacaMcta 110 /su42h.6770 umcl07h.1870 umc31x.2700 umc31x.620010.8- 82-

16.9- umc t0&1. 11g4() 18.6-sg26h.4250 7.6- 20.4-

bn/5.71d.6660 bnl6.25h.41208,4- isu5ge.4740

EactMcag.170·22.4- 11.6-G4p.320 122- 9.3-

EacaMcta.330 72-4.6- EaacMctg.250 EactMcag.150 EacaMcat. 170 •

csc37e.2880 EaggMcta430


umc23d.387O H2.390 f-"- t-~oo i'"-""8.3-122-

19.1- 19.0- 4.0_ umc32h.3040 162-0.0 csc5d. 12820 umc63d.19806.5f.. umcI24e.4140 csr133d.2570

isu95d.5020 EacaMcta. 140 • 12.4- 8.1-9.9- 9.0-

7.6__ umc103e.8250sg426x.2360 N20.450

EaggMctg.160· R15p.510EacaMcac.455

Linkage Group 21 Linkage Group 22 Linkage Group 23 Linkage Group 24 Linkage Group 25l'~'" r: l~'" ~_~W l--9.3- 9,2- 12.6- 102-umc23d.4720 20.9- umc149x.6700 bn1Z49x.4180 •t.o bnI16.06d.2270 •

13.7- 10.6-1.07- umcl68h.5030

9,2-

umc63d.2520 EacaMcta.260 ti 1 isu123h.4040 • EactMcta. 160 •sg54X.1300

isu123h.3130

Linkage Group 26 Linkage Group 27 Linkage Group 28 Linkage Group 29 Linkage Group 30t~'~'-..-t-"~~t='~~oo t: t'~~isul06x.238017.8- 8.8- 16.4- 15.8- 13.7-

3.4- NZ490'520.390 EacaMcac.330 2.1- umcI17h.6420

EacaMcac.290 isu123x.1700 .• sps-ld.7180

Linkage Group 31 Linkage Group 32 Linkage Group 33 Linkage Group 34 Linkage Group 35l-~oo'" t-,·- l'-- !-~"~-"~"7.0- 4.0.••••• umc34d.565015.5- 14.5- 4.0_ umc45a.6010 14.1-.

2.0 isu69d.41304.3_ isu64h.3750EaggMctg.340 4.8 f- umc6d.5240

sg26h.2560 EaggMctg.330 • smp-l h.2840EaggMcta.210

Continued on next page

Sugarcane genetic maps 131

Figure 2 Continued

Linkage Group 36 Linkage Group 37 Linkage Group 38 Linkage Group 39 Linkage Group 40~-t--- ts>~ ~~'M_ .~_""'..,o ~--isu45e.4750 7.0- 9.3-13.3- 4.8_ EactMcta.265 11.6- 00 EacaMcac.360

92-EacaMcac.305 CG6p.3TO i3 - csb23h.4300

EaacMcag.260 EacaMcat.l90 811.320


~u",,~ ~ E~"",~ 'E ~&~'~ "'-t ...",~oo t=Ma~,~11.3- 7.1- 6.6- 7.1_ umc29.5730 10.0-2.0 bnI8.45h.3480 csb22x.7570

EaggMcac.220 2.1 umc51h.3800 4.6- csb22h.4790 L6.450 EactMcag.410isu48d.5610


~"Q'ro ~~- ~_.~o tumcl6x:5300 t umc7x.26709.8- as- 6.2 - 7.3- 7.2-

umc65x.3660 umc12d.5260 umcl5d.9020 isul46d.4870EI9.220


~~'~- 1.0 ~umCl07h.l0760 1.0 ~iSUI46d.5170 as ~ isu7ge.5560 tumc130d.52605.1-6.0- umc161x.5820 6.0- isul46x.4OTO 4.8-00 bnI9.44h.4OTO 12 EactMcat430 EacaMcat.450io isuI4Ox.13840 EaacMcag.520 R15p.610 EaGtMcat490

umc62d. 1940


'-' ~"'''.''''' itee: 4.3-:ll= umcl 039.2690 ao ~CSr969.7790 ao _~CSb69d.6310 2.7-~ Ml0.8001.1j Wl1.260 isuI23x.l0040 isu38d.3000 umc43B.2850 G4p280

2.3 A20.310Wl1.210


2.3 _~umcl06d.I2820 2.2_~umcI66x.3150 2.0~ csrI33d.4450 1.0~CSr969.8250 0.0~ EacaMcta.190bnI6.25h.8570 umc83h.4040 csrI33e.7500 sps-ld.5090 EaacMcag.220

and eoupling phases should oeeur at approximately equalfrequencies. If ehromosome pairing were eompletely ran-dom, as in polysomie polyploids, then markers linked inrepulsion phase at a maximum of 10 eM would require amapping population of at least 750 individuals for theirdeteetion, assuming a polysomie oetaploid with striet biva-lent pairing (Wu et al., 1992). Deteetion of repulsion phaselinkages in both genomes with a population size of only100 individuals strongly suggests that these genomes areneither fully polysomie nor disornic. This result agrees witha previous study of a subset of 44 individuals from thispopulation (Al-Janabi et al., 1994a) and with Mudge et al.(1996), who studied the same eross with arbitrarily primedPCR markers. This strongly suggests partially preferen-tial ehromosome pairing, at least for some linkage groups,Linkages in repulsion imply that a saturated map for eup-loid S. officinarum or S. robustum will have less than 2n =80 linkage groups.

ACKNOWLEDGMENTS

The authors thank Michael McClelland, Rebecca Doerge

and Ron Sederoff for their encouragement. We thank those whograciously shared probes and other information, in particularMichael Lee for ISU prabes and unpublished data. Thanks to:the Hawaiian Sugar Planters' Association (HSPA, Aiea), in par-ticular Paul Moore and K-K- Wu, for supplying field data andplant material; Perkin-Elmer/ABI Division for supplying AFLPkits for beta-testing; Keira Maiden for technical assistance; JoshuaKohn, Ivor Royston and the Sidney Kimmel Cancer Center forvaried assistance. c.T.G. was supported by a fellowship frornthe Brazilian National Research Council of the Ministry of Sei-ence and Technology (MCT/CNPq/RHAE) No. 260007/95-1.B.WS.S., RJ.H., and G.R.S. were supported in part by a grantfrom Copersucar Technology Center (Piracicaba, Brazil) and theMauritius Sugar Industry Research Institute (Réduit, Mauritius).

RESUMO

Uma progênie de 100 indivíduos FI obtidos de umcruzamento entre cana-de-açúcar (5. officinarum 'LA Purple') eseu suposto progenitor (S. robustum 'Moi 5829') foi analisadautilizando marcadores moleculares em dose única. Marcadores dotipo AP-PCR,RFLP e AFLP,gerando um totalde 642 polimorfismos,foram mapeados em ambas espécies. O mapa genético de LA Purplefoi composto de 341 marcadores, distribuídos em 74 grupos de

structure of modern sugarcane cultivars iSaccharum spp.) by mo-lecular cytogenetics. MoI. Gen. Genet. 250: 405-413.

Guimarães, C.T., SilIs, G.R. and Sobral, B.W.S. (1997). Comparativemapping ol Andropogoneae: Saccharum L. (sugarcane) and its rela-tion to sorghum and maize. Proe. Natl. Aead. Sei. USA 94: 14261-14266.

Honeycutt, R.J., Sobral, B.W.S., Keim, P. and Irvine, J.E. (1992). A rapidDNA extraction method for sugarcane and its relatives. Plant MoI.Biol. Rep. 10: 66-72.

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McCarty, D.R., Shaw, J.R. and Hannah, L.C. (1986). The cloning, ge-netic mapping, and expression of the constitutive sucrose synthaselocus of maize. Proe. Natl. Aead. Sei. USA 83: 9099-9103.

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Ripol, M.I. (1994). Statistical aspects of genetic mapping in autopolyploids.M.S. thesis, Cornell University, Ithaca, NY.

Sobral, B.W.S. and Honeycutt, R.J. (1993). High output genetic mappingin polyploids using PCR-generated markers. Theor: Appl. Genet. 86:105-112.

Sobral, B.W.S., Braga, D.P.V., LaHood, E.S. and Keim, P. (1994). Phylo-genetic analysis of chloroplast restriction enzyme site mutations inthe Saccharinae Griseb. subtribe of the Andropogoneae Dumort. tribe.Theor. Appl. Genet. 87: 843-853.

Vos, P., Hogers, R., Bleeker, M., Reijans, M., Lee, T., Hornes, M., Frijters,A., Pot, J., Peleman, J., Kuiper, M. and Zabeau, M. (1995). AFLP:a new technique for DNA fingerprinting. Nucleie Aeids Res. 23: 4407-4414.

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132 Guimarães et aI.

ligação e 1.881 cM, enquanto que o mapa de ligação de MoI5829 continha 301 marcadores ao longo de 65 grupos de ligaçãoe 1.189 cM. A transmissão genética nessas duas espéciesapresentou polissomia incompleta devido a detecção de 15% dosmarcadores em dose simples ligados em fase de repulsão e 13%desses em MoI 5829. Devido a essa polissornia incompleta, osmarcadores em dose múltipla não puderam ser mapeados porfalta de um modelo genético para descrever tal segregação. Omapeamento de sondas' de RFLP, conservadas entre espéciespróximas evolutivamente, permitirá que os mapas genéticosgerados sejam utilizados como poderosas ferramentas nomelhoramento e em estudos de ecologia, evolução e biologiamolecular dentro das Andropogoneas.

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genetic mapsof saccharum officinarum l. and saccharum …

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