Copyright 0 1989 by the Genetics Society of America Y Chromosome Evolution in the Subgenus Mus (Genus Mus) Priscilla K. Tucker,' Barbara K. Lee and Eva M. Eicher The JacksonLaboratory, Bar Harbor, Maine04609 Manuscript received November 30, 1987 Accepted for publication January 20, 1989 ABSTRACT A 305 base pair DNA sequence isolated from the Y chromosome of the inbred mouse strain C57BL/10 was used to investigate the pattern and tempo of evolution of Y chromosome DNA sequences for five species in the subgenus Mus, including Mus spretus, Mus hortulanus, Mus abbotti, Mus musculus and Mus domesticus. Variation in hybridization patternsbetween species was character- ized by differences in fragment lengths of both intensely and faintly hybridizing fragments, whereas variation in hybridizationpatterns within species was characterizedprimarily by differences in fragment lengths of faintly hybridizing fragments. Phylogenetic analyses were conducted based on fragment size variation within and among species. Phylogenetic relationships inferred from these analyses partly agreewith the phylogenetic relationships obtained from biochemical and mitochondrial DNA data. We conclude that a set of DNA sequences common to the Y chromosomes of a closely related group of species in the subgenus Mus has evolved rapidly as reflected by sequence divergence and sequence amplification. N the classical model of sex chromosome evolution, DNA sequences, AC11 (NISHIOKAand LAMOTHE I heteromorphic sex chromosomes are hypothesized 1986) and YBlO (EICHERet al. 1989), isolated from to have evolved from a pair of homologous chromo- the Y chromosomes from two inbred mouse strains somes by the suppression of crossing over between the hybridize to genomic DNA from males ofsome species sex chromosome precursors and the subsequent loss in the subgenus Mus and distinct hybridization pat- of gene function onthe sex chromosome that becomes terns characterized by differences in fragment length the Y chromosome (MULLER 1914). OHNO(1967) and hybridization intensity were observed for each recalled this hypothesis to explain vertebrate sex chro- species. These data suggest that Y chromosome-spe- mosome evolution including the evolution of the cific DNA sequences rapidly evolve in closely related mammalian X and Y chromosomes. The X and Y species. chromosomes of mammals exhibit extreme hetero- In this report we present additional evidence for morphism and, with rare exception,recombination the rapid evolution of DNA sequences found exclu- between them is limited to a small region (SOLARI sively on the mouse Y chromosome from an investi- 1974). Thus, the majority of the Y chromosome is gation of the tempo and mode of Y chromosomal monosomic and inherited from father to son. sequence change at both the species and population Recent molecular investigations of the human levels. We alsoinvestigated how a paternally inherited (GOODFELLOW,DARLING andWOLFE 1985) andmouse character contributes to an understandingof the phy- Y chromosomes (SINGH,PURWM and JONES 1981; logenetic relationships within the genus Mus. We used JONES and SINGH198 1;PHILLIPS et al. 1982; EICHER, pYB 10 (EICHERet al. 1989) as a probein combination PHILLIPSand WASHBURN 1983; LAMARand PALMER with eight restriction enzymes to identify species-spe- 1984; BISHOPet al. 1985; NISHIOKAand LAMOTHE cific patterns of hybridization from a large sample of 1986; NALLASETHand DEWEY 1986) identifiedDNA mice in the subgenus Mus, including Mus spretus, Mus sequences specific to or enriched on the Y chromo- hortulanus (= M. spicilegus, BONHOMME1986), Mus some. Additionally, using Y-specific or Y-enriched re- abbotti (= M. spretoides or M. macedonicus, BONHOMME peated DNA sequences as probes, restriction fragment 1986), Mus musculus and Mus domesticus. Throughout length polymorphisms have been identified among Y this study we recognize M. musculus and M. domesticus chromosomes of human populations (CASANOVAet al. as distinct species rather than subspecies or semispe- 1985) as well as among Y chromosomes of laboratory cies based on data from the hybrid zoneinvestigations inbred mouse strains and wild mice (LAMAR andPAL- of these two taxa (HUNT andSELANDER 1973; SAGE, MER 1984; BISHOPet d. 1985; NISHIOKAand LA- WHITNEYand WILSON1986; SAGEet al. 1986). MOTHE 1986; EICHER et al. 1989). For example, two MATERIALSAND METHODS ' Currentaddress: Museum of Zoology, University of Michigan, Ann Mice: Wild mice used in this study came from either Arbor, Michigan 48 109. established laboratory colonies or from field trapping. Spe- Genetics 122: 169-179 (May, 1989) 170 P. K. Tucker, B. K. L.ee and E. M. Eicher cies, number of individuals sampled, original collecting lo- minimum of two laboratory raised mice from each taxa were calities and source of taxa used in our study are listed in analyzed. Table 1. DNA preparation: High molecularweight mouse ge- nomicDNA was prepared from either frozen tissue or RESULTS frozen nuclear pellets. DNA from liver, kidney, spleen, and testis was prepared following the method of JENKINS et al. Species-specific patterns of hybridization were ob- (1982). Frozen nuclear pellets were incubated at 65" over- served when Southern blots containing male genomic night in 10 ml of extraction buffer (50 mM Tris, pH 8.0, DNAs from M. abbotti, M. hortulanus, M. spretus, M. 100 mM EDTA, 100 mM NaCI, 1% SDS) and 0.5 mlof musculus, and M. domesticus (Table 1) were probed proteinase K (10 mg/ml) in 10 mM Tris, pH 7.5, prior to extraction with phenol following the method of JENKINSet with pYB10. A total of 180 different restriction frag- al. (1982). ments were identified by single digests of male ge- Restriction endonuclease digestions and Southern blot nomic DNA using eight different restriction enzymes, preparation: Restriction enzyme digests of genomic DNAs representing four (HaeIII, TaqI), five (Hinfl)and six were performed following the procedures of the supplier (BglII, EcoRI, HindIII, PvuII, PstI) base cutters. No BRL (Bethesda Research Laboratories, Inc.). Ten micro- grams of genomic DNA were digested for 4 hr and separated hybridization was observed when Southern blots con- by size in 1% agarose at 30 V for up to 20 hr. DNA was tainingfemale genomic DNAs from M. abbotti, M. transferred to Zeta-Probe nylon membranes (Bio-Rad) in hortulanus, M. spretus, M. musculus and M. domesticus 0.4 M NaOH overnight without pretreatment (REED and (Table 1) were probed with pYBlO (data not shown). MANN1985). This verifies the observation by EICHERet al. (1989) DNA labeling The probe pYB10, derived from the Y chromosome of C57BL10 (EICHERet al. 1989), was labeled that YBlO sequences are found exclusively on the Y with [a-"PI-dCTP (Amersham) following MANIATIS, chromosome in these five species. The hybridization FRITSCHand SAMBROOK(1982) using the T4polymerase kit patterns observed with PstI (Figure 1) typify all single supplied by BRL. The specific activity of labeled DNA for digestsusing theeight restriction enzymes listed all experiments was greater than 0.5 X lo9 cpm/pg. above.Three basic trendsemerged. First, there is Hybridization conditions and autoradiography: Nylon membranes were prewashed at 65" for 1 hr in 0.1 % SSC overall less hybridization toDNA from M. spretus and and 0.5% SDS and prehybridized at 65" for 4 hr in 100 ml M. hortulanus than toM. abbotti, M. domesticus and M. of 4 X SSCP [ 1 X SSCP = 121 mM NaCI, 15 mM Nan citrate, musculus. Second, forsix enzymes, HaeIII, TagI,BglII, 15 mM Na2HP04, 5 mM NaH2P04],10 X Denhardt's solu- HindIII, PvuII and PstI, at least one restriction frag- tion and 1% SDS. Membranes were hybridizedovernight at ment is shared amongall five species and these shared 65" in 20ml of 4 X SSCP, 2 X Denhardt's solution, 1% SDS, 0.2 pg/ml denatured sonicated salmon sperm DNA, fragments typically vary in intensity of hybridization and approximately 1.0 X lo6 cpm/ml denatured radioac- among species. Third,between speciesvariation is tively labeled probe. Following hybridization, the nylon characterized primarily by the presence and absence membranes were rinsed twicein 2 X SSC/O. 1% SDS at room of both intensely and faintly hybridizing restriction temperature for 15 min and three times in 0.1 X SSC/O. I % fragments, whereas within species variationis charac- SDS at 65" for 30 min. The stringency conditions at 65", terized primarily, but notexclusively, the presence 0.1 X SSC, and a 39% G-C content (determined from DNA by sequence analysis of YB10,EMBL accession number and absence of faintlyhybridizing restriction frag- X 12900) were calculated to be approximately 98% (SHAW ments.Intensely hybridizing fragments, including et al. 1984). The membranes were exposed to Kodak XAR- shared fragments that vary ia hybridization intensity 5 film by varying lengths of time at room temperature or at between species, are tabulated for each enzyme and -70" with Dupont Cronex Lightening Plusintensifying screens. each species in Table 2. Analysis of autoradiographs: The approximate size of We conducted a phylogenetic analysis (PAUP, ver- each restriction fragment was ascertained using a custom- sion 2.4, SWOFFORD,1985) of these five Mus species ized computer program written for the Apple Ile and a using the 180 restriction fragments as characters.TWO HIPAD digitizer (Houston Instrument). Phylogenetic anal- minimum length trees were produced from 170 apo- yses using parsimony (PAUP, version 2.4, SWOFFORD1985) were conducted for two sets of taxa using restriction frag- morphic (derived) characters (Figure2). This analysis ments lengths as characters. Fragments of equal molecular substantiates the observed species-specific patterns of weight generated by each restriction enzyme were assumed hybridization because each species branch, including identical between taxa. Each individual taxon was scored M. spretus, M. hortulanus, M. abbotti, M. musculus and for the presence or absence of all restriction fragments M. domesticus, is clearly defined by a series of restric- identified in a given setof taxa. For all phylogenetic analyses, variation in copy number atnong shared restriction frag- tionfragments including the majority of intensely ments was not scored as separate characters and thus the hybridizingfragments.