Copyright 0 1994 by the Genetics Society of America X A Detailed RFLP Map~ of Cotton, Gossypium hirsutum Gossypium barbadense: Chromosome Organization and Evolution in a Disomic Polyploid Genome Alesia J. Reinisch," Jian-mjn Dong,* Curt L. Brubaker,t David M. Stelly," Jonathan F. Wendelt and Andrew H. Paterson" *Department of Soil and Crop Sciences, Texas A&M University, College Station Texas 77843-2474, and +Department of Botany, Iowa State University, Ames, Iowa 50011 Manuscript received January6, 1994 Accepted for publication July12, 1994 ABSTRACT We employ a detailed restriction fragment length polymorphism(RFLP) map to investigate chromo- some organization and evolution in cotton, a disomic polyploid. About 46.2% of nuclear DNA probes detect RFLPs distinguishing Gossypium hirsutum and Gossypium barbadense; and 705 RFLP loci are assembled into 41 linkage groups and 4675 cM. The subgenomic origin (A us. D) of most, and chro- mosomal identityof 14 (of 26), linkage groups is shown. TheA and D subgenomes show similar recom- binational length, suggesting that repetitive DNA in the physically larger A subgenome is recombina- tionally inert. RFLPs are somewhat moreabundant in the D subgenome. Linkage among duplicatedRFLPs reveals 11 pairs of homoeologous chromosomal regions-two appear homosequential,most differ by in- versions, and at least one differs by a translocation. Most homoeologies involve chromosomes from different subgenomes, putatively reflecting the n = 13 to n = 26 polyploidization event of 1.1-1.9 mil- lionyears ago. Several observations suggest that another,earlier, polyploidization event spawned n = 13 cottons, at least 25 million years ago. The cotton genome contains about 400-kb DNA per cM, hence mapbased gene cloning is feasible. The cotton map affords new opportunities to study chro- mosome evolution, andto exploit Gossypium genetic resources for improvementof the world's leading natural fiber. HE genus Gossypium L. has long been a focus of raploids contain two distinct genomes, which resemble T genetic, systematic and breeding research. Gos- the extant A genome of G. herbaceum (n= 13) and D sypium comprises about 50 diploid and tetraploid spe- genome of G. raimondii Ulbrich (n= 13), respectively. cies indigenous to Africa, Central and South America, The A and D genome species diverged from a common Asia, Australia, the Galapagos, and Hawaii(FRYXELL ancestor about 6-1 1 million years ago (WENDEL1989). 1979, 1992). Cultivated types derived from fourspecies, The putative A X D polyploidization event occurred namely G. hirsutum L. (n= 2x = 26), G. barbadense L. in the New World, about 1.1-1.9 million years ago, (n= 2x = 26), G. arboreum L. (n = x = 13), and G. and required transoceanic migration of the maternalA herbaceum L. (n= x = 13), provide the world's leading genome ancestor (WENDEL1989, WENDELand ALBERT natural fiber, cotton,and are also a major oilseed crop. 1992), which is indigenous to the Old World (FRYXELL Cotton was among the first species to which the Men- 1979). Polyploidization was followed by radiation and delian principles were applied (BALLS1906), and has a divergence, with distinct n = 26 AD genome species now long history of improvement through breeding,with sus- indigenous to Central America (G. hirsutum), South tained long-term yield gains of 7-10 kg lint/ha/yr America ( G. barbadense, G. mustelinum Miers ex Watt), (MEREDITHand BRIDGE1984). The annual world cotton the Hawaiian Islands (G. tomentosum Nuttall ex See- crop of ca. 65 million bales (of 218 kg/bale) , has a value mann), and the Galapagos Islands ( G. danuinii Watt) of ca. US$15-20 billion/yr. (FREXELL1979). Diploid species of the genusGossypium are all n = 13, Variation in ploidy among Gossypium spp., together and fall into 7 different "genome types," designated A-G with tolerance of aneuploidy in tetraploid species of Gos- based on chromosome pairing relationships (BEASLEY sypium, has facilitated use of cytogenetic techniques to 1942; ENDRIZZIet al. 1984). A total of 5 tetraploid explore cottongenetics and evolution. Among 198 mor- (n = 2x = 26) species are recognized. All tetraploid phological mutants described in cotton, 61 mutant loci species exhibit disomic chromosome pairing (KIMBER have been assembled into 16 linkage groups, through 1961). Chromosome pairing in interspecific crosses be- the collective results of many investigators. Using nul- tween diploid and tetraploid cottons suggests that tet- lisomic, monosomic, and monotelodisomic stocks, l lof Genetics 138 829-847 (November, 1994) 830 A. J. Reinisch et al. these linkage groups have been associated with chro- DNA probes: Mapped DNA probes included PstI-genomic mosomes (ENDRIZZIet al. 1985). fragments from G. raimondii (prefix G: 75 probes), G. her- Our objectives wereto use low copy DNA markers to baceum subsp. africanum Watt (Mauer) (accession A,-73;pre- fix A 192 probes), and G. hirsutum accession “TM-1” (pre- investigate cotton genome and chromosome organiza- fured M: 21 probes; and P 78 probes), low copy genomic tion at themolecular level, in a cross of G. hinuturn X restriction fragments selected from a libraly of G. barbadense G. barbadense. We have established a detailed restriction cv. “Pima S6” DNA cut with a mixture of five different blunt- fragment length polymorphism (RFLP) map of cotton, cutting fourcutters (prefmed LXP: 1 probe and PXP:40 identifymg genomic origins of, and homoeologies probes; to be described elsewhere; X. Zhao and AHP,in prepa- ration), genomic probes derived from G. hirsutum cv. “Tam- = among, most ofthe linkage groups in tetraploid (n 2% cot GNCH” and containing Not1 sites (prefixed pVNC), and = 26) cotton, and characterizing the nature and fre- cDNAs (143 probes) from a library prepared from drought- quency of rearrangements which distinguish homoe- stressed tissue ofG. hirsutum accession ”T25.”All probes were ologs. Identification of homoeologous chromosomal re- prepared by PCR amplification of plasmid or phage DNA, gions reveals the approximate locus of DNA probes using M13 primers (SP010and SP030, Operon, Alameda, Cali- monomorphic in one subgenome but polymorphic in fornia), followed by chromatography through homemade Sephadex G50 (Sigma) spuncolumns (SAMBROOKet al. 1989). the other, and representsa means to greatly increase the DNA extraction, electrophoresis, blotting, and hybridiza- informativeness of genetic maps in polyploids such as tion: DNA extractions followed PATEMONet al. (1993).Replica cotton. Characterization of the comparative organiza- blots wereprepared using each of the restriction enzymes used tion of homoeologous chromosomes promises to in- for polymorphism screening (below). DNA electrophoresis, crease the markerdensity of molecular maps in disomic blotting, probe labeling, Southern hybridization, and autora- diography were as described by CHITTENDENet al. (1994). ex- polyploids, facilitating both genetic and physical cept that after liquid hybridization, filterswere washed at 2X, mapping applications. lX, and 0.5X SSC (instead of three washes at 0.1X). Individual DNA probes were screened for polymorphisms MATERIALSAND METHODS distinguishing the grandparents (G. hirsutum race “palmeri” Genetic stocks: The genetic mapping population com- and G. barbadense acc. “K101”) using 4-6 restriction enzymes prised 57F2 individuals froma cross between single individuals (EcoRI,EcoRV, HindIII, XbaI in all cases,and BamHI, and CfoI of G. hirsutum race “palmeri” (see BRUBAKERand WENDEL in a subset of cases), and filters were washed for 20 min 1993) and G. barbadense acc. “K101.” These accessions were each in 2X, lX, and 0.5X SSC, 0.1%SDS at 65” prior to selected because they are largely homozygous, and relatively autoradiography. primitive, thus free from the interspecific introgression that Data analysis Linkage maps and related statistics were de- characterizes somepopulations of these two species from sym- termined using MapMaker 1.0 (LANDER et al. 1987; provided patric portions of their indigenous ranges (PERCYand WENDEL by L. PROCTOR,DuPont Co.) running on a Macintosh Quadra 1990; WENDELand ALBERT 1992; BRUBAKERet al. 1993), as well 700 under System ’7.01. A LOD (base 10-log of the ratio be- as cultivated types (PERCYand WENDEL 1990;G. WANCand A. tween odds of linkage and odds of non-linkage) score of 4.0 H. PATERSON,unpublished data). Self-pollinated progeny from was used toinfer linkage in two-point analyses: sincethe cotton the “palmeri“ and “K101” parents were used for preliminary RFLP map spans nearly 5000 cM, the standard LOD 3.0 used surveys of DNA polymorphism, prior to mapping. for smaller genetic maps is not sufiiciently stringent. lnitial Genomic identityof RFLP alleles (see Figures2 and 3) were frameworks of markers were evaluated using the “compare” inferred based upon comparison of G. barbadense and G. hir- function, and only orders preferred by a LOD of at least 2.0 sutum parents to accessionsrepresenting the diploid progeni- (e.g.,a 100-fold difference in likelihood) over alternate orders tor genomes of tetraploid Gossypium, i.e., the A genome dip were accepted. New markers were added to the frameworks loids G. arboreum (accession 447) and G. herbaceum using the “try” function, and the maximum likelihood orders (accession 4 A,97), and the D genome diploids G. triEobum of each linkage group ultimately verified by the “ripp1e”func- (Mocino & Sese ex DeCandolle) Skovsted (un-named acces- tion. Segregation ratios were calculated SASusing (SM Institute, sion) and G. raimondii (un-named accession). 1989).