a high-density genetic recombination map of sequence - genetics

Copyright 2003 by the Genetics Society of America

A High-Density Genetic Recombination Map of Sequence-Tagged Sites forSorghum, as a Framework for Comparative Structural and Evolutionary

Genomics of Tropical Grains and Grasses

John E. Bowers,* Colette Abbey,† Sharon Anderson,† Charlene Chang,† Xavier Draye,†

Alison H. Hoppe,† Russell Jessup,† Cornelia Lemke,* Jennifer Lennington,†

Zhikang Li,† Yann-rong Lin,† Sin-chieh Liu,† Lijun Luo,† Barry S. Marler,*Reiguang Ming,† Sharon E. Mitchell,‡ Dou Qiang,† Kim Reischmann,†

Stefan R. Schulze,* D. Neil Skinner,* Yue-wen Wang,†

Stephen Kresovich,‡ Keith F. Schertz†

and Andrew H. Paterson*,†,1

*Plant Genome Mapping Laboratory, University of Georgia, Athens, Georgia 30602, †Plant Genome Mapping Laboratory,Department of Soil and Crop Science, Texas A&M University, College Station, Texas 77843 and

‡Institute for Genomic Diversity, Cornell University, Ithaca, New York 14850

Manuscript received February 5, 2003Accepted for publication May 5, 2003

ABSTRACTWe report a genetic recombination map for Sorghum of 2512 loci spaced at average 0.4 cM (�300 kb)

intervals based on 2050 RFLP probes, including 865 heterologous probes that foster comparative genomicsof Saccharum (sugarcane), Zea (maize), Oryza (rice), Pennisetum (millet, buffelgrass), the Triticeae(wheat, barley, oat, rye), and Arabidopsis. Mapped loci identify 61.5% of the recombination events in thisprogeny set and reveal strong positive crossover interference acting across intervals of �50 cM. Significantvariations in DNA marker density are related to possible centromeric regions and to probable chromosomestructural rearrangements between Sorghum bicolor and S. propinquum, but not to variation in levels ofintraspecific allelic richness. While cDNA and genomic clones are similarly distributed across the genome,SSR-containing clones show different abundance patterns. Rapidly evolving hypomethylated DNA maycontribute to intraspecific genomic differentiation. Nonrandom distribution patterns of multiple locidetected by 357 probes suggest ancient chromosomal duplication followed by extensive rearrangementand gene loss. Exemplifying the value of these data for comparative genomics, we support and extendprior findings regarding maize-sorghum synteny—in particular, 45% of comparative loci fall outside theinferred colinear/syntenic regions, suggesting that many small rearrangements have occurred since maize-sorghum divergence. These genetically anchored sequence-tagged sites will foster many structural, func-tional and evolutionary genomic studies in major food, feed, and biomass crops.

AS a model for the large genomes of many tropical cally important crops, may have shared a common an-cestor as recently as 5 million years ago (Sobral et al.grasses, sorghum [Sorghum bicolor L. Moench.; 748–1994), retain similar gene order (Ming et al. 1998),772 million base pairs (Mbp); Arumuganathan andand even produce viable progeny in some intergenericEarle 1991] is a logical complement to Oryza (rice;crosses (Dewet et al. 1976; P. L. Morrell and A. H.�420 Mbp; Arumuganathan and Earle 1991), a dis-Paterson, personal communication). By contrast, ricetant relative (tribe Oryzeae) that will be the first grassand the maize/sorghum lineage may have diverged �50genome to be completely sequenced (Goff et al. 2002;million years ago (Linder 1987) and show much moreYu et al. 2002). Sorghum is an especially importantchromosomal rearrangement (Paterson et al. 1995a).bridge to several economically important large-genomeAnalysis of the levels and patterns of genomic diversitycrops in its own tribe (Andropogoneae) such as maizewithin and between sorghum, sugarcane, rice, and(�2292–2716 Mbp) with which it may have shared com-maize (and others) promises to advance understandingmon ancestry between 11 (Gaut and Doebley 1997)of the biology and evolution of Poaceae grain and bio-and 24 (Thomasson 1987) million years ago. Sorghummass crops and reveal new opportunities for their im-and sugarcane, a large-genome (�2547–3605 Mbp)provement.polyploid that ranks among the world’s most economi-

Worldwide, sorghum is the fifth most important graincrop grown based on tonnage, after maize, wheat, rice,and barley (http://www.fao.org). Sorghum is unusually

1Corresponding author: Plant Genome Mapping Laboratory, 111 River-tolerant of low input levels, an essential trait for areasbend Rd., Rm. 228, University of Georgia, Athens, GA 30602.

E-mail: [email protected] such as northeast Africa and the U.S. Southern Plains

Genetics 165: 367–386 (September 2003)

368 J. E. Bowers et al.

that receive too little rainfall for most other grains. In tant quantitative trait loci (QTL) but lack the highmarker density needed for use in complex endeavorsthe more arid countries of northeast Africa, such as

Sudan, sorghum contributes 39% of the calories in the such as positional cloning of genes, genetic anchoringof bacterial artificial chromosome (BAC)-based physicalhuman diet (http://www.fao.org; 1999 statistics). In-

creased demand for limited fresh water supplies, cou- maps, or assembly of genomic shotgun sequence. Theonly other high-density sorghum map (Menz et al. 2002)pled with global climatic trends and expanding popula-

tions, suggests that dryland crops such as sorghum will is composed largely of amplified fragment length poly-morphisms; the difficulties associated with inferring or-be of growing importance.

Despite the likely growing importance of sorghum, thology of these arbitrary-sequence markers across taxaconstrain its value for comparative and evolutionary ge-its improvement has lagged behind that of maize, wheat,

and rice, each of which have more than doubled in nomics. Our map is currently being used to anchor BAC-based physical maps of both S. bicolor and S. propinquumaverage yield on a worldwide basis in the last 38 years

while sorghum yields have gained only 51% (average (Lin et al. 1999; Draye et al. 2001), to facilitate rapidgene isolation by map-based cloning and provide land-1961–1963 compared to 1999–2001; http://www.fao.

org). In sub-Saharan Africa, already home to many of marks for eventual genomic sequence assembly. Thegenetically anchored probes used in this map are alsothe world’s hungry and with a population projected to

double over the next 40 years (U.S. Census Bureau being hybridized to BAC libraries from rice, sugarcane,and maize, fostering comparative genomics across theestimates 2002; http://www.census.gov), sorghum yields

have gained only 6% over the last 38 years compared Poaceae.to 50% gains in wheat and maize (http://www.fao.org).

In the U.S., sorghum was introduced over 200 yearsMATERIALS AND METHODSago, possibly by Benjamin Franklin (Smith and Freder-

iksen 2000) and is now grown on 9–13 million acres. Laboratory procedures: The genetic population and molec-U.S. sorghum is principally used as an animal feed and ular methods are as previously described (Chittenden et al.therefore escapes direct notice by the general public, 1994), except that the mapping population was expanded

to 65 individuals from 56, drawing additional F2 progeny atbut is the 13th most valuable crop in the U.S. with arandom from residual seeds of the original cross. Briefly, DNAfarm-gate value ranging from $0.8 to 2.0 billion/yearwas extracted from young leaves by a published protocol(USDA 1992–2001 statistics). (Chittenden et al. 1994), �5 �g DNA per lane digested with

S. bicolor is native to Africa. One other euploid species 15 units of EcoRI, HindIII, or XbaI (Promega, Madison, WI),exists within the genus, S. propinquum, which is native electrophoresed and blotted onto Hybond N� (Amersham,

Arlington Heights, IL), rinsed in 2� SSC, and stored at 4�to Asia and contains many “weediness” traits such as rhi-until use. About 20–50 ng of PCR-amplified fragment waszomes, small seeds, and shattering. The genus also in-labeled with [32P]dCTP, hybridized to blots, washed, and ex-cludes S. halepense, a tetraploid (2n � 40) thought to posed to X-ray film as described (Chittenden et al. 1994).

be derived from naturally occurring crosses between DNA markers and sequences: Prefixes of DNA markers usedS. bicolor and S. propinquum (both 2n � 20). S. halepense is and their sources are as follows. Arabidopsis cDNA: AEST (R.

Scholl, Arabidopsis Biological Resources Center, Ohio Stateamong the world’s most noxious weeds, with widespreadUniversity), AHD and HMG (T. Thomas, Texas A&M); Barleydistribution. In the U.S., many local epithets for S. hale-cDNA: BCD (M. Sorrells and S. Tanksley, Cornell); Johnson-pense have largely been supplanted by the term “Johnsongrass rhizome cDNA: pHER, pSHR (Y. Si and A. H. Paterson,

grass,” first documented in an 1874 letter, referring to unpublished results); Maize PstI genomic clones: BNL, UMCColonel William Johnson, an Alabaman who sowed it (E. Coe and M. McMullen, University of Missouri); Maizeon his farm (McWhorter 1971). The first U.S. federal cDNA: CSU (Coe, McMullen); Millet Pst1 genomic clones: M

(M. Gale, John Innes Center); Oat cDNA: CDO (Sorrells,appropriation for weed research targeted JohnsongrassTanksley); Sorghum cDNA: HHU (Wyrich et al. 1998),(House Bill 121, 56th Congress, 1900).HHUK (Annen et al. 1998); Sorghum phytochrome genes:Cross-fertility between S. bicolor and S. propinquum PHY (L. H. Pratt and M.-M. C.-Pratt, University of Georgia);

has permitted us not only to benefit from high levels of Sorghum PstI genomic DNA: pSB, SHO (A. H. Paterson);DNA polymorphism between them to build the detailed Sugarcane cDNA: CDSB, CDSR (P. Moore, Hawaiian Agricul-

tural Research Center); Sugarcane genomic clones: SG (Sor-molecular map described herein, but also to conductrells); Rice genomic clones: RG and cDNA: RZ (S. McCouchgenetic analysis of many traits associated with grass do-and S. Tanksley, Cornell), C, G, and R (T. Sasaki, RGP, Japan).mestication (e.g., Paterson et al. 1995a,b). The genetic Sequences were obtained from the National Center for Bio-

map presented herein builds on and integrates much technology Information (NCBI) or developed in house by endearlier work (Chittenden et al. 1994; Lin et al. 1995; sequencing of probes using standard methods. In house se-

quencing used a software pipeline in which sequence data inAnnen et al. 1998; Wyrich et al. 1998; Draye et al. 2001).ABI trace file format were input into the programs PHREDSeveral other sorghum maps (Whitkus et al. 1992;(version 0.000925.c) and CROSS_MATCH (version 0.990329Ragab et al. 1994; Xu et al. 1994; Dufour et al. 1997;with minmatch � 12 and minscore � 20) to trim poor quality

Boivin et al. 1999; Peng et al. 1999; Subudhi and Ngu- and vector sequence. Residual vector and primer sequencesyen 2000; Haussmann et al. 2002) provide seminal data were trimmed manually and sequences of �50 nucleotides in

length were removed from further analysis. A list of GenBankon comparative genome organization and reveal impor-

369Sorghum STS Map for Comparative Genomics

TABLE 1

Summary statistics for the SB � SP linkage groups

SP SBdominant dominant

Linkage group (LG) Size (cM) Loci no. Probe no. No. % No. % Largest gap (cM)

A 130.1 333 328 32 0.10 38 0.11 4.6B 120.8 331 323 42 0.13 36 0.11 3.9C 118.5 499 476 34 0.07 122 0.24 3.1D 81.6 187 184 14 0.07 20 0.11 7.0E 84.7 146 139 10 0.07 23 0.16 7.8F 127.8 275 266 38 0.14 32 0.12 5.4G 107.0 196 190 24 0.12 26 0.13 7.0H 85.4 191 188 35 0.18 40 0.21 3.9I 107.0 216 209 25 0.12 32 0.15 4.6J 96.3 138 135 17 0.12 26 0.19 7.0Total 1059.2 2512 2438 271 395

accession numbers is available as supplementary documenta- RESULTStion at http://www.genetics.org/supplemental/.

Genetic map: The SB � SP map (Table 1; Figure 1Map construction: A framework map of �600 codominantand available at http://www.plantgenome.uga.edu/markers was constructed using the program MAPMAKER v2.0

on the PC, with error detection on (Lander et al. 1987). A sorghummap) is composed of 2512 loci on 10 linkagenew program written in Microsoft Visual Basic (J. E. Bowers groups that collectively span 1059.2 cM (Kosambi 1944).and A. H. Paterson, unpublished data) was then used to This is a 2236-locus (about sevenfold) increase com-insert additional markers into the framework. The algorithm

pared to our previously published map of 276 lociused by this program was to determine the genotypes of each(Chittenden et al. 1994), yet the recombinationalindividual for each interval between markers already placed

on the map, with multiple genotypes possible for individuals length has actually been reduced from 1445 cM to thein which crossovers were observed between the framework current 1059.2 cM largely by virtue of a sufficiently highloci. In cases where genotypes were uncertain due to dominant density of markers to distinguish errors from true dou-markers or missing data, the genotypes of the intervals were ble recombinants. The map is based on a total of 1376inferred from flanking loci, assuming that the minimum num-

detected crossovers (see materials and methods),ber of recombinations had occurred. An unmapped locus waswhich would correspond to 1386 potentially distincttested against the possible genotypes for all intervals already

on the map, in search of a perfect match. If such a match was map locations; we have identified markers at 853found the marker was assigned to the appropriate interval, (61.5%) of these possible locations. The largest gapand then the framework was recomputed. If no perfect match between two loci corresponds to only 7.8 cM or 10 cross-was found, a second pass was made looking for matches to all

overs and only seven intervals in the map were �5 cM.but one individual, followed by subsequent passes with higherOn the basis of the 65 F2 plants used, a single recombi-numbers of nonmatching individuals. Loci from these subse-

quent passes were rechecked for scoring errors in the individ- nation event yields an estimate of 0.77 cM between con-ual that did not fit the expected pattern. If the data were secutive loci, which defines the resolution limit of thedetermined to be correct the locus was then added to the map. Consequently, loci are plotted to intervals of thisframework map with a new recombination event not observed size (in the figure rounded to one decimal place).in the previous map. Individuals mapping to the ends of the

All of the restriction fragment length polymorphismchromosomes could not be placed with this approach and had(RFLP) markers tested could be placed on the mapto be added to the framework manually or with Mapmaker.

After the map had been constructed, it was manually edited although a small number (�20) that had initially beento reduce the number of recombinations by exporting the scored were determined in retrospect to be too faintlocations of crossovers observed in the map into a spreadsheet. for accurate scoring and were discarded. A similar num-Instances with multiple recombinations for an individual prog-

ber of markers with two segregating bands of nearly theeny plant were reordered if possible to reduce the total num-same migration rates, which could not be reliably distin-ber of recombination events observed. This step involved ex-guished from one another, were also discarded. Anothertensive checking of the raw data (films) for errors, with the

plants apparently responsible for double recombinations be- group of �20 markers that could not be mapped showeding rechecked. Ostensibly codominant markers that could not segregation ratios approaching 15:1 and were assumedbe placed on the map were split into two dominant markers to be caused by two loci with indistinguishable bandto attempt their mapping separately. Final map distances were

sizes and were therefore discarded.computed using Kosambi (1944) centimorgans (cM), andDuplicate probes were removed from the map by in-maps were drawn by another Visual Basic program written for

this purpose. spection of genomic hybridization patterns for coseg-


Figure 1.—Sorghum genetic map. Distances along the map are in Kosambi centimorgans. Marker prefixes are summarizedin materials and methods. Text color indicates loci that are codominant (black), dominant for the S. bicolor allele (blue), ordominant for the S. propinquum allele (green). Loci revealed by probes that contain SSRs are indicated by a percent sign. Ap-proximate centromere positions (determined as described in text) are indicated by an O. Space constraints prevent some markers


from being placed immediately next to their map location. In these cases the (superscripted, parenthetical) map location wasprinted in the smaller font followed by the list of markers mapping to that location, with a line of reduced length plotted at theproper location on the map. For chromosomal regions with high marker density some loci were listed at the bottoms of thefigures. Multiple markers mapping to the same chromosomal location show identical segregation patterns, and their physicalorder cannot be determined from present data.


Figure 1.—Continued.



regating loci and also by sequence comparisons of most found in the population vs. 262 adjacent crossovers, ahighly significant difference (Figure 2). The numbersprobes. Some probes used in past studies were shown

to be identical to newly mapped sorghum probes and of observed genotypes in the two categories differ sig-nificantly (P � 0.05) from the expected (equal num-in a few cases cDNAs from other species corresponded

closely to sorghum probes or to one another. In cases bers) for cases in which the two recombination eventswere separated by 0–10 cM, 10–20 cM, and 40–50 cMwhere RFLP markers had similar or identical sequences

and mapped to similar or identical loci, one of the and narrowly missed significance for the 20–30 cM(0.07) and 30–40 cM (0.06) spacings. Over intervals ofduplicates was removed from the map. In total, this

resulted in the removal of 336 markers at 386 loci (which �50 cM, no significant differences were found in thefrequency of double vs. adjacent crossovers.are not included in the 2050 probes and 2512 loci that

compose the map). The genetic locations and correspond- Segregation distortion: Five regions on the geneticmap showed segregation distortion significant at the 5%ing information for these loci remain available at our web

site (http://www.plantgenome.uga.edu/sorghummap). level. The apices of distortion in the five regions wereon LG B near cM 50.0, LG C near cM 46.2, LG D nearRecombinational interference: Recombinational in-

terference was assessed by comparing the frequency of cM 66.2, LG G near cM 26.2, and LG I near cM 0.0.Curiously, all five regions showed segregation distortionoccurrence of “double crossover” genotypes (i.e., aa–

ab–aa; bb–ab–bb) to “adjacent crossover” genotypes favoring the S. bicolor alleles. By far the most strikingcase was on LG C—the apex of the distortion was near(i.e., aa–ab–bb; bb–ab–aa) as a function of the size of

the interval that contains the two crossovers required to the locus CSU507 and comprised a segregation ratio of41:17:2 (homozygous S. bicolor :heterozygote:homozy-produce each genotype. In the absence of interference,

these two different classes of genotypes would be equally gous S. propinquum), significantly (P � 3 � 1012) differ-ent from the expected 1:2:1 ratio. In a larger set of F2probable; however, only 121 double crossovers were



progeny from the same cross (Lin et al. 1995; Paterson dw2 gene regulating plant stature (Lin et al. 1995; Pat-erson et al. 1995a), and pApo1 gene regulating apomixiset al. 1995a), we found similarly distorted segregation

(236:94:8) in this region. (R. Jessup, G. Burow, M. Hussey and A. H. Paterson,personal communication)]. The average number of lociPatterns of DNA marker distribution: We evaluated

the distribution of DNA markers across the sorghum in the deliberately enriched intervals plus the short ter-minal bins was 23, virtually identical to the averagemap by comparing intervals of exactly 10.0 cM in length,

starting from the top of each chromosome as drawn across the remainder of the genome (23.28); thereforeelimination of these anomalous regions has no effect(Figure 1), except that the last interval in each group was

either �15 cM or 5 cM to accommodate the varying on the analyses.Virtually every linkage group has at least one intervallengths of the linkage groups. On the basis of the total

number of loci per linkage group, the Poisson probabil- containing more loci than would be expected to occurby chance in 1% or fewer cases (A06-7; B01 and B08;ity distribution function was applied to identify intervals

that contained significant excesses or deficiencies of C02, -04, and -08; D02 and D04-07; E05; F06-8; G04 andG06-7; H04-05; I06; and J04 and -06).various classes of probes. We note that two regions (C04

and D04-07) were preferentially enriched for markers Significant marker deficiencies were associated with13 intervals, including A01*, A02; B10 and B12*; C06,because they contain genes that we seek to clone [C04,

the sorghum Sh1 gene regulating shattering of the ma- C07, and C12*; F01*, F05, F11, and F13*; G09; and H02.These included a disproportionately large number, 5ture inflorescence (Paterson et al. 1995b); and D04-

07, the Ma1 gene regulating photoperiodic flowering, (25%), of the terminal intervals (*), but the reduced



length of three terminal intervals (B12 � 5.8 cM; C12 � clone-derived loci over the intervals were closely corre-lated (r � 0.79).7.5 cM; F13 � 8.7 cM) contributed partly to their marker

deficiencies. The distributions of genomic and cDNA Distribution of dominant loci: A total of 666 (26%)



loci showed dominant inheritance, segregating as pres- dominant loci (nominally below the average of 2.7). Byfar the largest concentration of SP-derived dominantence of an allele from one parent and absence from

the other parent. A total of 395 (15.7%) of the dominant loci (23, 8.6%) was in interval H05—while this intervalis generally marker rich, the number of SP-derived dom-alleles were from SB and 269 (10.7%) from SP, a highly

significant difference (�2 � 23.9, 1 d.f., P � 1.1 � 106). inants in this interval is �50% higher than the numberof SB-derived dominants (15), the opposite of theirDistribution of dominant loci is shown in Figures 1

and 3. 50% lower abundance elsewhere in the genome. Severalother intervals are also preferentially enriched for domi-Among the 395 SB-derived dominant loci, 74 (18.7%)

are in the single 10-cM interval C05, far beyond the nant loci from one parent or the other: F07 containsan abundance of 9 (P � 0.0009) SP-derived dominantrandom expectation (�8 loci would have been expected

in only 1% of cases). The same interval also is enriched loci vs. 0 SB-derived dominants (P � 0.085); H01 con-tains an abundance of SB-derived dominants (P �for codominant loci (44), but contains only 2 SP-derived



0.0013) vs. only 1 SP-derived dominant (P � 0.25) and numbers) we were able to identify 130 simple sequencerepeat (SSR)-containing sequences (defining an SSR asI09 contains an abundance of 7 (P � 0.0009) SP-derived

dominants, vs. 1 SB-derived dominant (P � 0.29). 6 or more repeats of a dinucleotide or repeats stretching15 or more base pairs of longer repeat units). AlthoughEven after removing the dominant probes mapping

to interval C04 of LG C, a significant excess of S. bicolor- the distributions of genomic and cDNA clone-derivedloci were closely correlated (r � 0.79), the genomicderived dominant markers (321, vs. 267 S. propinquum

dominants, significant at the 5% level) still remains. distribution of the SSRs was only loosely related to thatof the entire population of mapped DNA probes (r �Curiously, this excess is explained almost completely by

one marker class, the pSB probes, which were derived 0.33), suggesting that SSRs may locate in different ge-nomic domains more frequently than low-copy probes.from S. bicolor hypomethylated (PstI-digested) genomic

DNA. The pSB clones detected 152 S. bicolor dominants The relationship between SSR distribution and probedistribution was somewhat closer (r � 0.43) after remov-and 102 S. propinquum dominants (exclusive of probes

mapping to LG C05). After removing the pSB clones, ing the strong biases in distribution of dominant loci,partly attributable to possible genomic rearrangementsthere remains a nonsignificant difference of 169 S. bi-

color dominants vs. 165 S. propinquum dominants for (see below). The map location of SSR-containing clonesis shown in Figure 1. Further characterization of a subsetnon-pSB probes outside of interval C05.

Simple sequence repeat-containing loci: On the basis of the SSRs has been described (Schloss et al. 2002).Distribution of duplicate loci: Among the 2050 probesof the sequences of 1933 probes (see http://www.plant

genome.uga.edu/sorghummap for GenBank accession mapped, a total of 357 revealed DNA polymorphisms at



multiple loci that could be mapped, with 279 detecting 2 act loci involved. On the basis of a chi-square contin-gency test, the distribution of duplicate loci over pairsloci, 58 detecting 3 loci, 13 detecting 4 loci, 6 detecting

5 loci, and 1 detecting 6 loci. The distribution of dupli- of linkage groups was not random (�2 � 224.06, with81 d.f.). Several pairs of linkage groups showed strikingcated loci across the genome is illustrated in Figure 4,

composed of 606 data points (keeping in mind that excesses of duplicated loci (A and G, C and G, C andH, E and H, E and I). Associations of individual linkage2-locus probes generate 1 point of intersection, 3-locus

probes generate 3 points, 4-locus probes generate 6 groups with multiple partners (for example G with Aand C), together with our prior observations (Chitten-points, 5-locus probes generate 10 points, and 6-locus

probes generate 15 points). A clickable web-based ver- den et al. 1994) and other work (Whitkus et al. 1992;Gaut and Doebley 1997; Gaut 2001), suggest that ifsion of this figure is available at http://www.genetics.

org/supplemental/, which displays the probes and ex- duplication in sorghum is due to paleo-ploidy, then



the polyploidization event must be very ancient, surely spondence with 0, 1, 2, or 3 other intervals in the ge-nome. A total of 74 duplicate loci are intrachromoso-predating the Sorghum-Zea divergence. Therefore we

reevaluated the data on the basis of smaller intervals, mal, not significantly different from the randomexpectation.breaking each sorghum chromosome into four “inter-

vals” of equal length in centimorgans. This yielded an Correspondence to gene arrangements in other taxa:Table 2 summarizes the sources of clones and loci thatoverall contingency chi-square of 2287.53 (with 1521

d.f., P � 7 � 1033), further supporting the notion have been mapped to date, illustrating the opportuni-ties to use this map as a basis for comparisons of manythat duplicated loci are not randomly distributed across

chromosome pairs. Among the 820 possible comparisons Poaceae taxa.As an especially important example of the utilization(including intrainterval comparison), a total of 22 pairs

(2.7%) of intervals shown in Table 2 showed positive of these data, Figure 5 illustrates comparative alignmentsof the sorghum and maize genomes based on 952 locideviations from the random expectation that were sig-

nificant at 0.005 (as measured by 1 d.f. chi-square), from the maize “bins” map (Gardiner et al. 1993; Daviset al. 1999). A clickable version of Figure 5 is availableabout 5.4 times higher than the random expectation.

Respectively, 12, 16, 10, and 2 intervals showed corre- (at http://www.genetics.org/supplemental/) that shows


Figure 2.—Summary of recombinational in-terference. The frequencies of adjacent crossovers(two crossovers occurring on different chromo-somes within the same individual) and doublecrossovers (occurring on the same chromosomein the same individual) are plotted vs. the dis-tance between the crossovers.

the specific probes and loci involved. This comparison 136 different functional molecular groups. The mostcommon molecular functional groups were “ATP bind-represents a considerable increase over previously pub-

lished data (Whitkus et al. 1992; Pereira et al. 1994; ing” (120 hits) and “Protein Kinase” (56 hits). Resultsof sequence similarity analyses are available at http://Ragab et al. 1994; Paterson et al. 1995a; Dufour et al.

1996). The distributions of loci over the 100 possible www.genetics.org/supplemental/.combinations of maize chromosomes and sorghum link-age groups were clearly not random (contingency �2 �

DISCUSSION790.04, with 81 d.f., P � 9 � 10117). A total of 19(19%) cells with the largest excesses (from 7.5 to 36.6) This genetically anchored set of sequence-tagged sites

provides transferable DNA markers suitable for a wideof observed data over random expectations account for520 (55%) of the corresponding points and 74% (582.84) range of investigations in structural, functional, and evo-

lutionary genomics in several major grain and biomassof the chi-square deviation from randomness, sug-gesting the correspondences illustrated in Figure 5 and crops. Although the map was created using the RFLP

method and has been applied to several goals by thislisted in Table 3.Marker sequence annotation: Multiple local align- technology (e.g., Lin et al. 1995; Paterson et al. 1995a,b;

Katsar et al. 2002; Ming et al. 2002), genetically mappedment searches using the programs blastn and tblastxwere used for sequence annotation against publicly sequence tagged sites such as these can be used to dis-

cover single-nucleotide or small insertion/deletion poly-available databases of the NCBI as of November 21,2002. The default matrix BLOSUM 62 and a cutoff of morphisms that can then be genotyped by many alterna-

tive technologies. This possibility increases the value of1 � 106 were used in all BLAST searches. The NCBIdatabase was subdivided into several taxon-specific groups these loci and reduces the costs associated with their

wider utilization. A total of 130 loci that contain simple-to allow for the efficient determination of not only thebest overall hit, but also the best hit among closely sequence repeats have the further advantage of being

relatively allele rich (Schloss et al. 2002), a benefit inrelated species, excluding unannotated expressed se-quence tag and genomic survey sequence database en- studies that require differentiation between closely re-

lated genotypes.tries. Additional analyses included the use of hiddenMarkov models to classify sequence data by protein se- This framework of genetically anchored sequence-

tagged sites will also provide a foundation for physicalquence signature. The program InterProScan (Zdob-nov and Apweiler 2001) was used to search to compare mapping and ultimately assembling a robust finished se-

quence of the sorghum genome. The present map per-the translated sorghum sequences against several proteindatabases (Pfam, SMART, and ProDom) and Genome mits us to assign loci to bins of �0.77 cM; on average,

this represents �300 kb of genomic DNA based on aOntology (Ashburner et al. 2000) numbers for each ofthese classifications were obtained. The results of these consensus genome size estimate of 750 Mbp (although

we have recently estimated the genome to be somewhatanalyses revealed that the 578 sorghum query sequencescould be classified into 205 distinct protein families and smaller, �690 kb; Peterson et al. 2002). To orient differ-


Figure 3.—Distribution ofcodominant and dominantmarkers along the sorghummap. For 10-cM intervals alongeach linkage group, the num-bers of codominant (solid),S. bicolor dominant (open),and S. propinquum dominant(shaded) loci are plotted.

ent loci within 0.77-cM bins, we are presently hybridizing less evenly distributed through the genome. By simplyhybridizing the 2050 mapped probes to the 10�-cover-the genetically mapped probes to BAC libraries for both

S. propinquum and S. bicolor. Since the two BAC libraries age BAC libraries, we expect to identify �20,000 BACsin each library, comprising �50% of the genome. Fur-each provide �10� coverage of the genome and are

composed of individual BACs that average �120 kb in ther, both libraries have been fingerprinted (http://www.genome.arizona.edu/fpc/sorghum/), permitting the re-length, this will permit us to resolve the order of closely

linked loci to an average resolution of �12 kb, assuming sulting “contigs” to be extended further. By selectiveBAC end sequencing and the use of comparative ap-that the breakpoints of individual BACs are more or


Figure 4.—Patterns of duplication withinthe sorghum genome. In this Oxford grid,each dot represents a genetically mappedlocus detected by probe that segregated attwo or more polymorphic loci in sorghum,with the x- and y-axis representing chromo-somal locations. Red circles along the axesrepresent the approximate locations of thecentromeres. The total number of probesmapping to each pair of sorghum linkagegroups is shown in each cell. Areas high-lighted in yellow represent regions of sig-nificant marker abundance between a pairof linkage groups (determined as describedin text). Note that some dots represent mul-tiple probes with the same genetic loca-tions; for a detailed list of exact informationfor each cell, see http://www.genetics.org/supplemental/.

proaches made possible by the alignment of our geneti- mapping of the locations of the sorghum centromeresis in progress by probing synaptonemal complex spreadscally mapped sequences to the nearly completed rice

sequence, a robust genetically anchored physical map with genetically mapped probes or their correspond-ing BACs by fluorescence in situ hybridization (D. G.is expected to coalesce.

Nonrandom patterns of DNA marker distribution Peterson and A. H. Paterson, unpublished data).Clearly, more information will be needed to explainprovide clues to the locations of interesting and impor-

tant features of sorghum genome organization. On most the multiple, dispersed marker-dense regions found onseveral linkage groups. For example, linkage group Bchromosomes, at least one significant concentration of

loci appears to correspond to the centromeric region. has one terminal concentration of markers and anotherinterstitial concentration. We have recently shown thatWe have recently applied overgo (Cai et al. 1998) probes

for sorghum centromeric repetitive sequences homolo- some sorghum chromosomes have cytologically distin-guishable knobs (D. G. Peterson and A. H. Paterson,gous to pHind22 and Cen38 (Miller et al. 1998; Zwick

et al. 2000) to the SP and SB BAC libraries. Co- unpublished observations), and future studies will inves-hybridization of these probes with genetically mappedRFLPs has associated concentrations of centromeric re-

TABLE 2peats with marker-dense regions of 8 of the 10 linkagegroups (LG A: DM064, cM57.7; B: DM007, cM57.7; C: Summary of probe sourcespSB1406, cM47.7; D: pSB580, cM47.7 and pRC162, cM60;

Genomic cDNA TotalE: CSU0462 and pRC182, cM47; G: R2447, cM64.6; I:5C04A07, cM69.3; and J: pSB0019, cM50.8). Due to the Arabidopsis 0 52 52repetitive nature of these probes and the possibility that Zea 62 189 251not all copies are centromeric, these data can be taken Triticeaea 0 65 65only as tentative indications of the possible locations of Pennisetum 23 171 194

Oryza 10 175 185the sorghum centromeres. For example, on two linkageSorghum 725 464 1189groups we found associations with mapped probes inSaccharum 11 103 114regions of normal marker density (LG A: pSB1075,TOTAL 831 1219 2050cM98.5; LG H: HHU49, cM68.5). Further, we found no

a Triticum, Hordeum, or Avena.association on one linkage group (F). More definitive


Figure 5.—Patterns of colinearity be-tween sorghum and maize. In this Oxfordgrid, each dot represents a locus detectedby a probe that was genetically mapped inboth sorghum (left) and maize (top), withthe x- and y-axis representing chromosomallocations in each taxon. The total numberof probes mapping to each pair of maizeand sorghum chromosomes is shown ineach cell. Lines highlight the regions forwhich we have inferred synteny betweenmaize and sorghum, as summarized in Ta-ble 3. Note that some dots represent mul-tiple probes with the same genetic loca-tions; for a detailed list of exact informationfor each cell, see http://www.genetics.org/supplemental/.

tigate whether these could account for some marker near the interval (H05) that contained by far the largestconcentration of SP-derived dominant loci (23, 8.6%).excesses or deficiencies. Linkage groups C, D, G, and J

also show multimodal distributions of marker density This suggests that the ribosomal DNA and a large flank-ing area may have moved in one of the two sorghumsthat warrant further study.

Differences in the abundance of dominant genetic since their divergence from a common ancestor, a hy-pothesis that we are further investigating (D. G. Peter-marker loci appear to suggest that a chromosome struc-

tural rearrangement has occurred since the divergenceof S. bicolor and S. propinquum from a common ancestor.

TABLE 3The single 10-cM interval C05 contains 74 (18.7%) ofCorrespondence of maize chromosomes to sorghumthe 395 SB-derived dominant loci found, far beyond the

linkage groupsrandom expectation (see above), and 71 of these coseg-regate at the single location cM 46.2 (along with 6 co-

Maize chromosome Sorghum linkage group(s)adominants, and one locus dominant for the SP allele).Curiously, this interval is also the apex of the most pro- 1 C, J, Cnounced segregation distortion found (41:17:2, fa- 2 B, D

3 E, Avoring bicolor homozygotes as described above). The4 J, F, HDNA sequences of some of the probes that detect5 C, FS. bicolor-dominant markers at LG C, cM 46.2 correspond6 I, Gto various portions of the ribosomal DNA [specifically 7 B

AEST602 matches 18s rRNA (GenBank accession no. 8 A, G, AX16077) at e � 10200 and C152 and pRC017 match 9 I, Gthe 25S ribosomal RNA gene (GenBank accession nos. 10 E, DM11585 and AY108843) at e � 10170 and e � 4 � 1071, a Proceeding from left to right along the maize chromo-respectively]. some as drawn in Figure 5. For identities of specific loci, see

These three probes also mapped as dominant markers supplementary documentation at http://www.genetics.org/supplemental/.for the S. propinquum allele on LG H at cM 32.3–40.0,


son, J. E. Bowers and A. H. Paterson, unpublished relationship with any of the factors studied herein. Cor-relations of allelic diversity with total mapped locusdata) and that is consistent with recent findings in rice

(Shishido et al. 2000) and legumes (Singh et al. 2001). abundance per interval (0.0006), codominant locusabundance (0.038), and SSR abundance (0.16) wereOn the basis of genotypes inferred from segregation at

nearby codominant loci, all 435 plants that have been remarkable in the lack of information they yielded. Animportant future investigation will be to study howstudied to date from this cross (including those from a

larger population used for mapping QTL; e.g., Lin et marker density and/or allelic diversity correlate withthe distributions of phenotypically significant variantsal. 1995; Paterson et al. 1995a) possess at least one

copy of the ribosomal DNA on either LG C or LG H. such as QTL.By virtue of a very high level of DNA polymorphismThese results are consistent with a requirement for a

copy of ribosomal DNA for survival of gametes. We have (Chittenden et al. 1994), the SB � SP cross has provenespecially facile for “comparative mapping” of DNAalso noticed some degree of enrichment of the two

affected genomic intervals for QTL that differentiate clones that have been previously mapped in other taxa.To foster opportunities to use the relatively small ge-between SB and SP [specifically for the number of seed-

ling tillers and regrowth on H05 (Paterson et al. 1995b) nome of sorghum to help advance genomics in thelarger genomes of many other tropical Poaceae, we haveand for the number of seedling tillers, three measures

of rhizomatousness, and seed weight on C04 (Paterson mapped 865 heterologous DNA clones from eight othertaxa (Table 2). In one example, we show herein theet al. 1995a,b)].

The finding that many S. bicolor hypomethylated (PstI- alignment of the sorghum genome to the four timesphysically larger genome of maize. Most maize chromo-digested) genomic probes lacked a homolog in S. propin-

quum suggests that there has been considerable and somes correspond to nonoverlapping regions of onlyone sorghum chromosome but most sorghum chromo-rapid divergence or deletion of low-copy DNA in these

taxa. In contrast to cDNAs and excepting the probes somes correspond to nonoverlapping regions of twomaize chromosomes, reiterating the recent duplicationmapping near the ribosomal DNA discussed above, a

total of 728 pSB probes detect 152 dominant loci that in maize. Sorghum is an especially valuable guide forgenomic analysis of Saccharum [sugarcane, one of thelack an S. propinquum allele vs. only 102 loci that lack

an S. bicolor allele, a highly significant difference. This world’s most important crops, with the 2001/2002 worldcane sugar crop forecast at a near record 126.8 millionsuggests that a portion of the sorghum genome may be

composed of rapidly evolving low-copy DNA, such as metric tons (FAS 2001)], which may have shared a com-mon ancestor as recently as 5 million years ago (Sobralhas been reported for tomato (Zamir and Tanksley

1988). However, this portion of the sorghum genome et al. 1994). The present data supplement and comple-ment our prior efforts in this regard (Ming et al. 1998,is likely to be relatively smaller in sorghum than in

tomato, as Cot analysis shows that sorghum has a much 2002). Other work in progress uses probes describedherein (Table 3) together with species-specific recombi-smaller low-copy DNA fraction (Peterson et al. 2002).

Finally, we note that the lack of an RFLP allele at a nation data to address the comparative organization ofsorghum and other tropical grasses including Penni-dominant locus does not necessarily reflect deletion of

the locus, but could be attributable to comigration with setum (R. Jessup, M. Hussey and A. H. Paterson, per-sonal communication), Cynodon (C. Bethel, E. Sciaramonomorphic loci, gain/loss of restriction sites creating

short or long fragments that are not captured on South- and A. H. Paterson, personal communication), Echi-nochloa (T. Fukao, A. H. Paterson and M. Rumpho,ern blots, or other artifactual reasons, which presumably

account for many of the 102 S. bicolor loci that are null unpublished data), and Panicum (A. Missaoui, A. H.Paterson and J. Bouton, personal communication).for these S. bicolor-derived probes.

The genomic distribution of mapped (i.e., polymor- Despite the clear value of the comparative approachfor fostering progress in study of gene arrangement inphic in SB � SP) loci shows little relationship to differ-

ences in levels of intraspecific allelic diversity in differ- complex genomes (e.g., Saccharum) or underexploredtaxa (e.g., Pennisetum, Cynodon, Echinochloa, and Pan-ent chromosomal regions (Dvorak et al. 1998; Hamblin

and Aquadro 1999). In a separate study (P. Morrell, icum), it is equally important to note that a remarkable45% of comparative data fell in regions other than thoseJ. E. Bowers and A. H. Paterson, personal communica-

tion), we have shown that allelic diversity is not randomly we infer to correspond between sorghum and maize.Many of these incongruities are likely to reflect nonchro-distributed across the sorghum chromosomes but is

highly structured. For 183 loci representing most of the mosomal rearrangement mechanisms that are becom-ing clear from microsynteny studies (Tikhonov et al.10-cM bins in this study, we have estimated allelic rich-

ness (not shown) from a worldwide sample of 55 land- 2000) and studies of ancient duplication (Bennetzen2000; Paterson et al. 2000) or, possibly, rapid diver-race and wild accessions representing the breadth of

diversity in the Sorghum genus (P. J. Morrell and A. H. gence or deletion of hypomethylated DNA as we reportabove. A few tantalizing hints of the possibility of morePaterson, personal communication)—curiously, these

estimates of allelic diversity showed remarkably little ancient duplication events in maize are suggested by


An anchored framework BAC map of mouse chromosome 11locus arrangements in several regions (e.g., maize chr.assembled using multiplex oligonucleotide hybridization. Geno-

1/sorghum LG A), but await more data to test with mics 54: 387–397.confidence. One sorghum linkage group (H) that is Boivin, K., M. Deu, J. F. Rami, G. Trouche and P. L. Hamon, 1999

Towards a saturated sorghum map using RFLP and AFLP mark-well populated with DNA markers (Table 1) shows re-ers. Theor. Appl. Genet. 98: 320–328.markably little correspondence to any maize chromo- Chittenden, L. M., K. F. Schertz, Y. R. Lin, R. A. Wing and A. H.

some (just a small portion of maize chromosome 4). Paterson, 1994 A detailed RFLP map of Sorghum bicolor �S. propinquum, suitable for high-density mapping, suggests ances-This hints at the possibility that large segments of chro-tral duplication of sorghum chromosomes or chromosomal seg-matin may have been lost during the maize-sorghum ments. Theor. Appl. Genet. 87: 925–933.

divergence; however, a conclusive test awaits more data. Davis, G. L., M. D. McMullen, C. Baysdorfer, T. Musket, D. Grantet al., 1999 A maize map standard with sequenced core markers,While our results clearly reinforce the evidence ingrass genome reference points and 932 expressed sequencesupport of the duplication of most regions of the maize tagged sites (ESTs) in a 1736-locus map. Genetics 152: 1137–1172.

genome, many questions remain about the levels, pat- Dewet, J. M. J., S. C. Gupta, J. R. Harlan and C. O. Grassl, 1976Cytogenetics of introgression from Saccharum into Sorghum.terns, and antiquity of chromatin duplication withinCrop Science 16: 568–572.sorghum itself. The patterns of distribution of duplicate

Draye, X., Y.-R. Lin, X. Y. Qian, J. E. Bowers, G. B. Burow et al.,loci in sorghum are clearly not random, with many small 2001 Toward integration of comparative genetic, physical, di-

versity, and cytomolecular maps for grasses and grains, using theislands of colinearity evident, and adjacent intervals of-Sorghum genome as a foundation. Plant Physiol. 125: 1325–1341.ten showing correspondence to syntenic intervals (A2,

Dufour, P., L. Grivet, A. Dhont, M. Deu, G. Trouche et al., 1996-3, and -4 to G3, -1, and -4; E3-4 to H2-1; F3-4 to I3, I1; Comparative genetic mapping between duplicated segments on

maize chromosomes 3 and 8 and homoeologous regions in sor-J2, -4 to I2-1). However, for �30% of the genome weghum and sugarcane. Theor. Appl. Genet. 92: 1024–1030.can discern no corresponding duplicated region, and

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Dvorak, J., M. C. Luo and Z. L. Yang, 1998 Restriction fragmentrice, in which the completed sequence (Goff et al. 2002; length polymorphism and divergence in the genomic regions ofYu et al. 2002) has largely borne out early hints (Kishi- high and low recombination in self-fertilizing and cross-fertilizing

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Hamblin, M. T., and C. F. Aquadro, 1999 DNA sequence variationWe honor the memory of coauthor Keith F. Schertz, who made and the recombinational landscape in Drosophila pseudoobscura :many of these discoveries possible while teaching several of us about a study of the second chromosome. Genetics 153: 859–869.sorghum and about much more. Science is richer for his efforts, and Haussmann, B. I. G., D. E. Hess, N. Seetharama, H. G. Welz andwe are poorer for his passing. We thank the USDA-National Research H. H. Geiger, 2002 Construction of a combined sorghum link-Initiative, National Science Foundation Plant Genome Research Pro- age map from two recombinant inbred populations using AFLP,

SSR, RFLP, and RAPD markers, and comparison with other sor-gram, International Consortium for Sugarcane Biotechnology, and U.S.ghum maps. Theor. Appl. Genet. 105: 629–637.Golf Association for financial support of various aspects of this work.

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