Anchor Probes for Comparative Mapping of Grass Species

Allen E. Van Deynze1,2, Mark E. Sorrells1 and Susan R. McCouch1*

1Department of Plant Breeding and Biometry, Cornell University, Ithaca, NY, USA, 14853

2 Calgene, 1920 Fifth Street, Davis, CA, USA, 95616


*corresponding author: ph: (607) 255-4020, Fax: (607) 255-6683, E-mail: srm4@cornell.edu



Introduction

Conventionally, species such as rice, wheat, barley, oat, maize, sorghum, and sugarcane have been studied separately. Comparative mapping using DNA proves can be used to combine genetic information from these related species. One of the prerequisites for comparative mapping is a genetic linkage map for each species. RFLP maps presently exist for rice (Oryza sativa L.) (Causse et al. 1994, Kurata et al. 1994a), hexaploid wheat (Triticum aestivum L. em. Thell) (Chao et al. 1989; Liu and Tsunewaki 1991; Devos et al. 1992; 1993; Xie et al. 1993; Nelson et al. 1995abc; Van Deynze et al. 1995a; Gale et al. 1995), barley (Hordeum species (Heun et al. 1991; Kleinhofs et al. 1993; Graner et al. 1991; 1994), oat (Avena species) (O'Donoughue et al. 1992; 1995; Rayapati et al. 1995; Van Deynze et al. 1995b), maize (Zea mays L.) (Burr and Burr 1991; Gardiner et al. 1993), sorghum (Sorghum species) (Chittenden et al. 1994), and sugarcane (Saccharum species) (da Silva et al. 1993).

Another requirement for comparative mapping is a set of probes that can be used to evaluate homoeology and conservation of linkage groups. In the course of surveying probes from an array of different libraries, many were found to hybridize well to multiple species of grasses. After systematic evaluation of these probes on seven species (rice, wheat, barley, oat, maize, sorghum, and sugarcane), a subset of those that gave good hybridization signal on a majority of species was designated as "anchor probes" for comparative mapping.

Consensus maps can be developed for related species with similar genomes. Such maps are based on conservation of linkage groups composed of homologous and homoeologous chromosomes. These maps merge information about closely related species and are useful for cross-referencing genetic information from more distantly related species. A consensus map has been developed for Triticeae species, based on a common set of markers mapped onto the respective linkage groups of T. aestivum, T. tauschii, and Hordeum species (Nelson et al. 1995ab; Van Deynze et al. 1995ac).

Comparative maps have been constructed for several Gramineae species. Cloned genes, random cDNA's and genomic DNA markers from maize were used to compare the maize and sorghum genomes (Whitkus et al. 1992; Melake Berhan et al. 1993). Substantial conservation of large segments of linkage groups was observed in these two species, with segments of most sorghum linkage groups showing homoeology to two independent segments in the maize genome. A similar study comparing maize, sorghum and sugarcane (Saccharum species) (Grivet et al., 1994), reported that the degree of conservation observed between sorghum and sugarcane was greater than between sorghum and maize, largely because there was less duplication in the sorghum and sugarcane genomes. Devos et al. (1993) demonstrated conservation of the wheat and rye genomes, with a few well-defined rearrangements distinguishing the rye genome.

Comparisons among more distantly related crops require a common set of probes. Probes from rice, oat, and barley cDNA libraries were used to analyze the genetic relationships among rice, wheat, and maize (Ahn and Tanksley 1993; Ahn et al. 1993), and among rice, Triticeae species, maize, and oat (Van Deynze et al. 1995abc). Wheat - rice comparisons using cDNA's (Kurata et al. 1994b), and comparisons between wheat group 7 chromosomes and maize chromosome 9 (Devos et al., 1994) have also been reported. These diverse comparative maps based on probes which hybridize across the cereals provide a basis for combining the genetic information that is available in these crops.

The objective of this study was to identify and characterize a set of DNA probes that would be useful for comparative mapping among several Gramineae species.



Materials and Methods

Selection of probes

Priority was given to cDNA rather than genomic DNA clones for this study because of the greater sequence conservation in these transcribed regions of the genome (S.N. Ahn, unpublished data), expecially important in hybridization experiments involving species that diverged from a common ancestor millions of years ago. The anchor probes were selected based on the following criteria: a) good hybridization signal in rice, barley, wheat, oat, maize, sorghum, and sugarcane, or in most of these species, b) single copy in rice, c) map position previously established in rice and maize, and/or in wheat, d) uniform genome coverage in rice with additions targetted to improve genome coverage in other crops. Probes were chosen based on the rice RFLP map because rice currently has one of the best characterized grass genomes (Causse et al., 1994; Kurata et al., 1994a). It is a diploid with a large proportion of single copy DNA (approximately 85% of the DNA behaves as single copy at high stringency, 0.5X SSC at 65¼C) (McCouch et al. 1988), and few patterns in the distribution of duplicated loci are evident (Causse et al. 1994). The BCD (barley cDNA), CDO (oat cDNA), WG (wheat genomic), and RZ (rice cDNA) probe libraries used in this study were described by Heun et al. (1991) and Causse et al. (1994), respectively.


RFLP and data analyses

Inserts from each of the probes were amplified using the polymerase chain reaction (PCR) with M13 primers and hybridized (stringency of final wash = 0.5X SSC at 65¼C) to membranes (Hybond N+) containing a single lane of rice (Oryza sativa, cv. IR36), oat (Avena sativa, cv. Ogle), barley (Hordeum vulgare, cv. SE16), wheat (Triticum aestivum, cv. Chinese Spring), sugarcane (Saccharum spontaneum, cv. SE 208), sorghum (Sorghum bicolor, cv. BTx406), and maize (Zea mays, cv. CO159) DNA digested with EcoRI with the concentration adjusted for genome size of each species (8 ug/lane for rice, 15 ug/lane for oat, 12 ug/lane for barley, 15 ug/lane for wheat, 12 ug/lane for sugarcane, 8 ug/lane for sorghum, and 12 ug/lane for maize). Loci detected as restriction fragment length polymorphisms with the same set of probes were placed on existing linkage maps for wheat (Nelson et al. 1995abc; Van Deynze et al. 1995ac), rice (Causse et al. 1994), maize (Ahn and Tanksley 1993) and diploid oat (O'Donoughue et al. 1992; Van Deynze et al.1995b). Polymorphic and nonpolymorphic markers were assigned to chromosomes or chromosome arms based on hybridization to nullitetrasomic (Sears 1966) and ditelosomic stocks (Sears and Sears 1978).

The same procedures for RFLP analyses (Heun et al. 1991; Causse et al., 1994) were used for each population. The individual linkage maps were developed by placing markers at a LOD threshold of 2.0 using Mapmaker v3.0 (Lander et al. 1987) with the Kosambi mapping function (Kosambi 1944). The consensus map for wheat, T.tauschii and Hordeum species (Van Deynze et al. 1995c), hereafter referred to as the Triticeae consensus map, was used to make comparisons among these species and rice, maize and oat.


Results

Probes

A set of 153 anchor probes (21 BCDs, 64 CDOs, 67 RZs and 1 WG) was chosen based on the criteria outlined in the previous section. The intensity of hybridization signal and copy number for each probe in each reference species were determined based on an evaluation of screening filters (Figure 1).

Figure 1. Autoradiogram of CDO460 hybridized to rice (IR36), oat (Ogle), barley (SE16), wheat (Chinese spring), sugarcane (SES208), sorghum (BTx406) and maize (CO159) digested with EcoRI in lanes 2-8, respectively; lane 1 lambda/HindIII size marker.



This data was complemented by the results obtained on mapping filters, and is summarized in Tables 1 and 2. The proportion of probes in the current anchor set giving good hybridization signals for rice and maize is biased upward because a large proportion of these clones had been previously mapped on these species.

More than 75% of oat cDNAs produced strong hybridization signals in the 7 species tested (Fig.2).

Figure 2. Proportion of clones providing good hybridization signal (stringency = 0.5X SSC at 65C) for selected barley (BCD), oat (CDO) and rice (RZ) cDNA clones.



For barley cDNAs, 100% hybridized to barley, wheat, oat and maize, about 90% to rice, 82% to sorghum, and only 58% to sugarcane. Rice cDNAs hybridized 100% to rice, 78-90% to the tropical crops, maize, sorghum and sugarcane, and 48-55% to wheat, barley, and oat genomic DNA.

Genome coverage

Anchor probes meeting the criteria outlined in the "selection of probes" section are well distributed throughout the rice linkage map except for distal portions of chromosomes 2, 7 and 12 (Figure 3-See end of document). Markers in these regions (Causse et al., 1994) consisted of rice genomic and cDNA clones that did not hybridize well to other species. Three probes in the anchor set, BCD454, CDO127 and CDO507, detected duplicate loci in rice. The anchor probes also provide uniform genome coverage for maize and oat (Figures 4 and 5-See end of document). In Triticeae, distal portions of chromosomes are underrepresented and there is clustering of marker loci near centromeres on 6 of the 7 chromosomes (Figure 6-See end of document). Although fewer anchor probes were mapped in wheat due to limited polymorphism, virtually all fragments (polymorphic and nonpolymorphic) were assigned to wheat chromosomes or chromosomes arms with nullitetrasomic and/or ditelosomic stocks (Tables 1 and 2).



Discussion

Identification of a set of anchor probes

The identification of a set of anchor probes facilitates comparative mapping efforts in the Gramineae family by combining genetic information from all species mapped, rather than limiting information to specific comparisons. Probes that are informative for comparative mapping will a) hybridize to the majority of target Gramineae species, b) be single copy in a reference species (such as rice), and c) provide good genome coverage in all species.

The set of anchor probes represents a collaborative effort between researchers studying rice, wheat, barley and oat with probes derived from each of these species. This set of probes provides good coverage of the linkage maps of rice, maize and oat. Linkage data is not available for 84 of the 153 probes for Triticeae species, mainly due to the low polymorphism in cultivated wheat (Figure 6-See end of document), but this is more than compensated by the use of aneuploid stocks that provide arm locations for nearly all hybridizing fragments (Table 2). For example, loci detected with CDO122 map to the same relative positions in homoeologous segments of rice chromosome 3, maize 1 and 5, and oat E (Figures 3 to 5-See end of document). If the positions of orthologous loci detected with this probe were conserved relative to Triticeae species, the distal portion of Triticeae chromosome 4S would be better represented (Van Deynze et al.1995c). The positions of these markers must be confirmed by mapping in additional populations of Triticeae species or by using the deletion stocks developed by Werner et al. (1992). Additional probes mapping to poorly represented regions in the Triticeae from other DNA libraries must be screened to meet the criteria and added to this set of anchor probes in the future.


Rice as model for comparative mapping in Gramineae

In establishing the anchor probe set, priority was given to probes detecting single copy sequences and providing good genome coverage in rice. The majority of the probes selected also detected low copy sequences in barley, wheat, oat, maize, sorghum and sugarcane (data not shown). The selected probes represented the genomes of oat, maize and Triticeae species. The rice genome also provides a good model for comparative mapping in monocots because it is a diploid with a small genome (0.45 pg per haploid cell) (Arumuganathan and Earle, 1991) and its well developed molecular maps, along with the availability of large-insert libraries, provide an abundance of publicly available DNA markers. Efforts to integrate the two principal genetic linkage maps of rice (Causse et al. 1994; Kurata et al. 1994a) are underway and will provide information on over 2,200 DNA markers with an average distance of 1 marker every 0.8 cM, and an average DNA/cM ratio of 250-300 kb/cM (Van Houten et al., 1996). The use of these markers and YAC (Umehara et al. 1994), BAC (Wang et al. 1995; Zhang et al., 1995) and cosmid libraries (Song et al., 1995), will facilitate comparative mapping and positional gene cloning in Gramineae species.

The probes in our current anchor marker set are being end-sequenced (W. Park, Biochemistry Dept., Texas A&M, College Station, USA; and J. Bennett, Division of Plant Breeding, Genetics and Biochemistry , IRRI, Los Banos, Philippines, personal communication), and plans to construct specific primers for them are underway.

The use of anchor probes for comparative mapping is an efficient way of establishing genetic relationships for comparisons among all the species being studied (Ahn and Tanksley 1993; Ahn et al. 1993; Van Deynze et al. 1995abc). Collaboration among research groups involved in mapping of related Gramineae species will contribute to extending the set of anchor markers. As the development of comparative maps is a dynamic process, we envision the expansion of this set of clones to include 1) cDNA clones from a wider array of species and libraries, 2) cDNA clones of known gene function which are of agronomic importance, and 3) clones from poorly represented regions of specific genomes that give single copy hybridization signal across a majority of Gramineae species tested.

All materials and information relating to this work are available to other researchers. The anchor probes described in this report are available from Susan McCouch (email: srm4@cornell.edu) and Mark Sorrells (email: mes12@cornell.edu), Department of Plant Breeding and Biometry, Cornell University, Ithaca, NY, USA, 14853-1901). The data, images of screening filters and linkage maps developed using the anchor probes are continuously being updated and are available on the World Wide Web [http://greengenes.cit.cornell.edu/anchors/].




Acknowledgements

We wish to thank Steve Tanksley, Bill Wilson, Daniella Braga, and Clare Nelson for critical reading of this manuscript, Andrew Paterson for DNA of Sorghum bicolor used in survey filters, Carole Morehouse for help formatting the manuscript, and the USDA Plant Genome National Research Initiative (USDA NRI Grant No. 94-37300-0324; USDA NRI/CGP Grant No. 94-37310-0661; and subcontract numbers 92-G0161-Cornell of USDA NRI Grant No. 92-37300-7550), and Copersucar Technology Center, the International Consortium for Sugarcane Biotechnology, for financial support for this project.



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Figures


Figure 1. Autoradiogram of CDO460 hybridized to rice (IR36), oat (Ogle), barley (SE16), wheat (Chinese spring), sugarcane (SES208), sorghum (BTx406) and maize (CO159) digested with EcoRI in lanes 2-8, respectively; lane 1 lambda/HindIII size marker.



Figure 2. Proportion of clones providing good hybridization signal (stringency = 0.5X SSC at 65C) for selected barley (BCD), oat (CDO) and rice (RZ) cDNA clones.



Figure 3. Position of anchor probes overlaid on the framework rice RFLP map (Causse et al. 1994). Loci that map at LOD <2.5 are assigned to intervals and indicated in parentheses.

***Rice Anchors: Chr. 1-3, Chr. 4-6, Chr. 7-9, Chr. 10-12





Figure 4. Position of anchor probes overlaid on a consensus map of Triticeae species (T. aestivum, T. tauschii, Hordeum spp.)(Van Deynze et al. 1995ac).

***Maize Anchors: Chr. 1-2, Chr. 3-5, Chr. 6-7, Chr. 8-10




Figure 5. Position of anchor probes overlaid on maize RFLP map (Ahn and Tanksley 1993). Loci that map at LOD <2.0 are assigned to intervals and indicated in parentheses.

***Fig. 5. Oat Anchors: Chr. A-B, Chr. C-E, Chr. F-G




Figure 6. Position of anchor probes overlaid on diploid oat RFLP map (Van Deynze et al. 1995b). Loci that map at LOD <2.0 are assigned to intervals and indicated in parentheses.

***Fig. 6. Triticeae Anchors: Chr. 1-2, Chr. 3-5, Chr. 6-7





Anchors sent: