Genetic maps of Saccharum officinarum L. and Saccharum robustum Brandes & Jew. ex grassl *

 

Claudia T. Guimarães¹, Rhonda J. Honeycutt², Gavin R. Sills³ and Bruno W.S. Sobral^4
1Universidade Federal de Viçosa - BIOAGRO, 36571-000 Viçosa, MG, Brasil. Send correspondence to C.T.G. E-mail: claudiag@alunos.ufv.br
² Sidney Kimmel Cancer Center, 3099 Science Park Rd #200, San Diego, CA 92121, USA.
³ Department of Crop and Soil Sciences, Washington State University, Pullman, WA 99164, USA.
⁴ National Center for Genome Resources, 1800 Old Pecos Trail, Santa Fe, 87505 NM, USA.
* Part of a thesis presented by C.T.G. to the Department of Genetics and Breeding, Universidade Federal de Viçosa, 1999, in partial fulfillment of the requirements for the Ph.D. degree.

 

 

ABSTRACT

Genetic analysis was performed in a population composed of 100 F₁ individuals derived from a cross between a cultivated sugarcane (S. officinarum 'LA Purple') and its proposed progenitor species (S. robustum 'Mol 5829'). Various types (arbitrarily primed-PCR, RFLPs, and AFLPs) of single-dose DNA markers (SDMs) were used to construct genetic linkage maps for both species. The LA Purple map was composed of 341 SDMs, spanning 74 linkage groups and 1,881 cM, while the Mol 5829 map contained 301 SDMs, spanning 65 linkage groups and 1,189 cM. Transmission genetics in these two species showed incomplete polysomy based on the detection of 15% of SDMs linked in repulsion in LA Purple and 13% of these in Mol 5829. Because of this incomplete polysomy, multiple-dose markers could not be mapped for lack of a genetic model for their segregation. Due to inclusion of RFLP anchor probes, conserved in related species, the resulting maps will serve as useful tools for 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 (Al-Janabi et al., 1993; daSilva et al., 1995). Polyploidy in Saccharum is widespread and is largely responsible for its genetic and taxonomic complexity. Studies using DNA markers and molecular cytogenetics revealed polysomic inheritance and octoploidy (x = 8) within S. spontaneum (2n = 64, from India) (Al-Janabi et al., 1993; daSilva et al., 1995; D'Hont et al., 1996). The basic chromosome number and level 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 segregate 1:1 in the progeny. More recently, daSilva (1993) and Ripol (1994) presented a methodology for mapping multiple dose markers in polysomic 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 genomic 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 genomic 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).

 

MATERIAL AND METHODS

Plant materials

Plant materials were kindly provided by the Hawaiian Sugar Planters' Association (Aiea, HI). The population consisted of 100 F₁ individuals produced by crossing S. officinarum 'LA Purple' as female with S. robustum `Mol 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).

DNA markers

RFLPs

Genomic DNA was extracted according to the method of Honeycutt et al. (1992). Fifteen mg of genomic DNA from parents and 100 progeny was restricted individually with DraI, EcoRI, HindIII, and XbaI, and resolved in agarose gels. The gels were blotted and Southern hybridization was performed according to daSilva (1993). After hybridization, blots were exposed to BioMax film (Kodak) at -80^oC 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 F₁ 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 Saccharum spontaneum 'SES 208' (daSilva et al., 1993, 1995), were also used as RFLP probes. Cloned genes from sucrose metabolism and transport pathways, including smp-1, a sugarcane membrane protein and putative glucose transporter (Bugos and Thom, 1993), sps-1, sucrose phosphate synthase from maize (Worrell et al., 1991), SS-1, maize sucrose synthase (McCarty et al., 1986), and HBr-1, 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 scoring of amplified products were performed according to Al-Janabi et al. (1993). Arbitrarily primed PCR products amplified with a³²P-dCTP were resolved in 5% polyacrylamide-50% urea gels in 1x 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 restriction fragment amplification

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 completion with EcoRI and MseI. Restricted genomic DNA fragments were ligated to EcoRI and MseI adapters, diluted 1:10, and pre-amplified using AFLP core primers, each having one selective nucleotide. Pre-amplification products were then diluted to 1:50 and used as a template for selective amplification using the combinations of MseI- and EcoRI-specific primers, each containing three selective nucleotides. EcoRI-selective primers were labeled with g³²P-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 (umc, bnl, isu, csb, csc, csr or sg), followed by the first letter of the restriction enzyme used (d, e, h, or x for DraI, EcoRI, HindIII, 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 1-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 (CG6-CG9), 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 EcoRI (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 (0), and analyzed for dosage among F₁ progeny using chi-square tests (P < 0.05), as described by Wu et al. (1992) and daSilva et al. (1995). Because of the double-pseudo-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 determined using MapMaker v 2.0 for the Macintosh (Lander et al., 1987) by coding the data as haploid (as the population is resultant from a double pseudo-testcross mating strategy). SDMs were grouped using a minimum LOD of 7.0 and a maximum recombination fraction (r) of r < 0.25 (Wu et al., 1992). Linkage groups were then ordered using multi-point analyses. Markers at r < 3 cM could not be ordered accurately because of the relatively 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 Al-Janabi et al. (1993).

 

RESULTS AND DISCUSSION

Linkage maps

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. In Mol 5829, linkage analysis of 301 SDMs generated 65 linkage 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. In Mol 5829, linked markers covered 1,189 cM, an average of 5.74 cM per marker.

 

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. The markers are shown on the right of the linkage groups; markers followed by an asterisk (*) are linked in repulsion phase.

 

 

Figure 2 - Genetic linkage map of Saccharum robustum `Mol 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.

 

Marker distribution and mapping output

The total number of polymorphisms between the two genomes analyzed was significantly different with c² = 4.49; P < 0.05 (503 for LA Purple and 438 for Mol 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 to the results of daSilva (1993), that found 73% of polymorphisms in S. spontaneum to be single dose.

 

Table I - Summary of marker data.

^aEach experiment consists of: RFLP, probe/restriction enzyme combination; AP-PCR, primer reaction; AFLP, EcoRI and MseI seletive primer combination. ^bSingle-dose markers (SDMs) determined using a c² test (P < 0.05), as described by Wu et al. (1992). LAP, Saccharum officinarum LA Purple; Mol, Saccharum robustum Mol 5829.

 

Markers generated by different methods were not uniformly distributed across the linkage groups. In LA Purple, 26% of the linkage groups had all three marker types, and in Mol 5829, just 9% of the linkage groups were covered by all types of markers. Lack of uniform 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 al. (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 pattern. The signal produced with maize genomic and cDNA probes suggests a high degree of DNA sequence similarity among these species, despite at least 25 million years of evolution since they shared a common ancestor (Al-Janabi et al., 1994b; Sobral et al., 1994). A similar result was reported by daSilva et al. (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 Mol 5829 were in repulsion phase and were assigned to 11 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 and coupling phases should occur at approximately equal frequencies. If chromosome pairing were completely random, as in polysomic polyploids, then markers linked in repulsion phase at a maximum of 10 cM would require a mapping population of at least 750 individuals for their detection, assuming a polysomic octaploid with strict bivalent pairing (Wu et al., 1992). Detection of repulsion phase linkages in both genomes with a population size of only 100 individuals strongly suggests that these genomes are neither fully polysomic nor disomic. 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 cross with arbitrarily primed PCR markers. This strongly suggests partially preferential chromosome 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 probes 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 from the Brazilian National Research Council of the Ministry of Science and Technology (MCT/CNPq/RHAE) No. 260007/95-1. B.W.S.S., R.J.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 F₁ obtidos de um cruzamento entre cana-de-açúcar (S. officinarum `LA Purple') e seu suposto progenitor (S. robustum `Mol 5829') foi analisada utilizando marcadores moleculares em dose única. Marcadores do tipo AP-PCR, RFLP e AFLP, gerando um total de 642 polimorfismos, foram mapeados em ambas espécies. O mapa genético de LA Purple foi composto de 341 marcadores, distribuídos em 74 grupos de ligação e 1.881 cM, enquanto que o mapa de ligação de Mol 5829 continha 301 marcadores ao longo de 65 grupos de ligação e 1.189 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 Mol 5829. Devido a essa polissomia 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.

 

 

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(Received March 16, 1998)