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

Transcription

1 Genetics and Molecular Biology, 22, 1, (1999) GENETIC MAPS OF Saccharum officinarum L. AND Saccharum robustum BRANDES & 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 F, individuais derived lrom a cross between a cultivated sugarcane (S. officinarum 'LA Purple') and its proposed progenitor species (S. robustum 'Moi 5829'). Various types (arbitrarily primed-pcr, RFLPs, and AFLPs) 01 single-dose DNA markers (SDMs) were used to construct genetic linkage maps lor both species. The LA Purple map was composed SDMs, spanning 74 linkage groups and 1,881 em, while the Moi 5829 map contained 301 SDMs, spanning 65 linkage groups and 1,189 em. Transmission genetics in these two species showed incomplete polysomy based on the detection 01 15% 01 SDMs linked in repulsion in LA Purple and 13% 01 these in Moi Because 01 this incomplete polysomy, multiple-dose markers could not be mapped for lack 01 a genetic model lor their segregation. Due to inclusion 01 RFLP ancho r probes, conserved in related species, the resulting maps will serve as uselul tools lor breeding, ecology, evolution, and molecular biology studies within the Andropogoneae. INTRODUCTION Saccharum L. is part of a polyploid complex within the Andropogoneae tribe. Cultivated forms of Saccharum (sugarcane) are most notably used for sugar and alcohol production worldwide, especially in the tropics. Sugarcane is the most genetically complex crop for which genome mapping has been achieved (AI-Janabi et al., 1993; dasilva et al., 1995). Polyploidy in Saccharum is widespread and is largely responsible for its genetic and taxonornic complexity. Studies using DNA markers and molecular cytogenetics 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 ploidy have not been conclusively determined for other Saccharum species. Due to its genetic peculiarities, molecular genetic markers cannot be applied to sugarcane as they are to most plants. Use of DNA markers has recently allowed genetic mapping in polyploids (dasilva and Sobral, 1996). A novel genetic approach to direct mapping of polyploid plants was proposed by Wu et al. (1992). This approach is based on single-dose markers (SDMs). SDMs are present in one parent, absent in the other parent, and segregatel: 1 in the 'Universidade Federal de Viçosa - BIOAGRO, Viçosa, MG, Brasil. Send correspondence to c.t.g. Ennail: claudiag@alunos.ufv.br 2 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, NM, USA. * Pari of a thesis presented by c.t.g. to lhe Department of Genetics and Breeding, Universidade Federal de Viçosa, 1999, in partial [uljillment of lhe requirements for lhe Ph.D. degree. progeny. More recently, dasilva (1993) and Ripol (1994) presented a methodology for mapping multiple dose markers in polysornic polyploids, which greatly improved the accuracy of identification of homology groups (dasilva et al., 1993, 1995). Restriction fragment length polymorphisms (RFLPs) were the first DNA markers used to construct genetic maps of higher organisms (Botstein et al., 1980). DNA fingerprinting methods, based on amplification of random genornic DNA fragments by arbitrarily selected primers (Welsh and McClelland, 1990; Williams et al., 1990), have also been used for genetic mapping (Al-Janabi et al., 1993) among other applications (Welsh et al., 1991). More recently, amplified fragment length polymorphisms (AFLPs), a technique based on selective PCR amplification of genomic restriction fragments, have provided another very powerful tool for genornic research (Vos et al., 1995). When mapping with single-dose polymorphisms, all bands are scored as dominant markers, therefore the typical advantage of RFLPs, namely codominance of markers, is lacking. Thus, PCR-generated markers with an inherently higher data output per unit labor are good choices for generating and saturating linkage maps (Sobral and Honeycutt, 1993; Vos et al., 1995). However, RFLPs remain the most informative marker to determine homologous relationships among chromosomes within Saccharum and among grasses, including maize and sorghum. S. officinarum is a domesticated species, which is thought to have been derived primarily from S. robustum, a wild species in Papua New Guinea (Brandes, 1929). We herein report the development of SDM linkage maps for each of these species using RFLP- and PCR-based markers for progeny of an interspecific cross. These maps have also been used in comparative studies among sugarcane, sorghum and maize, and in the analysis of quantitative traits in these two species (Guimarães et al., 1997).

2 Guimarães et ai. 126 MATERIAL AND METHODS Plant materiais Plant materials were kindly provided by the Hawaiian Sugar Planters' Association (Aiea, HI). The population consisted of 100 FI individuals produced by crossing S. offieinarum 'LA Purple' as female with 5. robustum 'MoI 5829'. Cytological evaluation of the population showed that parents and progeny displayed strict bivalent pairing at meiosis and had 2n = 80 chromosomes, as described previously by Al-Janabi et al. (1994a). scoring of amplified products were performed according to Al-Janabi et ai. (1993). Arbitrarily primed PCR products amplified with (X32P-dCTPwere resolved in 5% polyacrylamide-50% urea gels in lx Tris-borate-EDTA, and visualized by autoradiography using BioMax film (Kodak) at room temperature for 1-3 days. Over 400 ten-mers of arbitrary sequence (Operon Technologies, Inc.) and four RY-repeat twelve-mers (CG6-5-'TCGCTGCGGCGG-3', CG7-5'-CTGCGGTCGCGG-3', CG8-5'-CAGCCGTAG CGG-3', and CG9-5' -CCGCGACTGCGG-3') were screened against the mapping parents. Selective restrietion fragment amplifieation DNA markers RFLPs Genomic DNA was extracted according to the method of Honeycutt et ai. (1992). Fifteen ug of genomic DNA from parents and 100 progeny was restricted individually with DraI, EeoRI, HindID, and XbaI, and resolved in agarose gels. The gels were blotted and Southem hybridization was performed according to dasilva (1993). After hybridization, blots were exposed to BioMax film (Kodak) at -80 C for 3 to 7 days depending on signal intensity. One hundred and ninety probes were surveyed against parental DNA blots digested individually with the four enzymes to identify scorable polymorphisms. Subsequently, probes were hybridized to genomic DNA blots of the FI population that had been digested with the appropriate enzyme. Heterologous maize genomic clones (UMC - University of Missouri-Columbia, and BNL - Brookhaven Natl. Laboratory) and maize cdnas (ISU - Iowa State University) previously mapped in maize and sorghum were used as RFLP probes. Sugarcane genomic DNA (SG) clones and cdna clones from buds (CSB), cell culture (CSC), and roots (CSR), which were previously mapped in Saceharum spontaneum 'SES 208' (dasilva et al., 1993, 1995), were also used as RFLP probes. Cloned genes from sucrose metabolism and transport pathways, including smp1, a sugarcane membrane protein and putati ve glucose transporter (Bugos and Thom, 1993), sps-l, sucrose phosphate synthase from maize (Worrell et al., 1991), 55-1, maize sucrose synthase (McCarty et al., 1986), and HBr1, a maize phosphoglucomutase-encoding probe (kindly provided 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 Cycler 9600 (Perkin Elmer), using the protocol described by Sobral and Honeycutt (1993). Arbitrarily primed PCR products were resolved on either agarose or polyacrylamide gels. Agarose gel electrophoresis and recording and AFLPs (Vos et al., 1995) were generated using AFLP Analysis System I (Gibco-BRL). Two hundred and fifty ng of genomic DNA from parents and progeny was simultaneously digested to comp\etion with EeoRI and MseI. Restricted genomic DNA fragments were ligated to EeoRI and MseI adapters, diluted 1:10, and pre-amplified using AFLP core primers, each having one selective nucleotide. Preamplification products were then diluted to 1:50 and used as a template for selective amplification using the combinations of MseI- and EeoRI-specific primers, each containing three selective nucleotides. EcoRI-se\ective primers were labeled with 'f2p-atp before amplification. The thermal profile for both steps of amplification, primer labeling, and selective primer combinations were performed as recommended by the manufacturer. The selective amplified products were resolved by electrophoresis in denaturing polyacrylamide 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), followed by the first letter of the restriction enzyme used (d, e, h, or x for DraI, EeoRI, HindID, or XbaI, respectively), followed by a period and the molecular size (in base pairs). Size was a single-gel estimate calculated by linear regression and standardization against a l-kb ladder (Gibco, BRL) for each blot. Arbitrarily primed-pcr polymorphisms were named using the Operon denomination (from A to Z and from 1 to 20), or the RY-repeat primer designation (CG6CG9), followed by a period and the molecular size (in base pairs). The arbitrarily primed PCR polymorphisms that are followed by the letter p were resolved in denaturing polyacrylamide gels, while the others were resolved in agarose gels. AFLPs were coded by the EeoRI (E) and MseI (M) selective primer combination and the respective molecular size (in base pairs). Linkage analysis Polymorphisms were scored for presence (1) and absence (O), and analyzed for dosage among FI progeny

3 Sugarcane genetic maps 127 using chi-square tests (P < 0.05), as described by Wu et ai. (1992) and dasilva et ai. (1995). Because of the doublepseudo-testcross mating strategy used (reviewed in dasilva and Sobral, 1996), SDMs are identified in each of the parents, resulting in two maps: one for the male parent and one for the female parent. Linkage relationships among SDMs were deterrnined using MapMaker v 2.0 for the Macintosh (Lander et ai., 1987) by coding the data as haploid (as the population is resultant from a double pseudotestcross mating strategy). SDMs were grouped using a minimum LOD of7.0 and a maximum recombination fraction (r) of r < 0.25 (Wu et ai., 1992). Linkage groups were then ordered using multi-point analyses. Markers at r < 3 cm could not be ordered accurately because of the rel a- tively small sample size; however, the best possible order was always accepted, even if the LOD score supporting the order was not large. Map distance in centimorgans was calculated using the Kosambi mapping function. Linkages in repulsion phase were determined as described by AI- Janabi et ai. (1993). Linkage maps RESULTS AND DISCUSSION A total of 341 single-dose DNA markers were mapped in the LA Purple genome, yielding 74 linkage groups (Figure 1) with 58 unlinked SDMs. 1n MoI 5829, linkage analysis of301 SDMs generated 65linkage groups (Figure 2), while 93 SDMs remained unlinked under the chosen criteria. In LA Purple the linked markers spanned 1,881 centimorgans (cm) for an average of 6.65 cm per marker. 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 the two genomes analyzed was significantly different with X2 = 4.49; P < 0.05 (503 for LA Purple and 438 for MoI 5829) (Table I). S. officinarum showed a higher level of polymorphism for all marker types. Sixty-eight percent of the polymorphisms generated were single-dose. This percentage is similar tõ the results of dasilva (1993), that found 73% ofpolymorphisms in S. spontaneum to be single dose. Markers generated by different methods were not uniforrn1y distributed across the linkage groups. In LA Purple, 26% of the linkage groups had all three marker types, and in MoI 5829, just 9% of the linkage groups were covered by all types of markers. Lack of uniforrn distribution may be accounted for simply by the different numbers of each type of marker mapped on each genome (Table I) and the incomplete saturation of both genomes with markers. dasilva et ai. (1995) mapped 208 AP-PCR markers and 234 RFLPs in S. spontaneum 'SES 208', and they did not find significant deviation from a random distribution of the markers among linkage groups. Of the 190 maize probes surveyed against the parental sugarcane DNA, 131 probes produced a good hybridization pattem. The signal produced with maize genomic and cdna probes suggests a high degree ofdna sequence similarity among these species, despite at least 25 million years of evolution since they shared a common ancestor (AI- Janabi et ai., 1994b; Sobral et ai., 1994). A similar result was reported by dasilva et ai. (1993), in which 78% of maize probes surveyed produced a strong signal in S. spontaneum. Chromosome assortment in S. officinarum and S. robustum Both repulsion and coupling phase linkages were observed in S. officinarum and S. robustum genomes. Fifteen percent of LA Purple markers were detected in repulsion phase and were assigned to 17 linkage groups having at least one repulsion phase SDMs. Similarly, 13% of MoI 5829 were in repulsion phase and were assigned to l l linkage groups. If complete preferential pairing of homologous chromosomes (as in diploids and disomic polyploids) were observed in these species, then linkages in both repulsion Table I Summary of marker data. RFLP AP-PCR ARP Ali markers LAP Moi Total LAP Moi Total LAP Moi Total LAP Moi Total Number of experiments" Number of polymorphisms Number of SDMs b SDMs Iinked Total polymorphisms/exp SDMs/exp 'Each experiment consists of: RFLP, probe/restriction enzyme combination; AP-PCR, primer reaction; AFLP, Eco RI and MseI seletive 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.

4 128 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 twopoint analysis. The numbers to the left of the linkage groups represent the genetic distance in centimorgans as calculated by using the Kosambi function. The markers 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 p.340 urnc81x EacaMcat.210 EaggMcac.260 * EacaMcal.640. EacaMcac sg26d.5900 EacaMcal umc1ox.s umc29d. 1610" EacaMcat.400 sg2fid bn116.06dj150 EacaMctg.l10. umc 149x * umc1 03e.5560 umc113d csr15h csri5h.2/ isui03h umc45e.6630 ísu94e umc97h.4040 umc 103x EaggMcla csr 15h.7560 EaacMc/g.51O ~1f EacaMcta.260 ísu64h.80s EacaMcac.335 N/B smp-/h.38oo NZ580 PI/.340. Linkage Group 6 Linkage Group 7 Unkage Group 8 Linkage Group 9 Unkage Group 10 isu66h.4240 Ul1p.5TO V/TB60 isu/10m EactMcal300 D EacaMcta.280 K6.l30 umc csb69d umcll4e umcesx D7.510 EaacMclg.290 EactMcta330. umc66x umc114e EaggMclg.300 AZO.4IXJ umc13ge.6010 EacaMcac. 185 isu45e.56oo csb22h umcll4e csc37e bn/15.21h.9620 umc43e.31fjo umc51h.6720 umc65x bn/8.45h.6100 VI5A40 umc66x.4120 RI5p.520 J5.41O EactMcat.130 EaacMctg.540 Linkage Group 11 Linkage Group 12 Linkage Group 13 Linkage Group 14 Linkage Group 15 E.acMcag.200 EaggMcta.290 ísu66h.7280 EactMcat. 170 G4p umci47x RI5p.870 sg26d umc95d umci4x.3150 csr isu42h bnl6.25h.36/ al- H csbi5h.6540 X isu95d EaggMcta.150 :16- OO/5.62e EactMcag.580 sg426h.3270 EaacMctg.450 umc27h EacaMcac.365 umc83h4300 umc36h.4480 EaacMctg.275 EaacMcag.570 linkage Group 16 Linkage Group 17 Unkage Group 18 Linkage Group 19 Linkage Group 20 umci68h.102bo XI7.180 csb 10e.4190 umc47e.9530 bn/5.62e a9- bn/5.09x EacaMcat.520 umc94d umc32h EactMcta.350 umc121e.1930 EaacMctg EacaMcat260 EaacMclg EacaMcat isuj8d Ea.caMcac EacaMcat.225 umc85e.9530 AZO.6oo Q2.710 Linkage Group 21 Linkage Group 22 Linkage Group 23 Linkage Group 24 Linkage Group J4.800 csr120h.651o CG6p.440 umc 135h.9030 EaggMcac.340 csri2h bn/7.49x :. EacaMcac.360 umc 16x T- umc34d.42s umc44h sg54x.3350 umc 131ki.6660 EactMcag sg54x.1100 umc31 e umc34d.3230 EacaMcac.370 EaggMcta isu5ge,1850 Continued on next page

5 Sugarcane genetic maps 129 Figure J Continued i'''~ l"'~ i-= Linkage Group 26 Linkage Group 27 Linkage Group 28 Linkage Group 29 Linkage Group 30 bn/ csr12h urnc39x _ /su43x.2740 EacaMctg.21O 4.2_ uma EacaMcat. 140 smp-lh.4410 CSt r cs EaacMeag umc117e smp-lh EaggMcta.64O isu38d.3770 RI5p.650 EaggMcta.l90 l-'~t-"ro Linkage Group 31 Linkage Group 32 Linkage Group 33 Linkage Group 34 Linkage Group 35,"~E "~ l~~- l"m" EaaeMctg bn/8.45h CI umc51h.l ume ume49d.4580 umcl02xa umc88h _ cst91x T E cst o csri2h.3540 urnei cst91s.7940 EacaMcat.150 t~- l-"~.; t.: t-'&'~ Linkage Group 36 Linkage Group 37 Linkage Group 38 Linkage Group 39 Linkage Group ume sg426x.38oo W Kl0.980 cst M9.550 Linkage Group 41 Linkage Group 42 Linkage Group 43 Linkage Group 44 Linkage Group 45 tw", umcl04d tscasa.zeoo EaetMeag csr8h _ EaggMcta csr o EacaMcat isu42h.822o EaggMcac.390 EacaMcac. 150 csc le~- t-&'~ l_h~!-~ t"~ t~- t=,m,m t E '-= =t E -.'" Linkage Group 46 Linkage Group 47 Linkage Group 48 Linkage Group 49 Linkage Group umc36h.5030 csr EacaMera. 115 EaggMcac.290 EacaMcac.295 csb7s.5730 t~- t'."", t~"~'7~r~ Linkage Group 51 Linkage Group 52 Linkage Group 53 Linkage Group 54 Linkage Group 55 tterc t-oo '" sg99h sg99s.5530 EaetMcag.230 EaggMcta250 isu120x46b0 EaeaMcac r csr15h.6oo0 H6.55O t-'-- t=""'*3" t~'~' t"'''~.,- t->x- Linkage Group 56 Linkage Group 57 Linkage Group 58 Linkage Group 59 Linkage Group bn/5. 1Od.3080 EaetMcat.270 EaetMcat.260 / / Linkage' Group 61 Linkage Group 62 Linkage Group 63 Linkage Group 64 Linkage Group 65 t"'- 1.1 tisu87d.5190 il= tumc85h.l0280 csb i=w isu87x6oo NI5.670 CG6p.230 EacaMcat.3.50 EaacMctg.3.20 EaggMctg. 135 Linkage Group 66 Linkage Group 67 Linkage Group 68 Linkage Group 69 Linkage Group 70 :ij=csr/33s.2460 : :: isul05d.6660 EaacMctg _tX1.470 EacaMcac.24O 3.3 _:: ::umc85h _:: :: cst isul05d.4030 isu77x.2800 Linkage Group 71 Linkage Group 72 Linkage Group 73 Linkage Group _:: :: G4p ~ csb23h ~isU759.48oo 0.0~sg293X.13t50 EactMcag.280 X17A30 isu isu140x4150

6 130 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-point analysis. The numbers to the left of the linkage groups represent the genetic distance in centimorgans as calculated by using the Kosambi function. Markers 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 EacaMcta. 130 EactMcag.470 N sg54x.7030 EactMcta.220 '" 8, EactMcat.230 EaacMctg.270 G EaacMcag umcll4e isu75a umc36h umc34d.5310 C isu38d umc6d.2030 EactMcag.21O ~~y.: umc44h ,f umc6d EaggMcta csri csbloe isu48d.5340 EacaMcat csr133d.l csr133e.5010 csr133d 1570 isull0ah.541o Linkage Group 6 Linkage Group 7 Linkage Group 8 Linkage Group 9 Linkage Group isu32h.3460 umc14x umc55h umc 104d isu32h umc55x sg54x umc55h.7730 isu35a.461o 9.1 umc49d isu64x.5270 umc54d sg426x.3350 Dl EaggMcta EacaMcat N EacaMcac.365 TI2p.460 EactMcag.300 umc84h Linkage Group 11 Linkage Group 12 Linkage Group 13 Linkage Group 14 Linkage Group 15 EacaMcta 110 /su42h.6770 umcl07h.1870 umc31x.2700 umc31x umc t0&1. 11g4() sg26h csc37e.2880 bn/5.71d.6660 EaggMcta430 bnl6.25h ,4- isu5ge.4740 G4p EactMcag.170 EaacMctg.250 EactMcag.150 EacaMcat. 170 t-~oo i'"-"" Linkage Group 16 Linkage Group 17 Linkage Group 18 Linkage Group 19 Linkage Group 20 EacaMcta f-"- umc23d.387o H _ umc32h csc5d umc63d f.. umci24e.4140 csr133d.2570 isu95d.5020 EacaMcta umc103e sg426x.2360 N R15p.510 EacaMcac.455 EaggMctg.160 l'~'" Linkage Group 21 Linkage r: l~'" l-- Group 22 Linkage Group 23 Linkage Group 24 Linkage Group , ~_~W 102- umc23d umc149x.6700 t.o bni16.06d.2270 bn1z49x , umcl68h.5030 umc63d.2520 EacaMcta.260 ti 1 isu123h.4040 EactMcta. 160 sg54x.1300 isu123h.3130 t~'~'- Linkage Group 26 Linkage Group 27 Linkage Group 28 Linkage Group 29 Linkage Group NZ490' 2.1- umci17h.6420 EacaMcac.290 isu123x EacaMcac.330 sps-ld t-"~~t='~~oo t: t'~~ isul06x.2380 Linkage Group 31 Linkage Group 32 Linkage Group 33 Linkage Group 34 Linkage Group 35 l-~oo'" t-, - l'--!-~"~-"~" _ umc45a _ isu64h.3750 sg26h.2560 EaggMctg.330 smp-l h umc34d isu69d.4130 EaggMctg f- umc6d.5240 EaggMcta.210 Continued on next page

7 Sugarcane genetic maps 131 Figure 2 Continued ~-t--- ts>~ ~~'M_.~_""'..,o ~-- Linkage Group 36 Linkage Group 37 Linkage Group 38 Linkage Group 39 Linkage Group 40 isu45e _ EactMcta EacaMcac.360 i3 - csb23h.4300 EacaMcac.305 CG6p.3TO EaacMcag.260 EacaMcat.l t=ma~,~ Linkage Group 41 Linkage Group 42 Linkage Group 43 Linkage Group 44 Linkage Group t...",~oo _ umc bni8.45h.3480 csb22x.7570 EaggMcac umc51h csb22h.4790 L6.450 EactMcag.410 isu48d.5610 ~u",,~ ~ E~"",~ 'E ~&~'~ "' ~_.~o Linkage Group 46 Linkage Group 47 Linkage Group 48 Linkage Group 49 Linkage Group 50 Linkage Group 51 Linkage Group 52 Linkage Group 53 Linkage Group 54 Linkage Group bni9.44h.4oto 12 EactMcat430 EacaMcat.450 io isui4ox EaacMcag.520 R15p.610 EaGtMcat490 umc62d ~umcl07h.l ~isui46d.5170 as ~ isu7ge.5560 tumc130d.5260 ~~'~ umc 161x isul46x.4oto 4.8- ~~- ~"Q'ro tumcl 6x:5300 tumc7x as umc65x.3660 umc12d.5260 umcl5d.9020 isul46d.4870 EI :ll= umcl Linkage Group 56 Linkage Group 57 Linkage Group 58 Linkage Group 59 Linkage Group ao ~CSr ao _~CSb69d ~ Ml0.800 '-' 1.1j ~"'''.''''' Wl1.260 itee: isui23x.l0040 isu38d.3000 umc43b.2850 G4p A Wl1.210 Linkage Group 61 Linkage Group 62 Linkage Group 63 Linkage Group 64 Linkage Group _~umcl06d.i _~umcI66x ~ csri33d ~CSr ~ EacaMcta.190 bni6.25h.8570 umc83h.4040 csri33e.7500 sps-ld.5090 EaacMcag.220 and eoupling phases should oeeur at approximately equal frequencies. If ehromosome pairing were eompletely random, as in polysomie polyploids, then markers linked in repulsion phase at a maximum of 10 em would require a mapping population of at least 750 individuals for their deteetion, assuming a polysomie oetaploid with striet bivalent pairing (Wu et al., 1992). Deteetion of repulsion phase linkages in both genomes with a population size of only 100 individuals strongly suggests that these genomes are neither fully polysomie nor disornic. This result agrees with a previous study of a subset of 44 individuals from this population (Al-Janabi et al., 1994a) and with Mudge et al. (1996), who studied the same eross with arbitrarily primed PCR markers. This strongly suggests partially preferential ehromosome pairing, at least for some linkage groups, Linkages in repulsion imply that a saturated map for euploid 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 who graciously shared probes and other information, in particular Michael Lee for ISU prabes and unpublished data. Thanks to: the Hawaiian Sugar Planters' Association (HSPA, Aiea), in particular Paul Moore and K-K- Wu, for supplying field data and plant material; Perkin-Elmer/ABI Division for supplying AFLP kits for beta-testing; Keira Maiden for technical assistance; Joshua Kohn, Ivor Royston and the Sidney Kimmel Cancer Center for varied assistance. c.t.g. was supported by a fellowship frorn the Brazilian National Research Council of the Ministry of Seience and Technology (MCT/CNPq/RHAE) No /95-1. B.WS.S., RJ.H., and G.R.S. were supported in part by a grant from Copersucar Technology Center (Piracicaba, Brazil) and the Mauritius Sugar Industry Research Institute (Réduit, Mauritius). RESUMO Uma progênie de 100 indivíduos FI obtidos de um cruzamento entre cana-de-açúcar (5. officinarum 'LA Purple') e seu suposto progenitor (S. robustum 'Moi 5829') foi analisada utilizando marcadores moleculares em dose única. Marcadores do tipo AP-PCR, RFLP e AFLP,gerando um totalde 642 polimorfismos, foram mapeados em ambas espécies. O mapa genético de LA Purple foi composto de 341 marcadores, distribuídos em 74 grupos de

8 132 Guimarães et ai. ligação e cm, enquanto que o mapa de ligação de MoI 5829 continha 301 marcadores ao longo de 65 grupos de ligação e cm. A transmissão genética nessas duas espécies apresentou polissomia incompleta devido a detecção de 15% dos marcadores em dose simples ligados em fase de repulsão e 13% desses em MoI Devido a essa polissornia incompleta, os marcadores em dose múltipla não puderam ser mapeados por falta de um modelo genético para descrever tal segregação. O mapeamento de sondas' de RFLP, conservadas entre espécies próximas evolutivamente, permitirá que os mapas genéticos gerados sejam utilizados como poderosas ferramentas no melhoramento e em estudos de ecologia, evolução e biologia molecular dentro das Andropogoneas. REFERENCES Al-Janabi, S.M., Honeycutt, R.J., McClelland, M. and Sobral, B.W.S. (1993). A genetic linkage map of Saccharum spontaneum L. 'SES 208'. Genetics 134: Al-Janabi, S.M., Honeycutt, R.J. and Sobral, B.W.S. (l994a). 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