Copyright O 2005 by the Ceneucs Socicry of America 1101: 10.1534/genetics.105.048215

Comprehensive Molecular Cytogenetic Analysis of Sorghum Genome Architecture: Distribution of Euchromatin, Heterochromatin, Genes and Recombination in Comparison to Rice

J.-S. Kim,*,+M. N. Islam-Faridi,:?' P. E. Mein,*.+D. M. st ell^,^*: H. J. Price,: R. R. Kleins and J. E. Mullett7**32 *D@a?tment o/lImticulturnl Sciences, l~nstitvte/mPlant Gp?tomics and Bioterhnology and i~epa~$metatqf Soil and Crop SripncesJ Texas A&M Unir~mskty,College Station. Tans 77843, sl:h~~~-/l~,~JSouthern Plains A~ic?rltuml&s~anh Cmte?; College Slnticrn, Texas 77845 and **Depa?tmmt qf fUzochtvrhi~tryand Bi@F~y~ics,E*ras A&iI Uniz~crJity,College Statio?~,Texas 77843 Manuscript received July 13, 2005 Accepted for publication August 21, 2005

ABSTKAClT Cytogenetic maps of sorghum chromosomes 3-7, 1.1, and 10 were constructed on the basis of the fluorescence in silu hybridization (FISH) of -18-30 RAC probes rnapped across each of these chrornosornes. Distal regions of euchromatin and pericentromeric repons of heterochromatirl were delimited for all 10 sorghum chromosomes and their DNA content quantified. Euchromatic DNA spans -50% of the sorghum genome, ranging from -60% of chromosome 1 (SRI-01) to -33% of chromosome 7 (SBX-07). This portion of the sorghum genome is predicted to encode -70% of the sorghum genes (-1 gene mode1/12.3 kbp), assuming that rice and sorghum encode a similar number of genes. Heterochromatin spans -411 Mbp of the sorghum genome, a region characterized by a -34fold lower rate of recombination and -3-fold lower gene density coinparecl t,~euchromatic DNA. Thc sorghum and rice genomes exhibit a high degree of nlacrocolixlearity; however, the sorghuxrl genome is -%fold larger than the rice genorne. The distal euchromatic regions of sorghum chrornosornes 3-7 and 10 are -1.8-fold larger overall and exhibit, an -1.5-fold lower average rate of recombination than the colinear regions of' the honleologolls rice chromosomes. By contrast, the pericentromeric heterochromatic regions of' these cliromoso~nesare on average -3.6-fold larger in sorgllurn and recorr~bir~atiorlis suppressed -15-fold cornpared to the colinear regions of rice chromosomes.

FUSSES are ecologically well adapted, coveiing members of Pooideae, Ehrl~artoideac,and Bambusoi- G -20% of the earth's land surface (SCHANTZ1954). deae (KELLOGG2001). Rice was targeted for intense This group of plants is especially important to agricul- grass genomics research I~ecauseof its relatively small ture, corlt~ibutirlga large portion of the calories con- genome (<4YO Mbp), technologies for analysis of gene sunled in the human diet (EVANS1998). The grass family function (i.e.,SIIIMAMOT~ and KYOZLJKA2002; Av et a/. originated 45-70 million years ago (MYA) and today 2003; LI et 02. 2005), and its agricultural importance includes -10,000 species (KE:I,I.oGG2001 ) . Advances in (CANTRE:I.I.and REEVES 2002) . A rl~ultinationaleffort our understanding of pssphylogeny have helped place has reported a nearly complete sequence of the rice important functional differentiation that occurs within the genome, for which annotations and other sources of in- yrass family into evolutionary contest (GRASSPHYI,O(;E:NY formation indicate a non transposable element-related WORKINGGROIJI' 2000). Phyloge~leticirlfonnation is proteinsoding gene complement of -37,500--50,000 also useftll for guiding the selection and utilization of genes (RICE FULL-LENGTHcDNA CONSORTIUM2003, reference species for comparative genome research h ttp://~.n+'~r.tigr.org/ tdb/e'lkl /osal /pseudomolecules/ (THORNTONand DESAI,I,E2000; UKETA-VIDAI.et a/. info.shtm1; RENNETZENet nl. 2004; INTEI~ATIONAJ.RICE 2003). For example, grass species such as rice, wheat, GENOMESEQUENCING PROJECT 2005). barley, and oats carry out C3 photosynthesis as do other Sorghurn, maize, sugar-cane,and millet are grass spe- cks of the PACC clade that includes the Panicoideae (GRASSPHYLOGENY TORKI KING GROUP2000; KELLOGC Sequence data from this article have been deposited with the EMBL/ CenBank Data Libraries under accession nos. CZ682513-CZ682537. 2001) that diverged from rice -50 ?vNA (DOEBLEYet ccl. 'I'r~smdnddms: Southern Institute of Forest Genetics, USDA-Iiorest 1990). These gms species carry out C4 photosynthesis, Scnicc, Department of Ebresl Scicncc, Texas .&?r?l trniversity, College a11 importallt adaptation tllat illcreases the efficiellcy Station, TX 77843. of C02fixation in plants. C4 species coiltribute dispro- 2Cont.~$mrZingauthor: Depaltment of Biochemistry and Biophysics, Texas .&M Universitx College Station. TX 778432128. portionately to global productivity and agriculture, in E-mail: jmulle tataxnu .cdu that they compose only -3% of angiosperm species 1964 J.-S. Kin1 et al.

(EDM~AR~Sel al. 2004). The C4 grasses are particularly homepage.ht~nl).Moreover, even within the genus well adapted to regions of lower latitude that have Sorghum (Poaceae), chronlosoxrle numbers (211 = 10, higher average temperatures and are prone to drought 20, 30, 40) and genome size vary considerably (-8.1- (EDWARDSet nl. 2004). Among the C4 grasses, sorghum Sold) (PKICEet ul. 2005). Variation in genome size arnong has a relatively small genome coxitairling 818 Mbp of' the grasses is due primarily to differences in polyploicly, DNA distributed arnorig 10 chronrosomcs (PRICEet al. seg~nerltalduplications, and accumulation of rcpetitiw 2005). The importance of sorghum as a subsistence cereal sequences (SAGEIAIMAROOF et al. 1996; TIKI-IONOVet al. crop in the semiarid tropics (Do~cr~rr1988; NA'I-TONAL 1999; C24I.X 2002; LKVY and FE:I.~MAN2002; V.~NL)EPOET.F: RESEARCHCOUNCIL 1996), potential importarlce in bio- et al. 2003). The S. bicolor genome is approximately two- fuel production (GNANSOUNOUet nl. 2005), adaptation to fold larger in size and has two fewer chromosomes drought (DOGGKI-r1988; NATIONAL,KI:SEAKCH (:OIJNCII. relative to Chyztl, suti-cta. The difference in sorghum's chro- 1996), diverse germplasm (i.e., MENZ et (11. 2004), and mosome number relative to lice is due to two chromo- close relationship to rnaize (KELLOGG2001; SWIGONOVA sorne fusion events that occurred prior to the dil'cr . ge11ce et al. 2004) make this species a valuable target for grass of sorgh~~mfrom maize and Penniset~li(WILSON et al. genome research. 1999; KEI,I,OGG2001). The sorghum and rice genomes Extensive resources have beerr developed for sorghum are thought to encode a similar number of genes (l~ttp:// genomics (SORGIIUMGENOMICS PLANKING WORKSIIOP fungen.botany.uga.edu/f rojects/Sorgh~1n1/Sorgl1um PAK'I-ICIPANTS2005). Several linkage maps have been UnigeneSet.htm, http://1~ww.tigr.org/tdb/e2kl/osal/ constructed on the basis of ixrterspecific (i.e., BOWERS pseudomolecules/info.shtrnl). Moreover, rice and sor- et al. 2003) and intraspecific crosses (i.e., MENZet al. ghum chromosonles arc largely colinear indicating that 2002). The sorghum genome map has been aligned to sorghum's genome expansion relative to that of rice is the genome maps of other cereals revealing extensive not due to a large-scale genome duplication event (PENG irracrocolinearity, especially between sorghum, rice, and et (11. 1999;WILSON ~t al. 1999; KLEINet al. 2003). These maize (PENGet al. 1999; WILSONet al. 1999; KLEINet al. results suggest that the difference in S. bicolarand 0. sntiz~a 2003; PA'I'E:KSONet u1. 2004; D~:vos2005). Approxi- genome size could be due primarily to differential accu- mately 800,000 sorghum ESTs have been collected re- mulation of repetitive sequences in sorghum. vealing -22,000 unique transcript clusters (L. H. PUTT, In this study, the molecular cytogenetic architecture personal communicatioll; http://ww.fungen.org/) . of 7 sorghum chrolnosomes was analyzed, thereby com- Microarrays and qRT-PCR assays based on these sequen- pleting characterization of all 10 sorghum chromosomes. ces have been used to collect information on sorghum Colinear regions of sorghurn and rice chromosomes gene expression modulated by plant hormones in\rolwd were compared and the nature of the genome expan- in plant protection (SALZMANet al. 2005) and osmotic sion of sorghum is detailed and disclxssed. stress (RUCHANAKet al. 2005). In addition, the collec- tion of -500,000 methyl-filtered sorghum sequences tagged >90% of the sorghum genes (BEDELLet al. 2005). MATERIALS AND METHODS The architectrxre of sorghum chromosomes has also Mitotic metaphase chrornosorrle spreads were prepared been characterized in several studies. A molecular kar- from root tips of BTx623 seedlings according to the protocol yotype of thc sorghum genome was developed on the of KTM et nl. (2002), and pachptene chromosomes were pre- basis of fluorescence in sihr hybridization (FISH) of pared Srom immature anthcrs accorcling to the protocol of KIM et 01. (2005b). BACs used for FISH were derived from RACs derived from each sorghum clrronrosome (fix1 libraries prepared by Woo et a/. (1994), TAOarid ZHANG et ccl. 2002). Uaryotype-aided analysis of sorghum chro- (1998), and KLEIN et al. (2003). The BACS were located on the mosome size and DNA content recently allowed the sorghum linkage map as described by KI,E~N~t 01. (2003), and establish~netttof a rrnified sorghrxrn chromoson~enrxrn- RAC DNA used for FISH was isolated as previously described berirlg system (KIMet al. 2005a). In addition, the molec- (RMei al. 2002). A cen tromere-associated sequence (pCEN38) (ZWICK et nl. 2000) was used to locate sorghum cerltromeres as ular cytolo'g of three sorghum chromosomes has been prcklously described (ISIA&,I-FARIDIet al. 2002). In situ hybrid- analyzed in detail using genetically mapped BAC clones ization techniques were a modification of TSIAM-FARII)~d (11. and FISH (Isr,A~-FArirr>~el al. 2002; KIM et a/. 2005b). (2002). The chromosomal DNA on the glass slide w;ts dena- These sorghum chroinosomes were found to contain tured at 70" in 70% forrnarnide, 2X SSC for 1.5 rnin, followed distal regions of euchromatin and pericentromeric re- by dehydration in 70,85,9.5, and 100% ethanol for 2 mix1 each at -20". The hybridization rnixtt11-e (2.5 p.l/slide) contained gions of heterochromatin (XSI,AM-FAR~Iet al. 2002; KIM 50 ng of labeled probe DNA, a 10-fold excess of blockillg ef ul. 2005b). DNA (200- to 300-bp-digested gerlonlic DNA), 50% formam- Genome sizes and chromosome numbers of grzsses idc, and 10% dcxtran sulfate in 2X SSC. The mixture was range widely (ht tp://www.rbgke~v.orff.uk/cvaI/l~ot~~epage. denatured at 90" Sor 10 min, chilled on ice, added to the slide, htrnl) . For example, rice has an -370- to 490-Mbp ge- anct covered with a glass coverslip. Following overnight in- cubation at 37", slides were rinsed three tirnes at 37" in 2 X SSC nome distributed anlong 12 chromosomes and 'cv11eat for 5 inin each. Slides were blocked 5 min at room tempera- has an -16,900-Mbp genome distributed among three ture with 5% (w/v) RSA in 4X SSC plns 0.2% Twrcen 20. sets; of 7 chromosornes (htt~~://~~~~w.rbgke'~~.org.r~k/cd/Biotin-lat>elect probes were ctetected with 1% CyS-conjugated Sorghum Genome Architecture 1965

streptavidin and digoxygenin-labeled probes with 1% fluores- cein isothiocyanate (F1TC)conjugated antidigoxygenin anti- body. Slides were washed three times in 4X SSC plus 0.2% Tween 20 for 5 min at 37'. DAPI (4',6diamidino-2- phenylindole) in Vectashield antifade solution (Vector Labo- ratories, Burlingame, CA) was applied for counterstaining of chromosomes. Images were viewed through an Olympus AX- 70 epifluoresceilce microscope equipped with standard filter cubes. Images from a Peltiercooled 1.3-megapixel Sensys camera (Roper Scientific, Trenton, NJ) were captured with the MacProbe v. 4.2.3 digital image system (Applied Imaging, Santa Clara, CA). To assess the location of FISH signals, blue (DAPI signal from chromosomal DNA), green (FITC from BAC probes), and red (Cy3 from BAC probes) signals were measured from digital images using Optimas v. 6.0 (Media Cybernetics, Carlsbad, CA). Image capture and data analysis were done as previously described (KIM et al. 2005a). Sample sequencing of BAC DNA was performed as de- scribed previously (KLEINet al. 2003). DNA from nine phase 111-sequenced sorghum BACs was isolated and fingerprinted using a modified version of five-color-based high information content fingerprinting (HICF; KLEINet al. 2003; Luo et al. 2003). The nine BACs correspond to GenBank accession nos. AF114171, AF466199, AF466200, AF466201, AF369906, AY661656, AY661657, AY661658, and AY661659. To calculate the number of 75- to 500-bp DNA bands generated per kilobase pair of BAC DNA, all common vector bands were removed from each dye lane and the number of remaining; unique bands was divided into the total kilobase pairs in ea& BA~insert. This analysis FIGURE1.-Cytogenetic analysis of sorghum chromosome 3 showed that a 75- to 500-bp DNA fingerprint band is generated using genetically mapped BACs and FISH. (A) FISH signals on average every 1.405 ? 0.124 kbp of BAC-insert DNA an- alyzed by HICF. Using this information, two BAC contigs from on sorghum pachytene bivalents using an eight-probe cocktail the distal euchromatic part of the long arm of sorghum chro- with BAG mapped to the termini of sorghum chromosome 3. mosome 3 spanning BAC sbb20220 (211e12) to BAC sbb16652 (B) Pachytene bivalents showing a central darkly staining peri- centromeric region of heterochromatin plus interspersed (174d8) and BAC sbb22184 (232a8) to BAC sbb8411 (88ell) were calculated to contain -2.19 and ~2.36Mbp of DNA, regions of heterochromatin (arrows) and euchromatin. (C) FISH analysis showing hybridization of BAC probe 3-1 3 within respectively. the heterochromatic region. (D and E) Localization of BAC probes 3-12 and 3-14 at the euchromatin:heterochromatin boundaries and DNA associated with the centromere using RESULTS the pCEN38 probe. (F) FISH signals on pachytene bivalents Molecular architecture of sorghum chromosomes: In using a multiprobe cocktail composed of BACs mapping across sorghum chromosome 3. BAC numbers refer to those listed in previous work the architecture of sorghum chromo- supplemental Table 1 (http://www.genetics.org/supplemental/) . somes 1,2, and 8 was characterized using mapped BACs and FISH analysis (ISLAM-FARIDIet al. 2002; KIM et al. 2005b). In this study the molecular cytogenetic archi- mosomes were analyzed on pachytene bivalents (data tecture of the remaining seven sorghum chromosomes, not shown). For each bivalent examined, the cytogenetic 3-7,9, and 10, was characterized by FISH analysis using location of each fluorescent signal was determined by 18-30 BACs per chromosome. The BACs used in this locating the peak luminance value along the segmented study mapped at regular intervals across the linkage line that collectively spanned the FISH-adorned bivalent. maps of each chromosome (supplemental Table 1 at The peaks were then used to assign linear positions along http://www.genetics.org/supplemental/) (KLEINet al. the pachytene bivalent and create a cytogenetic map. The 2003). The relative order of BACs along each chromo- cytogenetic maps corresponding to the seven sorghum some arm was determined in some cases by dual-color chromosomes in this study are shown in Figure 2 (chro- FISH using pairs of adjacent BACs that map incremen- mosome diagrams denoted as Cyto). For comparative tally closer to the centromere as previously described purposes, the cytogenetic maps of chromosomes 1, 2, (ISLAM-FARIDIet al. 2002). In addition, multiprobe FISH and 8 characterized previously (ISLAM-FARIDIet al. 2002; cocktails were used to visualize the spacing and order of KIM et al. 2005b) are also shown in Figure 2. Overall, the groups of BACs that mapped and hybridized sequen- order of BAGFISH signals along the seven chromosomes tially along the chromosomes. Several examples of mul- analyzed was concordant with the location of these BACs tiprobe BAC FISH on a pachytene bivalent corresponding on the sorghum linkage maps (Figure 2, supplemental to sorghum chromosome 3 are shown in Figure 1, A and Table 1 at http://www.genetics.org/supplemental/). In F. Similar results were obtained when BAC-FISH cock- several cases, FISH analysis helped resolve the order of tails corresponding to each of the remaining six chro- DNA markers and their associated BAC clones that were J.-S. Kim et al.

FIGURE2.-Integrated linkage, cytogenetic, and physical maps of the sorghum genome and alignment to the physical maps of rice chromo- somes 1,2,ll,4,8, and 6. Sorghum linkage maps at the left are drawn using data from MENZet al. (2002) and cytogenetic maps are drawn on the basis of BAC signals observed on pachytene biva- lent~.Centromeres of chromosomes mapped us- ing the pCEN38 probe are shown as blue circles. Red squares and green circles represent the Cy3 and FITC signals, respectively, used in the detec- tion of multiprobe BAGFISH. Black stars repre- sent additional BACs used in single-probe FISH detection. Shaded boxes represent heterochro- matin and open boxes represent euchromatin in the sorghum cytogenetic and physical maps. Numbers to the left of the cytogenetic maps cor- respond to BAC numbers shown in supplemental Table 1 (http://www.genetics.org/supplemental/) used for FISH analysis. Physical maps of sorghum are drawn on the basis of calculations using the distribution of DNA content in heterochromatic and euchromatic regions as determined from BAG FISH measurements of mitotic metaphase cells (supplemental Table 2 at http://www.genetics. org/supplemental/). Physical maps of rice were drawn using data based on the TIGR Osal version 3 pseudomolecule (http://www.tigr.org/tdb/e2kl/ osal/pseudomolecules/info.shtml) . The sorghum BACs at the boundaries of sorghum euchroma- tin:heterochromatin were aligned to each rice physical map on the basis of gene sequence align- ments obtained from BAC sample sequencing (supplemental Table 3 at http:/www.genetics. org/supplemental/) .

located in the same "bin" on the linkage map. In addi- (Figure 1, A and B). In addition, a punctate staining tion, the analysis showed that the linkage maps of the pattern was observed on this chromosome near the tran- seven chromosomes span nearly the entire length of sition region between euchromatin and heterochroma- each chromosome arm (Figure 2). tin (Figure IB, dark-staining regions marked by arrows). In sorghum pachytene chromosomes, there is a sig- This indicates the presence of small, interspersed regions nificant difference in the intensity of DAPI staining of of heterochromatin within the euchromatic DNA near heterochromatin and euchromatin, making the bound- this boundary. Overall, only 4 of the 162 BACs used for aries of these regions relatively easy to detect. For FISH analysis were located in regions of heterochromatin example, on chromosome 3, lightly staining euchroma- (e.g., Figure lC, BAC probe 313 on chromosome 3). This tic DNA was present in the distal portion of each arm was due in part to the preferential selection of BACs with Sorghum Genome Architecture 1967

B SBI-03 0sChr. Of 581-04 0sChr. 02 SBI-05 0sChr. 11 Llnkage Llnlugr, Cyto Phys Phye Llnkw We Phys ptw

higher gene content (KLEIN et al. 2003) and because basis of measurements of mitotic chromosomes at meta- most BACs from heterochrornatic regions hybridized phase, when DNA density is most uniform (KIM et al. nonspecifically, due to their high content of repetitive 2005a). One objective of this study was to estimate the DNA (data not shown; KIMet al. 2005b). The location of amount of DNA located in the euchromatic and hetero- the centromeres in each sorghum chromosome was de- chromatic regions of each sorghum chromosome using termined using the probe pCEN38 (ZWICKet al. 2000). a similar approach. For this determination, BACs were We have previously shown that the pCEN38 probe hy- identified that hybridize at the boundaries between eu- bridizes to a region in the heterochromatin of sorghum chromatic and heterochrornatic DNA in each pachy- chromosome 1 adjacent to the nucleolus organizing re- tene bivalent. For example, BAC probes 312 and 314 gion (NOR) and that the heterochrornatic region of the hybridized to the boundaries between heterochromatin long arm of sorghum chromosome 1 is -1.9-fold larger and euchromatin on the short and long arms of chromo- than the heterochrornatic region of the short arm (ISLAM- some 3, respectively (Figure 1, D and E) . The BAC probes FARIDIet al. 2002; Figure 2). By contrast, hybridization of marking the euchromatin:heterochromatin boundaries the pCEN38 probe to pachytene bivalents corresponding on chromosome 3 were subsequently hybridized to mi- to the other nine chromosomeswas observed near the mid- totic metaphase chromosomes and the relative size of points of the respective heterochrornatic regions (Figure the euchromatic and heterochrornatic regions was de- lE, Figure 2). termined by analysis of multiple chromosomes (N= 20) The relative size and DNA content of each sorghum (supplemental Table 2 at http://www.genetics.org/ chromosome were quantified in a previous study on the supplemental/). The amount of DNA in the euchromatic . . 1968 J.-S. Kim et al.

C SBI-06 0sChr. U4 SBI-07 0s Chr. 08 SBI-08

Linkasa w0 Phys WY* Llnkage Cyfo Phya Phyo UnwP WO f'w

and heterochromatic regions delimited by the BAC used to calculate the amount of DNA in two BAC contigs probes was then calculated on the basis of the relative size that spanned portions of the euchromatic region on the of each region and the previous determination of the long arm of sorghum chromosome 3 (see MATERIALS DNA content in sorghum chromosome 3 (KIM et al. 2005a). AND METHODS). Next, BACs from the ends of each of This analysis indicated that the euchromatic portion of the these contigs were used in FISH analysis and the relative short arm of sorghum chromosome 3 contains -21.1 Mbp length of the chromosomal interval defined by the BAG of DNA, the euchromatic portion of the long arm contains FISH signals was determined by measurement of 14 SBI- -30.6 Mbp of DNA, and the heterochromatic region 03 pachytene bivalents. The combined HICF and FISH contains -38.2 Mbp of DNA (Table 1, Figure 2). analysis showed that on average each relative unit length The estimates of DNA content of the euchromatic of euchromatic DNA contained -0.648 Mbp (see supple- and heterochromatic regions from measurement of meta- mental Table 1at http://www.genetics.org/supplemental/) . phase chromosomes are based on the assumption that Using this conversion factor, the euchromatic portion sorghum chromosomes are uniformly compacted at this of the short arm of sorghum chromosome 3 was esti- stage. To test this assumption and our method for esti- mated to contain -19.4 Mbp of DNA (chromosome end mating DNA content, we used a second approach that to BAC probe 3-12) and the euchromatic portion of the relies on a combination of BAC contig fingerprinting long arm was estimated to contain -33.2 Mbp DNA (HICF) and BAGFISH analysis to determine the amount (chromosome end to BAC probe 3-14). Thus, if sor- of euchromatic DNA present in sorghum chromosome ghum chromosome 3 contains -89.9 Mbp of DNA over- 3. To calibrate the analysis, HICFwas carried out on nine all (KIM et al. 2005a), then the BAGFISH/HICF method phase 111-sequenced BAC clones containing DNA de- indicates that -52.6 Mbp of DNA is located in the distal rived from euchromatic regions of the sorghum genome. euchromatic regions and -37.3 Mbp of DNA is present The average number of -75- to 500-bp DNA bands de- in the pericentromeric heterochromatic region. These tected by HICF per megabase pair of sorghum DNA was estimates based on HICF and BAC FISH differ by <3% determined from this analysis. This information was then from the estimates based on BAGFISH measurements Sorghum Genome Architecture

D Set-09 SBI-10 0s Chr. 06;

Linkage CflO my0 Llnkage we Phy8 PhYs

on metaphase chromosomes (-51.7 Mbp for the euchro- The analysis of DNA content of euchromatic and matic region and -38.2 Mbp for the heterochromatic heterochromatic regions for sorghum chromosome 3 region). Thus, despite the inherent limitations associ- described above was extended to the other nine sor- ated with the two methods used for quantification of chro- ghum chromosomes. On the basis of previous estimates mosomal DNA in this study, both approaches gave similar of the DNA content of each sorghum chromosome (KIM estimates of the DNA content of the euchromatic and et al. 2005a), the amount of DNA in the euchromatic pericentromeric regions of sorghum chromosome 3. and pericentromeric heterochromatic regions of each

TABLE 1 Sorghum chromosome size and distribution of euchromatic and heterochromatic DNA

Chromosome Euchromatin Euchromatin Total Total size short arm long arm euchromatin Heterochromatin euchromatin Chromosome (Mbp) (Mbp) (Mbp) (Mbp) (Mbp) (%) SBI-01 SBI-02 SBI-03 SBI-04 SBI-05 SBI-06 SBI-07 SBI-08 SBI-09 SBI-10 Total: "The sample size for measurements was 20. 1.3Kin1 ut uI. TABLE 2 Ratio of DNA in colinear regions of homeologous sorghum and rice chromosomes

Chronlosorne Chrornosonle Euchrorrlatiri Euchromatin Euchrornatin Heterochrornatin pairs Sb: Of S&A/ Odil SbU/ 0s-LA Sb/Os Sb/Os SRI-03:0s-0 1 2.1 1.7 1.4 1.5 4.4 SBI-04:0~02 2.3 1.9 1.4 1.5 4.6 SB1-05:0s-1 1 2.8 3.1 2.0 2.4 3.4 SBI-06:0.44 2.1 1.5 1.8 1.7 2.9 SRT-07: 0~48 2.6 1.9 1.5 1.7 3.5 SBI-10:0s-06 2.2 2.0 1.8 1.9 2.6 Average 2.4 2.0 1.7 1.8 3.6 SA, chromosorne short arm; Ih,chromosorne long arm. "Data for Mbp/sorghurn chromosorne were taken from KTM et ni. (2005a). Data for Mbp/rice chronlosorrle were taker1 from the 'I'IGli Osal version 3 pseudon~olecule(h ttp ://~+wv.tigr.or~/tdb/e2kl /osal /pseudo~nolecules/ inro.shtm1). chromosome was detet-rnined (supplemental Table 2 at to a proportional or disproportional increase in euchro- http://\~~~enetics.org/supplernental/,Table 1, Fig rnatic chromosonle arms and pericerltrorxleric hetero- ure 2). Overall, the estimates of DNA in the euchro- chro~naticregions. To begin to address this question matic portion of the 10 sorghum chromosomes ranged we compared the relative size of the euchromatic and frorrl -71.3 Mhp for chroxrlosorrre 1 to -23.9 Mbp for heterochrornatic regions of six sorghum chrornosomes chromosorne 7 (Table 1). The amount of DNA in het- to the colirlcar regions of tho corresponding horneolo- erochromatic regions of the sorghum cl~romosomeswas gous rice chron~osomes.In related studies, sorghum estimated to vary from -49.2 Mbp in chromosome 7 to BACs distributed across all 10 sorghurn chromosomes -35.5 Mbp in chromosorrle 6 (Table 1).Overall, euchro- were sequence scanned and the resultiug sorghurn matin and heterochromatin each spanned -50% of genes used to align the sorghum and lice chronlosoilles the sorghum genome (Table 1, -40'7 and -411 Mbp, (KL,EINet al. 2003; P. E. KL,EIN,personal communica- respectively). tion). In this study, unique colinear gene sequences Comparative size, architecture, and gene density of were obtained from BACs mapped to the euchroina- sorghum and rice chromosomes: A genome size of 818 tin:heterochromatin boundaries of sorghum chromo- Mbp (PRICEet al. 2005) indicates that the sorghum somes 3-7 and 10 (supplemental Table 3 at httg:// genome is approximately twofold larger than the rice ww.genetics.org/supple~r~e~~t'al/),allo~tlng us to align genome (3'70-490 Mbp) (http://'~nt~v.~-bgkew,org.uk/the euchromatic and heterochromatic regions from cval/homepage.html; Uozu et al. 1997). This analysis these six sorghum chromosomes to the corresponding can be extended to individual chroi~zosoxx~estising our homeologous rice chromosomes. The arrlounts of DNA data and rice genome sequence data. (http://ww.tigr. in the lice segments colinear to the euchromatic region org/ tdb/e2kl /osal /pseudomolecules/info.shtml) . We of each sorghum chromosomal arm and to the pericen- compared the sizes of established homeologous sorghum tronleric heterochromatin were determined using data and rice chrornosornes (SBI-03:0~01, SBI-04: 0~02, SBI- based on the TIGR Osal version 3 pseudomolecule 05: 0s-11, SBI-06:(1.~04, SBI-07: 0s-08,SBI-08: 051 2, SBI- (http://wvw. tigr.org/ tdb/eZkl/osal /pseudornolecules/ 09: 05-05, SBI-1 0: 0s-06) ( i. e., MO~KI:et al. 1995; WII,SON info.shtm1) and con-ipared to the amount of DNA pres- ~t al. 1999). In addition, sorghunl chromosome 1 con- en t in coliaear regions of sorghum (Ti~k~le2). Amorlg tains DNA corresponding to rice chroinoso~nes3 and the six chromosome pairs analyzed, the cuchromatic 10, while sorghum chromosome 2 contains DNA corre- regions of sorghum averaged -1.8-fold larger in size sponding to rice chromoso~~les7 and 9 (~VJI.SON PI al. corrlpared to the colinear regions of rice (ranging from 1999; KELLOCC2001). Sizes of the l~omeologoussor- N1.4- to -3.1-fold). In contrast to the euchromatic re- ghum and rice chroinosomes (or chromosomal fusions) gions, sorghum pericentromcric heterochromatic regions were calculated on the basis of the estimated DNA were on average -3.6-fold larger in size relative to the content of each sorgllunl chronlosome (KIM et at. 2005a; colinear regions of rice (ranging from -2.6- to -4.6- Table 1) vs. the chromosome-specific rice sequence data fold) (Table 2). (http://mw.tigr.org/tdb/e2kl /osal /pseudomolecu~les/ Currently, -37,500-47,000 nontransposable eletnen t info.shtrn1). This showed that sorghum chromosomes (11011-TE) gene rnodels have been identified in the rice contain -1.7- to -2.8-fold more DNA than the homeol- gcnorrle sequence depending on the mctl~odsused to ogous rice chromosonle sequences. annotate gene models (INTERNATIONALRICE GENOME The increased size of sorghurn chromosomes corn- SEQ~IENC:INC;PKOJ~:CI' 200.3; http://mwv.rigr.org/tdb/ pared to homeologous rice chromosomes could be due e2k1/osal/pseudomolecules/info.~html) Using the Sorghurn Genorne 'lrchitecture

TABLE 3 Recombination in sorghum chromosomes

Euchromatin Euchromati~in Total short arm Iorlg arrn euchrornatin Heterochrornatin Chromosome (Mbp/cM) (Mbp/ cM) (Mbp/chl) (?vlbp/cM/l)

Average 0.253 8.7 upper estimate as a reference, the number of non-TE Recombination in sorghum and rice chromosomes: gene models/chrornosome ranges from -2600 for rice Alignment of the sorg1lun-l linkage and cytogenetic maps chronlosoine 9 to -6100 for lice chromosome 1 (http:// and identification of the euchromatic and heterochro- www. tigr.org/tdb/e2kl /osal/pseudomolecules/info. matic regions in each of the sorghum chromosomes shtml). Using these estimates, the number of non-TE allowed average rates of recombination in these regions gene models present in regions of the rice genome that to be determined (Table 3). In general, heterochro- are colinear with the euchromatic and heterochromatic matic regions of sorghum chromosomes showed much regions of the six sorghum chromosomes analyzed was lower rates of recombination (-8.7 Mbp/cNl) compared determined. Approxirrlately 25,400 non-TE gene mod- to euchromatic regions (-0.25l\$bp/chl) (Table 3). 1x1 els have been identified in the six rice chromoson~es addition, recombination in hcterochromatic regions of that are colinear to sorghum chromosomes 3-7 and 10 different sorghum chromosomes varied -15-fold from Fyith -18,500 of these in regions colinear to the sorghum -2 Mbp/cM (SRI-06) to -31 Mbp/cM (SRI-01 ). The euchromatic arms and the remaining -6900 located in euchronlatic region on the short arm of sorghum chro- regions colinear to sorghum heterochromatin. A~sum- mosome G showed a relatively low rate of recoxnbination ing there are a similar number of genes in coli~~earre- compared to other regions of euchrornatin (-2.3 Mbp/ pons of sorghum and rice, then the average gene density cN1 0s. an overall average of -0.25 Mbp/cM) (Table 3). of the sorghum euchromatic regions is predicted to be The rate of recombination across euchromatic DNA of -81 gene models/Mbp (18,500/227 Mbp) or -1 gene the other 19 chromosome arms varied -2.8-fold from mode1/12.3 klqr and the average gene density in the -0.44 Mbp/cM (SRI-01) to -0.16 Mbp/cM (SRT-07). sorghuirl heterochromatic regions is predicted to be The relative rate of recornbillation in colinear regions -29 gene models/Mbp (6,895 gene models/240.5Mbp) of the six pairs of homeologous sorghum and rice chro- or -1 gene mode1/34.5 kbp. If the lower estimate of the mosomes was compared (Table 4). The rate of recom- rice gene complen~ent(-37,500 genes) is used in this birration in the short ann of sorghum chrorxlosorxle 4 calculation, then the overall gcnc nunlbers and gene was slightly higher than that in the colinear region of density predicted for sorghum are lower by -20%. rice chromosome 2, whereas the rate of recombination

TABLE 4 Ratio of recombination in colinear regions of homeologous sorghum and rice chromosomes

Euchromatin short Erlchromatin long Total euchromatitl Hetcrochrotnatin Chrotnosome pair arm (Mhp/cM) artn (Mbp/cM) (Mbp/cM) (Mbp/cbl) SBI-03: 0.~01 1.5 1.1 1.2 25.9 SBI-04: 0s-02 0.9 1.7 1.3 9.2 SBI-05: 0$11 1.G 1.4 1.5 36.3 SBI-06: 0s-04 5.6 1.4 1.8 6.4 SBI-0'7:0>08 1.2 1.1 1.1 '7.3 SBI-10:0~06 1.2 1.3 1.2 7.2 Average 1.4 15.4 in most of the euchrornatic arms of' the sorghurn chro- 2001 ) . For example, analysis of pachytene stage chro- irlosorrles analyzed was slightly lower than that in the mosomes and FISH rnappirlg of two to five BACs per &I. colinear regions of rice (h1.l- to -1.7-fold lower re- tl-zcncatula linkage group indicated that the majority of the combination in sorghum). In contrast, the euchromatic genes in this genome are located in distal chromosomal portion of the short arm of sorghum chrornosorne 6 regions of euchromatic DNA (KCJI.IKOVAet al. 2001). showed an -5.6-fold lower rate of recornbination coin- Sorghum chromosomes coritain pericerltromeric re- pared to the colinear region of rice. Moreover, rates of gions of heterochromatin and regions of euchi-omatin reconlbination in the heterochromatic regions of the located in the distal portions of each chromosome arm. sorghum chromosomes analyzed were -15.4fold lower A similar arrangement of heterochrorrlatin and euchro- than those in the colinear regions of rice (ranging from matin is found in most chromosoinesof tomato (PETERSON -6.4- to -36.3-fold) (Table 4). et n1. 1999), iZI. ~runcalul(~(KUI,IKOVA et a2. 2001), and rice (CHENGet al. 2001a). In contrast, heterochromatin is distributed across a much greater portiorl of the rrlaize and wheat chromosomes (i.e., GILLet nl. 1991; CIIEN et al. 2000). Overall, euchromatin spans -50% of the sor- FISH analysis utilizing mapped BACs helped eluci- ghum genome ranging frorn -60% of sorghum chro- date the molecular architecture of sorghum chroino- mosome 1 (-71 Mbp) to -33% of sorghum chromosome somes 3-7,9. and 10. These results, combined with prior 7 (-24 Mbp). 'I'he amount of heterochromatin per analysis of sorghum chromosomes 1, 2, and 8 (ISLAM- chroix~osoxxieis Inore uriiform varying -1.4fold from FARIDIef nl. 2002; KIM et nl. 2005b), coinplctes an ovcr- -49 Mbp on sorghum chroinosomc 7 to -35.5 Mbp on all cytogenetic analysis of the organization of S. bicolor sorghum chromosome 6. DNA associated with sorghum chromosomes. In total, these studies utilized 220 ARCS centromeres was located near the inidpoint of the het- for FISH analysis ranging from 18 to 30 BACs per erocliromatin except for chrorriosome 1 where more chromosome. The BACs were selected to span the heterochromatic DNA was present on the long ann than sorghum linkage map and for their ability to generate on the short arm (ISLAM-FAKIIIIel nl. 2002). 'I'his dis- strong, localized FISH signals. As a consequexice of this tinctive feature could reflect an ancestral chrornosorne selection, most of the BACs were low in repetitive se- rearrangement, the possibility of which is suggested by quence content and located in euchromatin (KIM et nl. the relative locations of the NOR in S. proj!3inqu1ini 11s. S. 2005b), where gene density and recombination rate are bicolor (Bowws at nl. 2003). higher. A srnaller number of the BACs were located within The sorghurn genorrle has been estimated to be -2- heterochromatic regions indicating that while more fold larger than the genome of rice (PRICEet al. 2005). dif'ficult, these r-e'gions can also be analyzed by BAGFISH However, rice and sorghurn chromosomes are largely analysis. The analysis derrlonstrated that the sorghum colinear indicating that sorghum genome expansion linkage map derived from an in~aspecificcross of BTx623 relative to rice is not due to a large-scale genome dupli- and IS3620C (MENZet al. 2002) provides excellent cov- cation event (PENGet nl. 1999;WILSON et al. 1999; KLEIN erage of the sorghum genome. Overall, the ortfer of ri nl. 2003) even though thew is strong evidence for seg- BACs along the sorghurrr chroinosoines was concorcfant ineiltal and possibly a whole-genorne duplicatiorl in the with the location of these BACs on the sorghum linkage common progenitor of rice and sorghuill (PATERSON map, thereby confirming the high level of accuracy that r?t nl. 2003; Yr) et n1.2005). Several studies have identified was achieved during integration of the linkage and arld aligned eight l~orrleologouspairs of sorghum and physical maps. This study enables ii~for~nationon the rice chromosomes and shown that sorghum chromo- architecture of the sorghum chromosoi~lesto be cross- somes 1 and 2 represent fusions of DNA corresponding referenced to linkage, physical, and comparative maps to rice chrotnosomes 3 and 10 and 7 and 9, respectively of the sorghum genome (KLEINet al. 2000; MENZet al. (KELLOGG200 1) . Estirna tes of DNA coil tent per chro- 2002; KLEINet nl. 2003) and to an assessment of sor- mosome based on a genome size of 818 Mbp show that ghum diversity (MEN%et nl. 2004). This integrated set of sorghum chrotnosomes are -1.7- to -2.8-fold larger infbrrnation provides a solid foundation for genome than the corresponding homeologous rice chrotnosonles research on this important C4 grass species and should or chromosomal regions. However, the increase in DNA help provide a framework for the S. bit-olm sequencing content is not uniform across the homeologous sorghu~n project (SORGHUMGENOMICS PIANNINC; ?VC)RICSHOP and rice chrornosornes even thorxgh they show a high PARTICIPANTS2005; htt~~://~'~~j~.i.doe.gov/News/rie~~s~degree of macrocoliriearity (PENGet a/. 1999; WILSON 5-12-05.html). The integration of genetic and cytoge- d al. 1999; KLEIN et al. 2003). An analysis of six pairs of netic information was an important step in the rice and horneologous sorghum and rice chromosomes showed rl/ldicng) imrncatula genotxle sequencii~gprojects because that the euchrornatic portion of these sorghum chrorno- it provided information on the distribution of hetero- somes is -1 .$-fold larger than thc colinear regions of the chromatin, euchi-omatin,centromeres, and genes across l~omeologousrice chromosomes, whereas the pericen- these genomes (CHKNGel nl. 2001b; K~JIJK~VAel nl. trornet-ic heterochromatic regions are -3.6-fold larger. The increased size of' euchromatic and heterochro- sertion (SANMIGUKI,et al. 1996). The preferential inser- rrlatic regions of S. bicolor relative to 0. sati-nu raises ques- tion of retroelements into related sequences already tions about how and when this difference was established present in heterochromatic regions could have contrib- and its functional significance. Nlxme rous studies have uted to localized expa~lsionof heterochrornatin and to a shown that variation in genome size not related to poly- decrease in gene density in these regions observed in ploidy is due primarily to differences in repetitive DNA sorghum relative to ricc. It is also possible that the cur- (FL~VELLet a/. 19'74; Uozu et a!. 1997; BENNETZENet a!. rent differences in the size and distribution of hetero- 2003). In plants, retroelenlent DNiI accounts for a large chromatin in sorghum compared to rice and maize are portion of the repeat fraction. For example, in rice, the result of processes that decrease the DNA content of retroelement sequences account for at least -13% of genomes (BENNETZEKet al. 2005). Recent studies have the genome (SASAKIet al. 2002) compared to -33% for demonstrated that unequal homologous recombina- sorg11um (BEDELLet al, 2005) and at least -60% for the tion and illegitimate recombirlatiorl play major roles in maize genome (MESSINGet al. 2004; SANMIGCTELand elimi~iatingDNL4 from genomes (SHIRASUet al. 2000; BENNETZEN1999). While single LTR-retrotransposons DEWS et al. 2002). For example, it was estimated that are sometimes inserted in close proximity to genes located -190 hlbp of DNA has been rernoved from the rice in euchromatic regions, these seqrxences are preferell- genome over the past 5 rrlilliorl years (BENNETZENet a/. tially located in pericentroineric regions of heterochro- 2005). Differential action of retroti-ansposons could also matin (K~JMAKand BENNE'I'ZI.:N1999). This is consistent have caused differential genome expansion in this same witli sequence scanning of sorghum BACs mapping to time frairle because dating of 1,TR divergence in rice, heterochromatic regions, which showed lower gene sorghum, and maize indicates that the average age of density and a corresponding increase in retroelemen t- these elements is -1.5-2.5 MY in all three species (MA related sequences compared to BrlCs from euchromatic et al. 2004; BENIVKTZENet nl. 2005). Overall, the amount regions (KLEIN et (al. 2003). The increase in retroeleinent of heterochro~xlaticDNA in different sorghtrm chromo- content in sorghum compared to rice is also consistent somes ranged from -35.5 to -49 Mbp, suggesting that with the -3.6-fold increase in the size of the sorghum the processes adding and removing DNA from these re- heterochrorxlatic regions compared to the colinear re- gions are acting somewhat uniformly on all chromosomes. gions of ricc. The results obtained in this study were also used to The increase in size of sorghum heterochrornatic re- estimate the number of genes in the euchrornatic and gions compared to rice could be due to expansion and/ heterochromatic regions of the sorghum genome. In or dispersion of heterochroi~latirlalong the chrorno- one approach, gene density in sorghuxn was predicted somes. Two different methods indicated that the pericen- on the basis of alignment of sorghum chromosomal re- tromeric heterochromatic region of sorghum chromosome ,eons to rice because the organization of genes across 3 spans -38 Mbp. This -38-Mbp heterochrornatic region horneologous pairs of sorghum and rice chromosoares of sorghum chroinosome 3 aligns to a -8.6-Mbp colin- shows a high degree of macrocolinearit).: I11 this study, ear region of rice chromosome 1 that spans the centro- the euchromatic and heterochromatic regions of six mere (KI.E.:INet al. 2003). Earlier cytogenetic analysis of sorghum chrornosomes were aligned to colinear por- rice chromosomes revealed that heterocllroinatin cove1-s tions of the corresponding horneologous rice chrorno- - 18% of rice chromosome 1 pachytene-stage bivalen ts somes. If the gene content of rice and sorghum is similar or a minimum of -8.2 Mbp of DNA (estimated on the in colinear regions, then the euchrornatic portions of basis of data in CHENGel a1. 2001a). Therefore, the -4 the six sorghum chromosomes analyzed are predicted fold increase in the size of the pericentromeric hetero- to have an average gene density of 1 gene modc1/12.3 chromatic region of sorghum chrornosome 3 relative to kbp on the basis or an estimated gene complement of the colinear region of rice chron~osorne1 appears to be -4'7,000. This nleasrlrement of gene density is in rea- due prinlarily to an irlcrcase in the size of this region sonable agreement with published results on the anno- rather than to hetcrochroinatin dispersion along the tation of a small number of BAC sequences from sorghum chromosome. However, the boundaries between het- euchromatic regions (Mo~rs~rcl.:et 01. 2002; LAI ~t a/. erochromatin and euchromatiri are less distinct in rice 2004; SWIGONOVAet al. 2004; KIXINet al. 2005). Our than in sorghum. Therefore, further analysis of thc measurements indicate that euchromatic DNA spans location or heterochromatin on rice chroinosomes, like -50% of the sorghum genome (-40'7 Mbp). If a gene that conducted by 1,r et nl. (2005), will be needed to density of 1 gene model/l2.3 kbp is present throrxghorxt detennirle more precisely the relative distribution of the euchromatic regions of sorghum, then we predict heterochromatin in sorghum and rice. This comparison that -70% of the sorghum genes are located in the may also contribute to an understanding of' important euchromatic portion of the sorghum genome. Assum- differences in gene expressiorl in these regions of the ing sorghum and rice encode a sirrlilar set of genes, -30% genomes (LI et al. 2005). of the sorghuin genes reside in heterochromatic regions Retroelement sequences are often nested due to at -2.7-fold lower gene density (-1 gene mode1/34.:', mrxl tiple rounds of localized LTR-re tro transposon in- kbp). We note that the eatitnates above are based on a prediction of -47,000 lice gene models (http://www.tigr. heterochrc)matin spans most of the short arm. A similar org/ tdb/e2kl/osal /pseudornolecules/ii~fo.shtnli) and organization of heterochrornatin is present in the horneo- that a lower estimate of -37,500 rice gene models has logous rice chromosome 4 that is also associated with been predicted by another group using somewhat dif- reduced recombination (CHKNGet (11. 2C)Ola).The aver- ferent cri telia (IN?'E:RNA~T'IONAI,RXCK GKNOME SEQUKNCTN(; age rate of recombitlatior1 across the he terochrornatic PROJECT2005). Current procedures for predicting gene portion of the sorghurn gellome wru -34fold lower than models likely result in an overestimate of actual gene recombination withi11 euchromatic regions. This is not content in both sorghuin and rice (BE:NNETZENet nl. surprising because the general suppression c)f recombi- 2004). Therefore, a precise understanding of the sor- nation in heterochromatic regions is a long-s-stariding ghum gene complement ~2.111 require a complete sor- obsewation (MATIIER1939). Suppression of recombi- ghum genorne sequence and validation of predicted nation in heterochromatin is associated with accutnu- gene models. However, given these caveats, the cur-rent lation of repeated sequences arid the formation of analysis predicts that -70% of the sorghurn genes are modified chromatin structure in these regions (14\1RAb10~~ located in the distal euchromatic regions that span 2002). Recombination in the heterochrornatic region of -50% of the sorghum genorne with a gene density on sorghum 142s -15-fold lower than that in colinear regions average only -1.6-fold lower tlian that of rice. There- of rice. This is consistent with an increase in repetitive fore, sequencing these regions by a combination of DNA and associated heterochronzatin in sorghum com- whole-genome shotgun and BAG-by-BAC sequencing pared to the colinear regions of rice. However, it was methods appears straightforward. Small blocks of het- s~rrprisingthat the rate of recombination in hetero- erochi-omatin are located within the euchroinatic re- chromatic regions of different sorghum chroiz~osomcs gions, and knowledge of their location based on BAG also varied -15-fold. This may be explained in part by FISH analysis will be useful during sequence assernbly. differences in gene density within the hererochrornatic In contrast, the -50% of the sorghum genoirie repre- regions of different chrornosornes. However, other fac- sented by heterochromatin will be more ~hallen~gingto tors including differences in the arrangement of DNA sequence and assemble. These regions are predicted to segments and speciiic genes, and differential expansion have at least -2.7'-fold lower gene density than the eu- of repetitive DNA in the two parental lines used in link- chromatic arms. This is consistent with the reduced gene age map construction, could also contribute to the ob- density observed in sequence scan data from BACs that served variation (BRUNNER4t ul. 200.5). At present we map to the heterochrornatic regions (KI,EINel al. 2003). know little about the order arld distribution of genes While the distribution of genes within the heterochro- and repetitive DNA uitlliil sorghum heterochromatiu matin is unknown, islands of gcizic sequences do appear and the relationship between this and local rates of to exist in the pericentrorneric region. Most UACs from recombination. Studies similar to those conducted in this region used in FISH analysis resulted in a "chromo- maize (Fu et al. 2001; Fv et al. 2002; YAOet nl, 2002) will some painting" pattern consistent with the presence of be required to more fully explain the variation in recom- interspersed repetitive eleizzents (data not shown). How- bination observed here. While the exact causes of low ever, several RACs produced a strong, localized signal and variable recombination in pericentromeric hetero- cc~nsistentwith the existence of srnall sectors of relatively chromatin remain to be clarified, the impact of low high gene density (Figure 1C, BAC probe 3-13). Never- recombination on the evolution and linkage disequilib- theless, sequences from most of the BACs mapped to the rium of the sorghum genes encoded by this portion of pericen trolrleiic region are rich in repetitive sequences, the genoxxle is of great ir~terestarid practical sigilifica~~ce especially retrotransposon-derived sequences, relative to sorghum geneticists and breeders. to BACs mapped to euchroinatic DNA IN et al. 2003). We thank W. L. Rooney for providing plan t lnaterial for chro~nosolne Because recombination is suppressed in the hetero- preparations. This work was supported in part by National Science chromatic regions arid sequence repeat content is high, Foundation Plant Genome Kesearcli Grants LfB1-0077713 (P.E.K and construction of robust BAGbased physical maps and J.E.M.)and DBI-03215'78 (P.E.K., R.R.K., arid J.E.M.),by the Pex~y sequence assembly in this part of the sorghum genome Adkissor1 Chair in -Agricultural Biology (J.E.M.), and by the U.S. 1)epartment of .Agricul ture's 12gricultural Research Senice (R.R.K). will be rnnclz rnore cl~allenging. The rate of recombination across tlle euchromatic portion of the sorghum genome is relatively high (-0.25 Mbp/cM), similar to the average rate of recombination LITERATURE CITED across rice chrornosonles outside of cerl trorneric re- gions (Wu et al. 2003). There was an -2.8-fold variation AN. S. Y., S. PARK,D. H. JEONG, D. 17. LEE, H. C. 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