A comparison of three Agrigenetics maize RFLP linkage maps
--Jennifer Shoemaker, David Zaitlin, Jeffrey Horn, Sandra DeMars, Jane Kirschman and Jan Pitas
The Agrigenetics RFLP linkage map of maize has undergone considerable change since it was first published four years ago (Murray et al., MNL 62:89-91, 1988). Several more cloned genes have been added, and the map has expanded with the inclusion of segregation data for an additional 78 public probes. A cytological marker, the gene cluster encoding the cytoplasmic 5S rRNA, was placed on the long arm of chromosome 2 (see Zaitlin, Steffensen and Zimmer in this issue), and a genomic SalI fragment (6-1-1) that lies adjacent to a Mu1 element (Qin, M and Ellingboe, A) has been mapped to a locus on chromosome 8. All of the 3-class RFLP probe segregation data has been reanalyzed, and we have chosen to present the map in a format that reflects the specific population in which each probe locus was actually mapped rather than as a grand composite (as was done previously). Differences between the current map and the 1988 version and discrepancies between the Agrigenetics linkage map and the maps developed at the University of Missouri and Brookhaven National Laboratories are discussed.
Mapping populations: Two-hundred ninety-one probes were mapped to 293 discrete loci in an F2 progeny set from A619 X Mangelsdorf, PC's Multiple Tester (map a in the Figure). Allelic segregation data were recorded from a minimum of 88 and a maximum of 99 individuals for each probe. The six recessive phenotypic traits (from Mangelsdorf's Tester) were all scored in the field as 2-class data for 90 plants. Map b was calculated from the segregation scores for 87 probes (89 loci) in 93 F2 progeny from B68 Ht X B73 Ht rhm. In addition to the two F2 populations, we mapped 115 probes to 120 loci in a set of 200 related inbred lines (a recombinant inbred family; see Burr, B and Burr, FA, Trends Genet. 17:55-60, 1991) from the cross De811 X B73 Ht rhm. Also, three single genes for resistance to fungal leaf diseases (Rp1, Ht2, and rhm) have been mapped in other populations and are not included here.
Molecular probes: The majority of the Agrigenetics proprietary maize RFLP probes were isolated from two specific cDNA libraries made from poly(dA)-containing maize RNA. Clones prepared from etiolated coleoptile mRNA in pSP64 (Melton et al., Nucl. Acids Res. 12:7035-7056, 1984) are given the prefix "c", while those prepared from root polysomal mRNA in pGEM-2 are designated "r" clones. A number of genomic clones are also represented. PstI-derived clones ("p") are in pGEM-3Z and XhoI fragments inserted into the SalI site of pGEM-3 are "x" clones. The pGEM plasmid vectors were obtained from Promega Corporation in Madison, WI. All of the PstI-generated clones from the public laboratories, those with BNL and UMC prefixes, were recloned into the plasmid pJKKmf(-) (Kirschman and Cramer, Gene 68:163-165, 1988), which, as with the pGEM family of plasmids, enables the synthesis of a labelled RNA transcript of high specific-activity ("riboprobe") from either of two bacteriophage promoters.
Mapping program: We have developed proprietary mapping software that enables us to construct genetic linkage maps from molecular probe segregation data in virtually any population of known structure. The basic program, Surveyor, employs orthogonal contrasts to detect segregation distortion not due to linkage. If population sizes are too small (~32 individuals for F2), no linkage is calculated. Currently, Surveyor does not use orthogonal contrasts when evaluating the data from a recombinant inbred population. Thus for the map labelled c, we deleted all probes and individuals that had greater than 10% missing data; the final population size was 182 individuals.
Surveyor uses maximum likelihood methods to calculate genetic linkage. Except in the simplest cases (e.g. BC1, F2), there is no "formula" for calculating linkage based on observed frequencies; instead, linkage values are calculated by numerically maximizing complicated likelihood formulae with the aid of a high-speed computer. Once the linkage values have been calculated, the set of probes is partitioned into linkage groups based on a similarity relation which depends on a tolerance parameter. Early in mapping, the researcher may explicitly define this parameter; as the linkage map becomes better defined, the researcher can specify the expected number of chromosomes and have the computer calculate a suitable value for the parameter. Each linkage group is then ordered in such a way that the total variance associated with the resulting order is minimized. We use a method called "simulated annealing" to search the space of all possible partition orders in an efficient manner. This process results in a maximum likelihood order for each linkage group. Graphical images are then generated by the Agrigenetics program Drawmap and can be directly ported to a Sun workstation for manipulation in Framemaker.
Figure 1. Agrigenetics maize RFLP linkage map. The chromosomes are oriented with the short arm at the top of the page, and the hatched termini signify that the actual ends of the arms (telomeres) are undefined at present. Linkage distances (refer to the scale) are given in percent recombination. Allelic segregation data for probes scored in each population were analyzed using the computer program Surveyor. Agrigenetics probe loci are shown on the right side of the figures, and loci detected by probes from public institutions (UMC and BNL), cloned genes, isozymes and phenotypic markers are shown on the left. Isozyme and gene loci are italicized, and the positions of phenotypic markers are indicated by dotted lines. Lower case suffixes indicate that a given locus is reiterated within the genome in at least one of the three populations (UMC44a on 10L and UMC44b on 2L, for example). For each chromosome, the three independent maps have been aligned at the first locus common to all three.
Features of the linkage maps: As of January 1992, 314 individual RFLP probes, the two isoenzymes Enp1 and Pgd1 (Mdh1 was removed because of the poor quality of the data), and six phenotypic markers (bm2, lg1, su1, y1, j1, and g1) have been mapped in three populations. Thirty-eight probes are from the University of Missouri, Columbia and 57 probes are from Brookhaven National Laboratories. The remaining 219 probes were developed at Agrigenetics. The maps include 16 cloned gene sequences of known identity, 11 of which are listed in Murray et al., 1988. Five genes have been added since 1988: a genomic clone of the maize aldolase gene (Dennis et al., J. Mol. Biol. 202:759-767, 1988) and two cDNA clones (OCSBF-1 and OCSBF-2) that encode protein factors that bind specifically to the octopine synthase (ocs) enhancer element of Agrobacterium tumefaciens Ti plasmids (Singh et al., Plant Cell 2:891-903, 1990) were all received from Liz Dennis, L; the maize phytochrome sequence (Phy2 on the map) was provided by Allen Christensen, A (Christensen and Quail, Gene 85:381-390, 1989) and cLC46E, a cDNA clone of C2 was from Udo Wienand, U (personal communication). A genomic clone specific for O2 (opaque-2), received from R. Schmidt, R (Schmidt et al., Science 238:960-963, 1987), was not mapped because there was no detectable sequence polymorphism around this locus in our three mapping populations.
UMC42 and UMC133 map to different positions as compared to the most recent UMC core RFLP map (Gardiner, J et al., MNL 65:54-56, 1991). We map these loci to chromosomes 6 and 1 respectively while the UMC group maps them both to chromosome 4. These discrepancies are most easily explained by the fact that the probes were mapped in populations that differed with respect to the parental inbreds and the number of individuals. We find that UMC57 maps to chromosome 7, while the UMC group mapped it to chromosome 10 in 1989 (J. Gardiner, J, personal communication), although the latest UMC map does not include this locus. A comparison of the Agrigenetics maps with a maize RFLP map provided by Ben Burr, B in April, 1991 reveals several differences with respect to BNL loci. In addition to small inversions on chromosomes 1 and 9 involving tightly linked loci, there is a rearrangement on chromosome 7 that includes BNL14.07, BNL7.61, BNL8.37 and BNL8.21. BNL6.10 and BNL6.22 are separated on chromosome 5 by at least 10 map units on the BNL map, while we find that these two probes map to one locus. On the short arm of chromosome 6 we define independent loci for UMC85 and BNL6.29 that are 2 map units apart (map c), while these two probes map to a single locus on the BNL map.
A comparison of the three Agrigenetics maps shows that they are essentially colinear. Except for some minor rearrangements, the locus orders are essentially preserved between map a (from A619 X Mangelsdorf, PC's Tester) and the composite map published in 1988. The most notable changes are the inversion on the top of chromosome 1 (BNL8.05 is now at the end of 1S, while c467 was the most distal on the 1988 map), and a few places where loci are very tightly linked or where probes that previously mapped to a single locus now define separate loci (r301, r190, r89 and c4 on 4a). These changes are due to probe additions as well as to improvements and corrections in the mapping program.
Astute observers will notice that there are occasions where all members of a reiterated pair or trio of loci are not represented on the maps. This is because an independently segregating secondary band could not be firmly placed (r45b and UMC8a are examples) or, as with c303b and c539b, too few individuals were reliably scored to calculate linkage (see above). Also, there are several instances in which a large region of a chromosome is missing and is represented by a hatched line. This indicates (i) that there were no polymorphic probes found in that region in a particular population (3b, 3c, and 10b), or (ii) that probes scored as polymorphic and previously known to map to the region did not show linkage to the other loci on the chromosome (1b).
The authors would like to acknowledge Greg Schulenberg, G and Cheryl
Morstad, C for their expert technical assistance, Kim Maly, K, Diana Mefford,
D and Signe Melton, S for help with the map drawing, and Michael Murray,
M for his support and guidance. We also thank Jack Gardiner, J and Ed Coe,
EH, Jr of the University of Missouri, Columbia and Ben Burr, B of the Brookhaven
National Laboratories for providing us with their probes.
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