‘bvisSIHJ RETURNING MATERIALS: Place in book drop to LJBRAflJES remove this checkout from _:—. your record. FINES win be charged if book is returned after the date stamped below. DDDNS'IRATICN 0F PATERNAL INHERITANCE 0F PLASTIIB IN PICE'A (PINACEAE) By Michael Stine A DISSERTATION Suhnitted to Michigan State University in partial fulfillment of the requirenents for the degree of IXXITOR 0F HHMBOPHY Plant Breeding and Genetics - Forestry 1988 DWSTRATION OF PATERNAL INHERITANCE 0F PLASTIIB IN PICEA (PINACEAE) By MICHAEL STINE Chloroplast DNA (chNA) was purified from Picea glauca, P. mans, P. engelmannii, and P. amorika, and was digested with several restriction endonucleases. Interspecific restriction fragment length polymorphisms (RFLPs) of ch A were identified, The RFLPs were identified as ch A'by the hybridization of cloned, 32-P labeled, petunia chNA to the polymorphic bands, and.by the lack of hybridization of a cloned.and labeled mtDNA probe from maize. Chloroplast DNA RFLPs that showed no intraspecific variation when examdned across the natural range for each species, were used as markers to follow the inheritance of plastids in interspecific hybrids. The inheritance of plastids was determined for Fl-hybrids from reciprocal crosses of P. glauca and P. pungens, P. glauca and P. amorflm, and Fl-hybrids of P. emelmannii x pungens. All 31 F1- hybrids examined showed.the chNA genotypes of the pollen parent, or the paternal species . C°pyright by MICHAEL STINE 1988 I would like to thank my major professor Dr. Daniel E. Keathley, and um coumittee members Drs. James Hanover, Kenneth Sink and Barbara Sears for guidance throughout this study. In particular I would like to thank Dr. Barbara Sears for her personal instruction on many of the techniques used in this study. Also, Dr. Harry E. Sonmer is acknowledged for initially calling my attention to the phenomenon of paternal inheritance in gymnosperms , and Mark Hilf for conversations on molecular approaches to the study. Thanks go to Dr. J. Palmer for the gift of the Petunia chNA library and for discussions on approaches for the purification of chNA from gymosperms, and to Dr. T. Fox for the use of the coxII probe from mize, and to Drs. James Hanover and the late Jonathon Wright, and their many students, for the numerous spruce hybrids, and provenance tests available for use in this study. Most of all I want to thank my parents and my wife Bonnie, for their love and support during the completion of this dissertation, and throughout the many years of graduate school . iv TABLE OF CONTENTS LIST OF FIGURES LIST OF TABLES INTRODUCTION CHAPTER I. Inheritance of plastids in interspecific hYbrids of blue spruce and.white spruce. Introduction Materials and Methods Results and Discussion Literature Cited II. Inheritance of plastids in reciprocal crosses of white spruce and Serbian spruce. Introduction Materials and Methods Results and Discussion Literature Cited III. Paternal inheritance of plastids in Engelmann spruce x blue spruce hybrids. Introduction 'Materials and Methods Results and Discussion Literature Cited Summary and Conclusions Appendix Page vi vii 10 16 30 32 33 34 36 44 46 47 48 50 57 59 63 Chapter I Figure Figure Figure Figure Figure Chapter II Figure Figure Chapter III Figure Figure Figure 3. 2. LIST OF FIGURES Identification of RFLPs that differentiate white spruce and blue spruce. White spruce provenance test. Blue spruce provenance test . Demonstration of autoradiographic techniques to detect underrepresented INAs . . Comparison of chNA RFLPs fran Fl-hybrids to parental species . Identification of MP3 that differentiate white spruce and Serbian spruce. Comparison of chNA RFIPs from Fl-hybrids to parental species. Identification of RFLPs that differentiate blue spruce and Engelmnn spruce. Conservation of diagnostic chNA RFLPs in Engelmrm spruce. Comparison of chA RFLPs from Fl-hybrids to parental species. vi Page 18 21 23 24 25 37 40 51 53 54 Chapter I. Table 1. Table 2. Chapter II. Table 1. Table 2. Chapter III. Table 1. Table 2. LIST OF’TABLES Parentage of white spruce and blue spruce hybrids. CpDNA RFLPs that differentiate white spruce fromxblue spruce. Parentage of white spruce and Serbian spruce hybrids. CpDNA RFLPs that differentiate white spruce from.Serbian spruce. Parentage of blue spruce and Engelmann spruce hybrids. CpDNA RFLPs that differentiate blue spruce from Engelman spruce. Summary and Conclusions Table 1. Table 2. Appendix Hybrid.spruces examdned.for for plastid inheritance. Conservation of interspecific RFLPs in "pure" species. Table A1. Location of blue spruce, white spruce, and hybrid trees used. Table A2. Location of white spruce, Serbian spruce, and hybrid trees used. vii Page 11 19 35 38 49 50 60 60 63 64 INIWDUCI‘ION Most angiosperms show maternal inheritance of plastids, with about one third showing some degree of biparental inheritance (Sears 1980, Whatley 1982) . In contrast, for most gymospenns, there has not been direct genetic evidence which would establish how plastids are inherited. This is primarily due to their long generation time and the lack of available plastid mutants. Most studies have therefore relied on either light, or electron microscopy to examine the fate of plastids following fertilization (Maheswari and Konar 1971; Willemse 1974). Delayed ferti- lization, following pollination, is a comon characteristic of many gymnosperms (Wilson and Burley 1983), which nukes it difficult to follow the fertilization process. Furthermore, microscopy destructively samples the fertilized archegonium cell linking it impossible to follow the fate of plastids at later points in time. So, even if plastids from the pollen tube are shown to enter the cell, it is not possible to determine whether the chNA remains viable, whether the plastids are capable of division, or if differential sorting out of plastids from the two cyto- plasms occurs. The report by Ohba et a1. (1971) was the first report in which the inheritance of a plastid mutant was followed in sexual crosses to estab- lish the inheritance of plastids in a gymnosperm. In this report, between 90 and 99% of the progeny showed the plastid phenotype of the pollen parent, with up to 5% chimeric progeny; thus, indicating probable 2 biparental inheritance. It should be noted that in reciprocal crosses , the rates of paternal transmission varied, with the mutant form showing 90% paternal transmission, and the wild-type showing a 99% paternal transmission rate, respectively. The use of restriction analysis of chloroplast DNA (chNA) to follow the inheritance of plastids has become increasingly popular (Metzlaff et a1. 1981; Bowman et a1. 1983). This technique has an advantage over the more traditional methods of following either natural or induced chloro- plast mutants , in that most chNA RFLPs should represent neutral muta- tions (Beckmann and Soller 1986). In addition to providing informtion on the transmission pattern of plastid DNA, RFLP analysis of the chNA will provide groundwork for research in a nunber of areas. These techniques are readily amenable for the use in phylogenetic analysis, either by mapping structural changes in the molecule (Palmer 1985) , or by the sequencing of specific genes located on individual fragemnts (Zurawski and Clegg 1987) . Many basic questions in gymnosperm taxonomy still exist as illustrated by Florin (1963) describing seven families and 52 genera of conifers, while Silba (1984) and Hart et a1. (1987) maintain Florin’ 8 seven families, but Silba (1984) accepts 60 genera, and Hart et a1. (1987) 63 genera. Meyen (1984) differs even more from Florin (1963) by placing Ginkoales into a differ- ent class from Conifierales, and reduces Taxales to family rank. One of the major problems in the stub? of evolution and taxonomy of plants is choosing characteristics that show the proper level of varia- tion to resolve groups at the desired level. mpping of RFLPs of chNA has most cannonly been used to resolve species and genera (Palmer 1986) , but based on recent results these techniques may provide useful and needed informtion on gymnosperm taxonmy at the level of family or higher. Until recently, Ginkgo bdloba was the only gymnosperm.for which a map of the chNA existed (Palmer 1985) . The map of the Ginkgo chNA molecule reveled a chNA structure which included a large inverted repeat region, which has been found in all angiosperms (except the Fabaceae subfamily Papilonoideae), three ferns, and the two bryophytes so far studied (Palmer 1985). Strauss et a1. (1988) have demonstrated that Douglas-fir (Pseudotsuga menziesii) and Monterey pine (Pinus radiate) both lack the inverted repeat structure, which would.represent a.maJor difference from.Gango. Thus, the mapping of ch A RFL s will very likely provide much needed information on the relationships of the gymna- sperms, and augment the existing morphological data. The study of population structure of tree species will also benefit by the determination of plastid and.mitochondrial inheritance. When the phenomenon of paternal inheritance of chNA, and the probable Internal inheritance of mitochondrial DNA (Neale and Sederoff 1987) is better understood, it may be possible to follow two relatively simple genetic systems in a single species, and to determdne how the dispersal mechanism (seed versus pollen) affects gene flow. Traditional tree breeding will benefit if enough variation in the chNA of individual species exists to allow for the identification of races or sub-species. In addition to understanding natural populations, this would help to identify seed sources of planted or exotic species for which records of their origin do not exist. Advanced.generation breeding will be aided by the understanding of chNA inheritance in the identification of hybrid progeny, either from 4 controlled crosses or naturally ocurring putative hybrids. With the . paternal transmission of plastids, coupled to the relatively easy task of identification of the species of the tree bearing the ovulate stroboli, all hybrid progeny should show the chNA type of the putative pollen donor. The utility of this technique will be limited until it is deter- mined whether chNA is strictly paternally inherited, or if there is some level of maternal or biparental inheritance. The mefulness of this technique for identifying hybrids will be inversely proportional to the level of biparental or maternal inheritance of chNA. For the study reported here, it was originally planned that RFLPs would be identified that differentiated each species to be used, and than individual trees would be crossed. 'Ihe chNA restriction patterns of the progeny were then to be compared directly to those of their parents. During the course of this study, very few female stroboli were produced by the species of interest, and it was not possible to complete the reciprocal crosses needed to produce interspecific hybrids. It was then decided to utilize existing Picea hybrids at Michigan State University (NBU) . The Department of Forestry at P80 has had a long-standing interest in spruce hybrids (Wright 1955, Hanover and Wilkinson 1969, Bongarten and Hanover 1982, Schaefer and Hanover 1985, Ernst et a1. 1988) and as such, a substantial nunber were readily available for study. The trees used in this study are listed in Tables A1 and A2. Due to unavoidable mortality, many of the parents of individual hybrids were not available, and thus the direct comparison of the hybrids to their parents was impossible. This necessitated finding RFLPs that differentiated individual species, but were conserved within a single species. This was accomplished.by sampling trees from.throughout the natural range of each species, and then checking for conservation of the interspecific RFLPs. Once interspecific RFLPs that are conserved within a species were identified, representatives of each parental species were compared to the interspecific hybrids. In all 31 Fl-hybrids examined, the chNA restric- tion pattern of the paternal species was observed. The bands produced by the endorestriction digests of the DNA samples were demonstrated to be ch A by: 1) using methylation sensitive restric- tion endonucleases, which freely cut chNA, but only rarely cut nuclear DNA; 2) hybridization of cloned chNA to the polymorphic bands; 3) failure of a cloned.mtDNA prdbe to hybridize to restriction fragment bands visible in the gels. The remainder of this dissertation will present the results of these studies in separate chapters for each hybrid.systemm Chapter I presents the analysis of blue spruce and.white spruce hybrids, and the provenance tests of each species. Chapter II shows the results of Serbian spruce and white spruce hybrids, for which the Serbian spruce parents of all the hybrids examined.were available for study. Chapter III presents a.more limited study of four Engelmann spruce x blue spruce hybrids, and three putative, naturally occurring interspecific hybrids. A sumry and conclusion is presented at the end of the dissertation. LI'I'ERATIE org Beckmann JS, Soller M (1986) Restriction fragment length polymorphisms and genetic improvement of agricultural species. Euphytica 35:111-124 Bongarten BC, Hanover JW (1982) Hybridization among white, red, blue and white x blue spruces. For Sci 28:129-134 Bowman CM, Bonnard G, Dyer TA (1983) Chloroplast DNA variation between species of m ticm and Aegilops. Location of the variation on the chloroplast genome and its relevance to the inheritance of the cytoplasm. Theor Appl Genet 65:247-262 Ernst SG, Keathley DE , Hanover JW (1988) Inheritance of isozymes in seed and bud tissues of blue and Engelmann spruce. Genome 29:239-246 Florin R (1963) The distribution of conifers and taxad genera in time and space. Acta Horti Berg. 20:121-312. Hanover JW, Wilkinson RC (1969) A new hybrid between blue spruce and white spruce. Can J Bot 47:1693-1700 Hart JA (1987 ) A cladistic analysis of conifers: preliminary results. J Arnold Arbor 68:269-307 Maheswari P, Konar RN (1971) Pinus Council of Scientific and Industrial Research, New Dehli, 107p Metzlaff M, Borner T, Hagemann R (1981) Variations of chloroplast DNAs in the genus Pelargoniun and their biparental inheritance. Theor Appl Genet 60:37—41 Neale DB, Sederoff RR (1988) Inheritance and evolution of conifer genomes. In: Hanover JW, Keathley DE (eds) Genetic Manipulations of woody plants. Plenun Press, New York, pp 251-264. Ohba K, Iwakawa M, Okada Y, Murai M (1971) Paternal transmission of a plastid anomaly in some reciprocal crosses of sugi , cryptomeria japonica D. Don. Silvae Genet 20:101-107_ Palmer JD (1985) Isolation and structural analysis of chloroplast DNA. Methods Enzymol. 118:167-186. Schaefer PR, Hanover JW (1985) A morphological comparison of blue and Engelmnn spruce in the Scotch creek drainage, Colorado. Sivae Genet 34:105—111 Sears BB (1980) Elimination of plastids during spermatogenesis and fertilization in the plant kingdom. Plasmid 4:233-255 Silba S (1984) An international census of the coniferae. Phytologia Mem 7:1-73. Strauss SH, Palmer JD, Howe GT, Doerksen AH (1988) Chloroplast genomes of two conifers lack a large inverted repeat and are extensively rearranged. Proc Natl Acad Sci (USA) 85:3898-3902. Whatley JM (1982) Ultrastructure of plastid inheritance: green algae to angiosperms. Biol Rev 57:527-569. Willem-e MIM (1974) Megagametogenesis and formation of neocytoplasm in Pinus sylvestris L. In: Linskens HF (ed) Fertilization in higher plants. North-Holland, Amsterdam. pp 97-102 Wilson MF, Burley N (1983) Mate choice in plants: Tactics, mechanisms, and consequences. Princeton University Press, Princeton, New Jersey. 251 p. Wright JW (1955) Species crossability in spruce in relation to distribution and taxonomy. For Sci 1:319-349 Zurawski G, Clegg MI‘ (1987 ) Evolution of higher-plant chloroplast DNA- encoded genes: Implications for structure-function and phylogenetic studies. Ann Rev Plant Physiol 38:391-418. CHAPTERI Inheritance of plastids in interspecific hybrids of blue spruce and white spruce. ABSTRACT Chloroplast DNA (chNA) was purified from blue spruce (Picea mans Engelm.) and white spruce (P. glauca (Moench) Voss), and was digested with several different restriction endonucleases . Restriction f ragnent length polymorphisms (RFLPs) were identified that differentiated the chNA of both species. Intraspecific conservation of the RFLPs that differentiated each species was confirmed by examining trees from across the natural range of each species. Ten Fl-hybrids were examined, and the chNA from each showed the banding pattern of the paternal species. Cloned Petunia chNA containing part of the rbcL gene hybridized to poly- morphic bands, while a cloned maize mtDNA probe of the coxII gene, failed to hybridize to any band. INTRODUCTION Most angiosperms exhibit maternal inheritance of the plastids, with approximately one third.having some degree of biparental inheritance of plastids (Sears 1980). The classic method of studying this character- istic is to follow the inheritance of a plastid mutant (either natural or induced) in reciprocal crosses. There is evidence that among gymosperms the inheritance of the cytoplasmic organelles may be either strictly or largely paternal. Ohba et al. (1971) followed the inheritance of induced chloroplast mutants in sugi (Cryptomeria japonica D. Don) atd determined that the plastids were inherited paternally approximately 90 to 99% of the time, providing the first genetic evidence for predominantly paternal transmission of plastids. Other evidence for paternal inheritance of cytoplasmic organelles in the Coniferales comes from microscopy studies of fertilization. Cytoplasmic organelles were seen moving through the pollen tube (Maheshwari and Konar 1971), or in which the neocytoplasm which formed following fertilization appeared to exclude the maternal organelles (Willemse 1974). Restriction analysis of chloroplast DNA (chNA) and.mitochondrial DNA (mtDNA) is a fairly recent tool for the study of organelle molecular biology. By comparing restriction fragment length polymorphisms (RFLPs) of either chNA or mtDNA, it has been possible to ascertain the inheri- tance of organelles with a high degree of certainty in Palargoniun (Metzlaff et al. 1981) atd in Triticun atd Aegilops (Bowman et al. 1983). The main advantages of this technique are that there is no need to either find or induce chloroplast or mitochondrial mutants, there is no question whether or not the mutation affects the inheritance of the organelles, 10 and there is no ambiguity as to whether there is differential sorting out of the organelles following fertilization. Recently, there have been several reports that used RFLP analysis of chNA to follow the inheritance of chloroplasts in members of the Coni- ferales. Neale et al. (1986) reported paternal transmission of plastids in 33 of 36 progeny from intraspecific crosses of Douglas-fir (Pseudo- tsuga menziesii (Mirb) Franco), the other three showed non-parental RFLP types. Szmidt et al. (1987 ) have reported firding the paternal pattern of chNA in 3 out of 6 interspecific Fl-hybrid progeny of Larix, with one showing the maternal pattern , atd two showing nonparental patterns . Paternal inheritance of chA has also been demonstrated in interspecific Fl-hybrids of lodgepole pine (Pinus contorta Dougl. ex. Loud.) x jack pine (P. banksiana Lamb.) (Wagner et a1. 1987). Neale atd Sederoff (1988) also reported paternal chNA transmission in redwood (Sequoia sempervimns D. Don Endl.). We report here methods of chNA purification from Pioea, the analy- sis of the DNA samples, demonstrating that it is chNA atd not nuclear DNA (nucDNA) or mtDNA, identification of RFLPs that differentiate both species, atd the paternal inheritance of chNA in interspecific hybrids. MATERIALS AND METHODS Plant @erial All trees used in this study are located in the Kellogg Experimental Forest, 7060 N. 42rd St., Augusta, MI 49012. The accession nunber, plantation number, std the row atd colunn nunbers for each tree are 11 listed in Table A1, and the parentage of the hybrids is listed in Table 1. The blue spruce are located in plantation 70.22, which is a rangewide provenance test established in 1970. The white spruce used are from a rangewide provenance test (plantation 63.05) that was established in 1963. The hybrids of blue atd white spruce are located in plantation 70.21, which was established over the four year period, 1970-1973. Detailed records for each tree are available from the Michigan Coopera- tive Tree Improvement Program (MICHOCI‘IP) at Michigan State University. Table 1 . Parentage of blue atd white spruce hybrids. Hybrid Female Parent Pollen Parent Accession Species Accession Species Accession Nunbert Nunber Nunber! 720007 P. glauca 60.25-3-9 P. plmgans 310676 720008 P. glauca 60.01-NN-10 P. mans 310677 720009 P. glauca 60.25-3-5 P. pungens 310678 720010 P. glauca 60.25-3-7 P. pungans 310679 720011 P. glauca 60.06-41 P. pungans 310678 720017 P. glauca 60.06-40A P. pungens 310679 720058 P. pungans Pp—5 P. glauca 190061 720059 P. pungans Pp-l P. glauca 190561 720060 P. pungans Pp-l P. glance 190562 720061 P. ptmgens Pp-l P. glauca 190560 X Add 67,000,000 to obtain the complete MICHGYI‘IP accession mmbers. Trees were sampled by cutting off branch tips containing the cur- rent, and previous season’s growth, with the cut erds placed into distilled water. Each tree was sampled by removing branches from the entire perimeter of the tree, from 1.0-1.5 m above the grourd. This was done to maximize the likelihood of identifying chimeric itdividtals. The cut branches were stored in the dark, at 4° C, for up to two weeks. 12 Chloroplast DNA Isolation The chloroplast DNA (chNA) isolation procedures were modified pro- cedures of Palmer (1985) atd Stine et al. (1988). Needles from irdivid- ual trees were cut from the branches atd washed thoroughly with distilled water. Any diseased or damaged needles were discarded. Both the current atd previous season’ 8 needles were used. All of the subsequent steps were carried out either on ice or at 4° C. All centrifugation runs were done at 4° C unless stated otherwise. All glassware and pipettes were silanized, prior to use, as outlined in thniatis et al. (1982). Between 75 and 100 g of needles were placed in a Waring blender with 10 to 20 volunes (w/v) of semi-frozen homogenation buffer (8% w/v sorbi- tol, 0.15% w/v polyvinylpyrrolidone MW = 40,000 (PVP), 0.1% w/v bovine serun albunin fraction V (BSA), 10% w/v polyethylene glycol (PEG 6000), 8 M EDTA, 1 11M ascorbic acid, 3 m cysteine, 50 M Tris (pH 7.5), 5 m mercaptoethanol) atd were homogenized for 45 secords . Throughout the chNA preparation, each step was monitored microscopically to ascertain the chloroplast’s purity. The homogenate was filtered through a 100 micron mesh wlon screen atd then through two layers of Miracloth (Calbiochem). The filtrate was centrifuged for 15 minutes at 2000 x g (Sorvall “-3 rotor). The pellet was then susperded in approximately 20 ml of wash buffer (homogemtion buffer minus the PVP) and then layered onto four sucrose step gradients . The sucrose step gradients were prepared by the procedures of Stine and Keathley (1987) and were composed of five layers containing 80%, 62.5%, 45.0%, 27.5% and 10.0% w./v. sucrose in 50 n1“! Tris, pH 7.5, 25 11M EDTA and 6.0% w./v. sorbitol. The samples were centrifuged for 10 13 minutes at 18,000 x g in a vertical rotor (Sorvall SV-288) with slow acceleration atd deceleration. The chloroplasts were removed from the 27.5-45.0% sucrose interface with a Pastuer pipette. The chloroplasts were then diluted slowly with 2 to 3 volunes of 50 nfl Tris (pH 8.0)/20 nfl EDTA, atd centrifuged at 18,800 x g for 10 minutes in a fixed angle rotor (Sorvall 88-34). The pellet was susperded in 2.0 ml of NET buffer (15 11M NaCl, 100 m EDTA, 50 M Tris, pH 9.0) atd 200 ul of predigested pronase (1.0 mg/ml) was added. The mixture was left on ice for fifteen minutes, after which sarkosyl was added, to a final con- centration of 1.0% w/v. The lysis mixture was gently shaken for 2 to 3 hours at 4° C. Two voltmes of a solution of 40 M Tris, pH 8.0, saturated with cesiun chloride (0801) were then added to the chloroplast lysate, atd the solution was centrifuged at 85,000 x g for 1.5 hours at 19° C in a swing- out rotor (Sorvall AH—650) , in an ultracentrifuge. The proteins atd other debris on the surface of the lysate were removed. The cleared lysate was then transferred to clean centrifuge tubes, bisbenzimide (Hoechst dye 33258) was added to a final concentration of 0.1 mg/ml atd the concentration of 0801 was adjusted to a refractive irdex of 1.3965 1 0.0005. This mixture was then centrifuged for 14 hours in a vertical rotor (Sorvall TV-865) at 155,000 x g atd 19° 0. Following centrifugation, the DNA batd was visualized with UV light (366 nm), atd was removed with a pipette. The bisbenzimide was extracted from the DNA solution with isopropanol saturated with NaCl atd water. This step was repeated until no fluorescence was detected using UV light. The DNA solution was then extracted two additional times. Two volunes of sterile double distilled water atd three volunes of isopropanol were 14 added, atd the DNA was precipitated at —20° C over night. The DNA samples were then centrifuged in a microcentrifuge at 13,000 x g at 25° for 10 minutes. The DNA pellet was washed 3X with 70% (v/v) ethanol, centrifuging for 5 minutes at 13,000 g after each washing. The DNA pel- let was dried under a stream of filtered air atd then dissolved in sterile TE buffer (10 nfl Tris (pH 8.0), 1 II“ EDTA). The hydrated DNA samples were then stored at 4° C for subsequent digestion with restric- tion enzymes. Restriction A_n_alysis atd Agarose Electgphoresis Digestion of DNA samples with restriction etdonucleases was carried out according to the directions supplied by the manufacturer of each A enzyme. The DNA fragnents were separated by statdard agarose gel elec- trophoresis techniques as outlined in Maniatis et al. (1982) . The samples were loaded so as to give approximately equal intensity CpDNA batds, with the total anmmt of DNA per lane varying. Agarose gels (0.8% or 1.0% w/v) were used with TBE buffer (0.089 M Tris, 0.089 boric acid, 2.0 nM EDTA, pH 8.0). Ethidiun bromide at 0.5 ug/ml was incorporated into both the gel and the TBE buffer. Following electrophoresis, the DNA bands were visualized on a 302 nm UV light transilluninator, atd photo- graphed using Polaroid type 55 film. To estimate the size of the irdividual restriction fragments, lambda DNA cut with Hird III in combination with Eco RI was used as mole- cular markers in each gel. The distance of migration of each lambda DNA fragment was plotted in semi—log fashion against the known size of the 15 fragment, atd the size of the chNA fragments were then estimted based on their migration. Southern Transfer The DNA in the agarose gels was then transferred to nitrocellulose filters (PEI bratd, 0.45 an pore size) using the procedures of Southern (1975) atd described in Mniatis et al. (1982). Probe Premtion A pBR322 clone bank of Petunia chNA, described in Sytaln and Gottlieb (1986) was provided by J. Palmer c/o D. Neale, Pacific Southwest Forest atd Range Earperiment Station, 1960 Addison Street, Berkeley, CA 94701, atd a p32322 clone (pZmEI) of the cytochrome oxidase subunit II (coxII) gene from mize mitochondria, described by Fox atd leaver (1981) was supplied by T. Fox. The cloned chNA fragments were supplied as stab cultures in Luria—Bertani media with antibiotics atd agar. The cloned DNA was purified using Triton X-100 detergent lysis procedure of Ausubel at al. (1987). The nomenclature of the cloned chNA fraanents used here follows that of Sytsma atd Gottlieb (1986). Ratdom Primed Libel ing The procedures for ratdom primed labeling of DNA are those as described by the manufacturer (Boehringer Mannheim Biochemicals) atd supplied with the ratdom primed labeling kit. Approximately 2.0 ug of probe DNA was labeled in each reaction. 16 Hybridization atd Autoradiogrgmy The Southern filters were hybridized according to the procedures of Maniatis et al. (1982), atd were incubated overnight at 65° C with gentle shaking. The hybridized filters were washed according to procedures of Thomashow et al. (1980). The hybridization fluid was removed atd the filters rinsed twice at 23° c with 2x 330. This was followed by four washes with 3x 330, 0.2: sns atd 5.0 mM mm at 65° c for so minutes each. For stringent washing (for the Petunia chNA probes), this was followed by one so minute wash at 65° c with 0.3x 330, 0.2% see atd 5.0 mM EDTA. Two brief 23° c rinses with 2x 330 were used to remove the 3138. The filters were allowed to air dry on Whatmn 3M paper. When dry, they were wrapped with cellophane. Autoradiography was carried out at either 23° C or at -70° C with DuPont Cronex Lightning Plus intensifying screens. Kodak X-Omat AR x-ray film was exposed for between 1 atd 36 hours. For subsequent hybridization with different probes, the initial probe was washed off by the procedures of Gatti et al. (1984) followed by those of Thomashow et al. (1980) . R_E_SULTs AND DISCUSSION The research described here includes the. developnent of methods to purify spruce chNA, the identification of chNA RFLPs, atd the damn- stration of the pattern of inheritance of chNA in the spruce hybrids. The use of chNA RFLPs, allowed the pattern of chloroplast inheritance patterns to be deduced, and avoided many of the problems of trying to use microscopy to follow the fate of chloroplasts following fertilization. In addition, if biparental transmission of the plastids is followed by 17 differential sorting sorting og the plastids in cell divisions subsequent to fertilization, this approach will be able to identify the final plas- tome type in the mature plant. The first step was to Obtain reasonably pure samples of chNA from the trees to be sttdied, atd to digest them with various restriction endonucleases. Figure 1 shows the pairwise comparison of blue spruce and white spruce ch A cut with the enzymes Cla I and Ava I. These two enzymes were chosen because they are methylationesensitive and infre- quently cut nuclear DNA, but freely cut the non-methylated chNA. The use of methylation sensitive enzymes to selectively cut chNA.in samples containing nucDNA has been reviewed.by Palmer (1985). By using methyl- ation sensitive enzymes, it is easy to visualize the ch A bands, even with varying levels of nucDNA contamination. The methylation insensitive enzymes Eco RI, Bam HI and Hind III will cut the DNA.samples in this study, but the varying levels of backgrourd fluorescence from the nucDNA makes it difficult to consistently visualize the ch A bands. Interspecific RFLPs that differentiate blue spruce chNA and.white spruce chNA are shown in Fig. 1A, Digestion with Cla I and.Ava I results in several polymorphic bands (labeled with arrowheads), and are 1isted.in Table 2. The broad.band of fluorescence near the top of each lane is most likely nuclear DNA (nucDNA) that was copurified.with the chNA. Those bands labeled with a "*" represent Cla I fragments of 4.45 or 2.9-kb in size (white spruce and blue spruce respectively) that are used in the following figures to demonstrate paternal inheritance of chNA. 18 A. B. Cla I Aval Cla | Ava I 1 2 3 4 1 2 3 4 Fig. 1. Identification of RF'LPs that differentiate white spruce atd blue spruce. A. Restriction patterns of chA from white spruce (lanes 1 and 3) and blue spruce (lanes 2 atd 4). RFLPs that differentiate blue spruce atd white spruce, are idicated by arrowheads. For RFLPs labeled with "X" see text. Framents were electrophoretically separat in 0.8% agarose, TBE buffer, 1.5 V/cm, 8 hours. B. Hybridization of -P labeled probe P16 to some of the RFLPs identified in Fig. 1A. 19 Table 2. CpDNA RFLPs that differentiate white spruce from blue spruce. Fragment White Blue (kb) Spruce Spruce Cla I 2.9 + ++ C13 I 208 + - + = present - = absent H = stoichiometrically double intensity It was necessary to demonstrate that the RFLPs present in Fig. 1 were chNA atd not nucDNA or mtDNA. Several approaches to this problem were taken. The first was described earlier, the use of methylation sensitive enzymes that infrequently cut nucDNA. The second approach was to probe the putative chNA samples with known cloned chNA fran another species. Figure 18 shows an autoradiografll produced by probing a Southern filter, from the gel shown in Fig. 1A, with a Petunia chA fragment (P16) of 4.1 kb in size. The probe is from the large single copy region atd contains part of the gene for the large subunit of ribulose bisphosphate carboxylase—oxygenase (rbcL) . P16 strongly hybrid- ized to the "t" fragments in Fig. 1A, atd weakly hybridized to a 1.21-kb white spruce Cla I fragment atd to a 1.33-kb blue spruce Cla I fragment. This probe also hybridized to Ava I fragments of 6.0 or 5.2—kb (white spruce or blue spruce respectively). Another probe (P3) also hybridized to the same batds as P16 in addition to several other fragments (data not shown). P3 is 21.0—kb in size atd borders P16 on the chNA molecule atd contains the remainder of rbcL gene. The clone bank used represents approximately 92% of the petunia chloroplast genome, atd all 13 cloned fragments hybridized to visible 20 fragments on the filters under high stringency washing conditions. In no instance did the probes hybridize to regions on the filter that did not correspond to visible bands on the gels, damnstrating the bands repre- sent chNA. Due to the presence of chNA sequences in mitochondria (Sederof f 1987 ) , it is possibile these RFLPs represent mtDNA. This was addressed by using a highly conserved mtDNA probe (pZmEl) which contains the coxII gene. The Southern filter from the gel shown in Fig. 1A was also probed with pZmEl, and it failed to hybridize to any band visible in Fig. 1A under low stringency washing conditions (data not shown). Using identi- cal hybridization techniques, pZmEl will hybridize to Southern filters containing positive control lanes (cloned pZmEl DNA) (data not shown), and to total DNA preparations from these spruce species (David, personal commication) . To establish whether the Cla I RFLPs shown in Fig. 1 are conserved within a species, eight trees from across the natural range of each species were examined. For white spruce, chNA was isolated from trees from British Columbia, Saskatchawan, Phnitoba, North Dakota, Ontario, New York, New Hampshire and Labrador, and was digested with Cla I. ‘No intraspecific variation in the W3 listed in Table 2 was observed (Fig. 2A). The gel from Fig. 2A was probed with P16 and is shown in Fig. 23. No variation in the 4.45-kb fragment ("It") is apparent. The weakly hybridizing band is variable, and existing as a 1.21-kb band in trees from Saskatchawan, Manitoba and South Dakota, and as a 1.33-kb band in the remining trees . 21 Fig. 2. White spruce provenance test. A. Cla I restriction patterns for single trees from British Columbia, Saskatchawan, Punitoba, S. Dakota, Ontario, New York, New Hampshire, Labrador (lanes 1-8 respec- tively). Arrowheads and "X" indicate bands identified in Fig. 1. Frag- ments were electrophoretically separated in 0.8% agarose, TBE buffer, 1.0 V/cm, 12 hours. B. Hybridization of P16 to a Southern filter from the gel shown in Fig. 2A. 22 For blue spruce, two trees from different counties in Arizona, Colorado, and Utah, and one tree each from New Mexico and Wyoming were examined as described for white spruce. Again, as Fig. 3A demonstrates, no variation was observed in the RFLPs listed in Table 2. When the Southern filter from this gel was probed with P16, no variation was observed in either the 2.9-kb band ("It") , nor the weakly hybridizing 1 .33-kb band. Based on the results of the provenance tests (Figs. 2 and 3), the RFLPs listed in Table 2 that differentiate blue spruce from white spruce, were considered to be invariant within a species. Thus, we felt confi- dent in that these species specific chNA restriction patterns were also valid for the parents of the hybrids. To determine the level of biparental inheritance of chA which was detectable by autoradiography, samples of blue spruce and white spruce chNA cut with Cla I were mixed and separated in a gel (Fig. 4A) atd probed with P16 (Fig. 4B). The amount of blue spruce chNA was kept constant, and the white spruce chNA was diluted. In Fig. 4B the 4.45-kb band can still be seen when diluted loo-fold (lane 5). In overexposed autoradiographs, it was still visible when diluted IOOO-fold. Thus, we should be able to recognize the transmission of chNAs fran the mternal parent down to approximately one part per thousand. Figure 5A shows the Cla I restriction patterns of six Fl-hybrids compared to the parental species. All six hybrids are from different controlled crosses , and there are no parents in cannon between then. In the six hybrids shown, and four additional (not shown), the restriction pattern was that of the paternal species. A Southern filter from this gel was probed with P16, and is shown in Fig. 5B. The blue spruce x 23 1234567812345678 9 o Fig. 3. Blue spruce provenance test. A. 018. I restriction patterns of chNA from single trees from Arizona (lanes 1-2), New Mexico (lane 3), Colorado (lanes 4-5), Wyoming (lane 6), and Utah (lanes 7-8). Arrows and "3:" indicate bands identified in Fig. 1. Fragments were electrophoreti- cally separated in 0.8% agarose, TBE buffer, 1.0 V/cm, 12 hours. B. Hybridization of P16 to a Southern filter from Fig. 3A. 24 A. B. 123456123456 Fig. 4. Demonstration of autoradiographic techniques to detect under- represented DNA. Cla I restiction framents of chNA from blue spruce (lane 1), white spruce (lane 2), equal amounts of each (lane 3), blue spruce plus 10-fold, loo—fold and 1000-fold dilutions of white spuce (lanes 4-6 respectively). Fragments were electrophoretically separated in 0.8% agarose, TBE buffer, 1.5 V/cm, 8 hours. B. Hybridization of P16 to a Southern filter from the gel shown in Fig. 4A. 25 A. B. W wa WxB BW wa WxB B 12345678 1234567? Fig. 5. Comparison of chNA from Fl-hybrids to parental species. A. Cla I restriction patterns of parental species and hybrids. lane 1, white spruce; lanes 2—4 blue spruce x white spruce hybrids (720058, 720060, and 720061 respectively); lanes 5—7 are white spruce x blue spruce hybrids (720007, 720008, and 720011 respectively); lane 8, blue spruce. The restriction fragments were separated in 1.0% agarose, TBE buffer, 1.25 V/cm, 11 hours. B. Hybridization of probe P16 to Southern filter of gel shown in Fig. 5A. 26 white spruce hybrids (lanes 2-4) showed no variation in the 4.45-kb band ("3") and.no evidence of the 2.9-kb band ("I“) that would.indicate some level of maternal transmission of chNA, The filter was then over- exposed, and no evidence of the 2.9-kb band.was seen. In.these hybrids, the weakly hybrizing band is either 1.21 or 1.33-kb in size, representing the variation demonstrated in Fig. 2. The white spruce x blue spruce hybrids (lanes 5-7) show the paternal chNA type (2.9-Rb band). Lane 5 (720007) shows what is apparently a low level of maternal transmission of the 4.45-Rb band. The only hybrids that showed.any level of putative maternal bands are white spruce by blue spruce. Several "pure" blue spruce also showed low levels of the 4.45-Rb band, indicating possibly heteroplasmic or chimeric individuals. Since only the 4.45-Rb band.was present in low levels, and never the 2.9-kb band in "pure" white spruce or blue spruce x white spruce hybrids, the possiblity of low levels of the 4.45-Rb band being due to partial diges- tion of the DNA samples was investigated. Blue spruce chNA samples were digested.with Cla I according to the manufaturers instructions, except the length of digestion was varied. Samples were digested for 1 minute, 10 minutes, 1, 2 or 8 hours, and then electrophoretically separated.and transferred.to nitrocellulose filters. Prehing with P16 yeilded strong signals corresponding to both the 2.9 and 4.45-kb bands at 1 minute. The 4.45-kb band rapidly decreased in inten- sity with increasing time, and was barely visible in an overexposed auto- radiograph in the lane with the sampled.digested for 8 hours (data not shown). Since the chNA samples were routinely'dflgested for two to four hours, the apparent low level of the 4.45-kb band most likely represents a partial digestion product, rather than a low level of maternal 27 transmission of chNA. To confirm this hypothosis the chNA sample 720007 was redigested for eight hours, and the 4.45-kb band showed reduced intensity compared to the stored out sample used in the gel in Fig. 5 (data not shown). Thus, all ten hybrids examined showed only the paternal chNA restriction patterns . The difference between the blue spruce 2.9-kb band and the white spruce 4.45-kb band appears to be a Cla I restriction site mutation. We cannot confirm this however, as we did not prepare restriction site maps for these species. This was due to the high level of nucDNA in the samples which would have made cloning fragments difficult. Additionally, when using the Petunia probes for heterologous probing, hybridization of one probe to two or more bands in our gels does not necessarily imply that the fragments are adjacent to each other in spruce chNA molecules, as conifer chloroplast genomes have been shown to be extensively rear— ranged compared to angiosperms (Strauss et al. 1988) Another piece of evidence which indicates the RFLPs used in this study are not nuclear in origin, but represent organelle DNA, is the non- Mendelian pattern of the inheritance of the RFLPs. If the We are nuclear in origin Fl-progeny of reciprocal crosses should show identical patterns . Since the inheritance of the RFLPs appears to be strictly uniparental , in this case paternal , the W3 must represent cytoplasmic DNA. (he of the concerns of this study was the possibility of biparental inheritance of the plastids. If this occurred, the different plastid types might sort out into different sectors within one tree, or the whole tree might rennin heteroplasmic. By sampling the entire crown of each 28 tree at one height (not just one branch), and by using autoradiographic methods, it should be possible to identify chimeric or heteroplasmic individuals among the hybrids, as long as the least abundant DNA accounts for at least 0.1% of the DNA in the sample. In none of the 10 hybrids examined did there appear to be any indication of heteroplasnw or chi- meric individuals; only the pollen parent chNA type was found. This study differs from Neale et al. (1986) and that of Szmidt et al. (1987) , because only the paternal chNA restriction patterns were observed in the hybrids. Neale et al. (1986), used chNA RFLPs to demon- strate the paternal transmission of plastids in intraspecific crosses of Douglas-fir . However, three of 36 progeny examined showed nonparental patterns in their chNA restriction digests. Szmidt et al. (1987) also reported finding two nonparental opDNA restriction patterns, and 1 mter- nal pattern, out of a total of six interspecific hybrids of Larix. Mien examining the chNA of eight Fl-hybrids of Jack pine x lodgepole pine, Wagner et al. (1987), like this study, found only the paternal chNA type. The results of this study clearly show that the chNA in the hybrids of blue spruce and white spruce is inherited from the paternal parent. The mechanism for paternal inheritance of chNA in P1’cea is still un- clear, though studies using microscopy, indicate during fertilization in the Pinaceae the plastids from the pollen tube enter the egg cell, and the fennle plastids are excluded. Studies by Maheshwari and Konar (1971) and Willemse (1974) on fertilization in Pinus, have shown that the pollen parent plastids enter the cytoplasm of the egg during fertilization, and that the maternal parent chloroplasts are excluded from the neocytoplasm (Willemse 1974). Chesnoy and Thorns (1971') however, could not determine 29 the parentage of plastids in Pinus nigra Arnold. Owens and Simpson (1988) have shown that during fertilization of Douglas-fir (Pseudotsug‘a menziesii), pollen tube organelles enter the egg cell, and a small por- tion of the pollen cytoplasm migrates along with the male gamete to the female nucleus. For Biota, a member of the Cupressaceae, Chesnoy (1969) demonstrated by light microscopy that female plastids are excluded from the neocytoplasm following fertilization. The ability to hybridize blue spruce and white spruce is well estab- lished (Hanover and.Wilkinson 1969, Bongarten and.Hanover 1982). The trees used in this study have previously been shown to be hybrids by morphological characteristics and monoterpenes composition (Hanover and Wilkinson 1969 , Bongarten and Hanover 1982). In this study, the chNA in the hybrids was always that of the paternal species. This indicates that analysis of chA could aid in the verification of hybrids resulting from controlled pollinations. This would be especially useful if the range of morphological characteristics of the parental species overlap. 30 LITERATURE CITED Ausubel FM, Brent R, Kingston RE, Moore DD, Seidnnn JG, Smith JA, Struhl K (1987) Current protocols in molecular biology. John Wiley, New York, pp 10705-10707 Bongarten BC, Hanover JW (1982) Hybridization among white, red, blue and white x blue spruces. For Sci 28:129-134 Bounan CM, Bonnard G, Dyer TA (1983) Chloroplast DNA variation between species of Tri ticun and Aegilops. location of the variation on the chloroplast genome and its relevance to the inheritance of the cytoplasm. Theor Appl Genet 65:247-262 Chesnoy L (1969) Sur la participation du gamete male a la constitution du cytoplasme de l’embryon chez le Biota orientalis Endl. Rev Cytol Biol veg 32:273-294 Chesnoy L, Thomas M] (1971) Electron microscopy studies on gametogenesis and fertilization in gymosperms. Phytomorphology 21:50-63 Fox TD, Leaver CJ (1981) The Zea mys mitochondrial gene coding cytochrome oxidase subunit II has an intervening sequence and does not contain T‘GA codons. Cell 26:315-323 Gatti RA, Concannon P, Salser W (1984) Multiple use of southern blots. BioTechniques 2:148-155 Hanover JW, Wilkinson RC (1969) A new hybrid between blue spruce and white spruce. Can J Bot 47:1693-1700 Maheswari P, Konar RN (1971) Pinus Council of Scientific and Industrial Research, New Dehli, 107p Maniatis T, Fritch EF, Sambrook J (1982) Molecular cloning: A laboratory manual. Cold Spring Harbor Lab, Cold Spring Harbor, New York. Metzlaff M, Borner T, Hagemann R (1981) Variations of chloroplast DNAs in the genus Pelargoniun and their biparental inheritance . Theor Appl Genet 60:37-41 Neale DB, Sederoff RR (1988) Inheritance and evolution of conifer organelle genomes. In: Hanover JW, Keathley DE (eds) Genetic Manipulation of woody plants. Plenun Press, New York, pp 251-264 Neale DB, Wheeler NC, Allard RW (1986) Paternal inheritance of chloroplast DNA in Douglas—fir. Can J For Res 16:1152-1154 Ohba K, Iwakawa M, Okada Y, Murai M (1971) Paternal transmission of a plastid anomaly in some reciprocal crosses of sugi, Cryptomeria japonica D. Don. Silvae Genet 20:101-107 31 Owens JN, Simpson SJ (1988) (Abstract) An ultrastructural study of fertilization and cytoplasmic inheritance in Douglas-fir. In: Hanover JW, Keathley DE (eds) Genetic Manipulation of woody plants. Plenum Press, New York, p 483 Palmer JD (1985) Isolation and structural analysis of chloroplast DNA. Methods Enzymol 118:167-186 Sears BB (1980) Elimination of plastids during spermatogenesis and fertilization in the plant kingdom. Plasmid 4:233-255 Sederoff RR (1987) Mblecular mechanisms of mitochondrial-genome evolution in higher plants. Amer Nat 130(8):30-45. Southern E (1975) Detection of specific sequences amoung DNA fragments separated by gel electrophoresis. J Mol Biol 98:503-517 Stine M, Keathley DE (1987) A quick and easy method of preparing discontinous sucrose gradients for centrifugation. BioTechniques 5:732-733 Stine M, Sears BB, Keathley DE (1988) Inheritance of plastids in interspecific hybrids of blue spruce and.white spruce. In preparation. Strauss SH, Palmer JD, Howe GT, Doerksen AH (1988) Chloroplast genomes of two conifers lack a large inverted repeat and are extensively rearranged. Proc Natl Acad.Sci (USA) 85:3898-3902. Sytsma KJ, Gottlieb LD (1986) Chloroplast DNA evolution and phylogenetic relationships in Cflarkia sect. Pbripetasma (Onagraceae). Evolution 40:1248-1261 Szmidt AE, Alden T, Hallgren J-E (1987) Paternal inheritance of chloroplast DNA in Larix; Plant Molec Biol 9:59-64 Thomashow MF, Nutter R, Mentoya AL, Gordon MP, Nester EW (1980) Integration and organization of Ti plasmid sequences in crown gall tumors. Cell 19:729-739 wagner DB, Furnier GR, Saghai-Maroof MA, Williams SM, Dancik BP, Allard RW (1987) Chloroplast DNA polymorphisms in lodgepole and jack pines and their hybrids. Proc Natl Acad Sci USA 84:2097-2100 Willemse MTM (1974) Megagametogenesis and formation of neocytoplasm in Pinus sylvestris L. In: Linskens HF (ed) Fertilization in higher plants. North-Holland, Amsterdam. pp 97-102 CHAPTERII Inheritance of plastids in reciprocal crosses of white spruce and Serbian spruce. ABSTRACT Chloroplast DNA (chNA) was purified from Serbian spruce (Picea omorika (Panic) Purkyne) and white spruce (P. glauca (Moench) Voss), and was digested with several different restriction endonucleases . Restriction fragment length polymorphisms (RFLPs) were identified that differentiated the two species. Intraspecific conservation of the RFLPs that distin- guish the two spruce species was confirmed by examining the two Serbian spruces that served as parents of the hybrids. Because the white spruce parents of the hybrids were not available, eight trees from the natural range of white spruce were examined to determine the typical white spruce chNA RFLP pattern. The chNA from 17 Fl-hybrid trees had the banding pattern of the paternal species. Cloned Petunia chNA containing part of the rbcL gene hybridized to polymorphic bands; in contrast a cloned mize mtDNA probe of the coxII gene did not hybridize to any band. 32 33 INTRODUCTION Recently we demonstrated paternal inheritance of plastids in inter- specific hybrids of blue spruce (Fficea pungens Engelm.) and white spruce (Stine et al. 1988). In this report, we demonstrate paternal inheritance of chNA in hybrids of white spruce and Serbian spruce. This further illustrates how chloroplast inheritance in members of the Coniferales differs from that of the angiosperms, which show either maternal or bi- parental, but not predominantly paternal inheritance of plastids (Sears 1980, Whatley 1982). As in Stine et al. (1988), we used restriction analysis of chloro- plast DNA (chNA) to study chloroplast inheritance. This technique has been used successfully to determine the inheritance of the organelles with a high degree of certainty in Pelargoniun (Metzlaff et al. 1981) and in Triticum and Aegflops (Bowman et al. 1983), and is becoming a comnon method to study organelle inheritance (Palmer 1985). The main advantages of this technique are that there is no need to either find or induce chloroplast mutants, there is no question whether or not the mutation affects the inheritance of the organelles, and there is no ambiguity as to whether there is differential sorting out of the organelles following fertilization. All of the aforementioned are problematic with the long generation time of spruce, and the varying degrees of delayed fertili- zation following pollination (Wilson and Burley 1985). Recently, several other reports have used restriction fragment length polymorphism (RFLP) analysis of chNA to follow the inheritance of chloroplasts in the Coniferales. Neale et al. (1986) reported paternal transmission of plastids in 33 of 36 progeny from intraspecific crosses of Douglas-fir (Pseudotsuga menziesii (Mirb) Franco), with the remaining 34 three having apparently non-parental restriction patterns. Szmidt et al . (1987) reported finding the paternal chNA restriction pattern in three of six interspecific Fl-hybrid progeny of Larix, with one showing the maternal pattern, and the remaining two having non-parental restriction patterns. Paternal inheritance of chNA has also been demonstrated in Fl-hybrids of lodgepole pine (Pinus contorta Dougl. ex. Loud.) x jack pine (P. banksiana Lamb.) examined (Wagner et al. 1987), and hybrids of white spruce and blue spruce (Stine et al. 1988). Neale and Sederoff (1988) also reported paternal chA transmission in redwood (Sequoia sempezvirens D. Don Endl.). This article follows the same approach reported earlier for analysis of white spruce and blue spruce hybrids (Stine and Keathley 1988) , and reports the strictly paternal transmission of chNA in Fl—hybrids from reciprocal crosses of Serbian spruce and white spruce. MATERIALS AND PJIE'I‘HODS Plant Material All trees used in this study are located at the Kellogg Experimental Forest, 7060 N. 42nd St. , Augusta, MI 49012, or on the East Lansing cam- pus of Michigan State University (PBU) . The accession number, plantation nunber, and the row and colum nunbers for each tree are listed in Table A2 , and the parentage of the hybrids in Table 1 . The two Serbian spruce trees that were used to produce all of the hybrids are located on the the north side of Service Rd. between Farm Lane and Bogue St. on the DSU campus. The two white spruce trees listed in Table 1. were in plantation 70.21 at Kellogg Forest, but were removed in 1986, when this plantation 35 Table 1 . Parentage of white and Serbian spruce hybrids. Femle Parent Pollen Parent Hybrid Nunber Species Accession Species Accession Examined Nunberx Number! 710003 4 P. omorika 270002 P. glauca 190423 710004 4 P. glauca 190423 P. anorika 270001 710005" 5 P. glauca 190424 P. omorilm 270001 P. florika 270002 710006 3 P. anorika 270001 P. glauca 190423 710007 1 P. glauca 190423 P. anorika 270002 x Add 67,000,000 to obtain the canplete MICHQYI‘IP accession numbers M 710005 hybrids resulted from pollination with bulked pollen from 270001 and 270002 was converted to a seed orchard for Fz-hybrid seed production. The hy- brids of Serbian and white spruce are located in Michigan Cooperative Tree Improvement Program (MICHCXYI‘IP) plantation 78.01, and were planted in 1980. Complete records on these trees can be obtained from MICEKXJI'IP, Michigan State University, East Lansing, MI 48823. The entire perimeter of the crown of each tree was sampled at 1.0- 1.5 m above ground, and between 75 and 100g of the current and previous season’s needles were sampled. The chNA was isolated by differential centrifugation following the procedures for chNA preparation and analy- sis described by Stine et al. (1988) . Digestion of the DNA samples with restriction endonucleases followed the instructions of the various unnu- facturers. Standard procedures for agarose gel electrophoresis, and Southern transfer were used (Phnniatis et al. 1982) . lambda DNA cut with Hind III and Eco RI was used to provide molecular size narkers. Petunia chNA clones, described in Systma and Gottlieb (1986) , were provided by J. Palmer, c/o D. Neale, Pacific Southwest Forest and Range Experiment Station, 1960 Addison St. , Berkeley, CA 94701. The cloned probe pZmEl, 36 described in Fox and Leaver (1981) , which contains the gene for cyto- chrome oxidase subunit II from mize mitochordria DNA (mtDNA) , was pro- vided by T. Fox. The probes were labeled by random primed labeling according to the manufacturer (Boehringer Mannheim Biochemicals), and were hybridized to the filters according to the procedures of mniatis et al. (1982) . The filters were washed as described by Stine et a1. (1988). grams A_N_D DISCUSSION This study is a further illustration of the phenomenon of paternal transmission of chloroplasts in members of the coniferales. Since genetic evidence of the inheritance pattern of organelles in most species of this family is still lacking, it was necessary to rigorously demon- strate that the RFLPs that showed paternal inheritance , are in fact chNA. The RFLPs were shown to be chNA by several methods. The We were generated using restriction enzymes that are methyl- ation sensitive. These enzymes cut chNA which is not methylated, but only rarely cut methylated nuclear DNA (Palmer 1985) . The enzymes Cla I and Ava I were chosen because they were useful in differentiating chNA from white spruce ard blue spruce (Stine et al. 1988). Since blue spruce is more closely related to white spruce, than is Serbian spruce (Wright 1955, Liu 1982) , we predicted that these enzymes would successfully dif- ferentiate white spruce chNA from Serbian spruce chNA. Several interspecific chNA RFLPs produced by digesting chNA with Cla I and Ava I that differentiate Serbian spruce chNA ard white spruce chNA are shown in Fig. 1A. Diagnostic bards are labeled with arrowheads and are listed in Table 2. The broad bard of fluorescence near the 37 A. B. Cla I Aval Cla I Ava I 1 2 3 4 1 2 3 4 11 "Jr Fig. 1. Identification of RFLPs that differentiate white spruce and Serbian spruce. A. Restriction patterns of chNA from white spruce (lanes 1 and 3) and Serbian spruce (lanes 2 and 4). RFLPs that differ- entiate Serbian spruce and white spruce, are identified by arrows; "It" framents, see text. Fragments were electrophoretically separated in 0.8% ag se, TBE buffer, 1.0 V/cm, 8 hours. B. Hybridization of cloned, -P labeled, Petunia chNA (probe P16) to RFLPs indicated in Fig. 1A. 38 Table 2. CpDNA RFLPs that differentiate white spruce from Serbian spruce. Fragment White Serbian (kb) spruce spruce Cla I 5.2 + - Cla I 5.15 - + Cla I 4.45 + - Cla I 2.9 + ++ Cla I 2.8 + - Ava I 6.1 + - Ava I 5.2 - + + = present - = absent ++ = a stoichiometrically double intensity hard top of each lane is probably nuclear DNA. Those bards marked with a "I!" represent Cla I fragments of 4.45 ard 2.9-kb (white spruce ard Serbian spruce respectively) which were used to demonstrate the inheritance of chNA by autoradiographic methods. The RFLPs were identified as chNA by probing with cloned Petunia chNA. The clone bank used represents 92% of the petunia chloroplast genome, and all 13 cloned fragments used, hybridized to visible fragments on the filters. In no case did the probes hybridize to regions on the filter that did not correspord to visible bards on the gels. Figure 13 shows an autoradiograph produced by probing a Southern filter from the gel shown in Fig. 1 with a 4.1 kb Petunia chA fragment. This cloned fragment (P16) is from the large single copy region of the Petunia cp genome , ard contains part of the large subunit of ribulose bisphosrhate carboxylase-oxygenase (rbcL) gene. Another probe (P3) , which contains the rest of the rbcL gene on a fragnent of 21.0 kb in size, also hybrid- izes to these RFLPs, in addition to several other non-polymorphic bards 39 (data not shown). These results help to demonstrate that these RFLPs represent chNA . The third approach to identify these RFLPs, was to probe using a highly conserved mtDNA probe, in order to demonstrate the presence or absence of mtDNA in our chNA preparations. This was necessary because DNA sequences which are putatively of chloroplast origin are cournonly found in the mitochordrial genome (Sederoff 1987) . The coxII gene from maize mitochondria (probe ernEl) failed to hybridize to the lanes con- taining chNA digests (data not shown). The probe does hybridize to filters with a positive control lane (pZmEl), ard to filters with total DNA preparations of white spruce (David, personal communication) . Thus indicating that the chNA preparations do not contain mtDNA. The two Serbian spruce that were used as either the female or pollen parent in all of the hybrids were analyzed for their Cla I chNA restriction patterns. The white spruce parents were not available for analysis in this study. The Cla I chNA RFLPs in white spruce were pre- viously checked by the analysis of the chNA from eight trees from throughout the natural range of white spruce, and were shown to be con- served (Stine et a1. 1988). Figure 2A shows the Cla I restriction patterns of five Fl-hybrids compared to their Serbian parents ard an unrelated white spruce. All five hybrids shown are from different controlled crosses. A total of 17 hybrids were examined. In the ten white spruce x Serbian spruce hybrids, only the Serbian parent restriction pattern was fourd. The seven Serbian spruce x white spruce hybrids showed a typical white spruce chNA restriction pattern. A southern filter from this gel was probed with P16 (same as used in Fig. 13), and the autoradiograph is shown in Fig. 2B. 40 A. B. S WxS SxWW S WxS SxWW 123456731234567? Fig. 2. Comparison of Cla I digests of chNA from Fl-hybrids to their parental species. Restriction patterns of chNA digested with Cla I. Lanes 1—2, Serbian spruces 270001 and 270002 respectively; lanes 3-5, white spruce x Serbian spruce hybrids 710004, 710005 and 710007 respect- ively; lanes 6-7, Serbian spruce x white spruce hybrids 710003 and 710006 respectively; lane 8, white spruce. Arrows indicate polymorphisms. Fragments were electrophoretically separated in 0.8% agarose, TBE buffer, 1.0 V/cm, 14 hours. B. Hybridization of P16 to some of the RFLPs iden— tified in Fig. 2A. 41 The bands to which P16 hybridized represent the paternal chNA type. The filter was then overexposed to check for possible heteroplasnw or chi- meric Fl-hybrids. No evidence of maternal patterns were found (data not shown). All 17 hybrids were examined in the same manner ard no evidence for heteroplasmic or chimeric individuals was observed. The probe P16 hybridized weakly to a low molecular weight band in addition to the "x" fragments. In Fig. 2A lanes 1-7 show hybridization to a 1.33-kb bard, while lane 8 has a 1.21-kb bard. We demonstrated that this fragment shows intraspecific variation previously (Stine et al. 1988) . Further— more, the Serbian spruce and white spruce hybrids (lanes 1-7) are not related to the particular white spruce shown in lane 8, and thus the variation is certainly an.RFLP which is individual-specific, rather than species specific. Another piece of evidence which indicates RFLPs identified in this study are not nuclear in origin, but represent organelle DNA (chNA or mtDNA) is that they are uniparentally inherited. If the RFLPs were nuclear DNA, the RFLP patterns would be expected to be identical in F1- hybrid progeny from reciprocal crosses. Since the inheritance appears to be strictly uniparental, in this case paternal, cytoplasmic DNA is indicated. The hybrids used in this study are from a continuing spruce breeding program, and while they show intermediate foliage characteristics, the hybrid nature of these trees has not been critically determined. In this study, the chNA found in the hybrids was always that of the paternal species. Assuming correct species identification of the ferrule ard pol- len parents, ard the demonstration of conservation of chNA restriction 42 patterns within white spruce, analysis of chNA should prove valuable for verification of hybrids in continued.breeding of spruces. In this study, as in our previous study with hybrids of white spruce and.blue spruce (Stine et al. 1988), we only found the paternal opDNA in the progeny. wagner et al. (1987) also reported finding only the pater- nal chNA type in Fl-hybrids of Jack pine (Pinus banksiana Lamb.) and lodgepole pine (PL contorta.Dougl.). Neale et al. (1986) and Szmidt et al. (1987), however, reported finding nonparental chNAs in some progeny. Neale et al. (1986) , used chNA RFLPs to demonstrate predominantly pater- nal transmission of plastids in intraspecific crosses of Douglas-fir, although 3 of 36 progeny showed nonparental patterns in their chNA restriction digests. Szmidt et al. (1987) also report finding nonpar- ental chNA restriction patterns in 2 out a total of 6 interspecific hybrids of Larix, in addition to one maternal restriction pattern. Since recombination of chNA has not been demonstrated in sexual crosses of higher plants (e.g. Chin and Sears 1985), there is no reason to expect to find other than parental chNA types in the progeny, and the aberrant chNA patterns reported by Neale et al. (1986) and Szmidt et al.‘(1987) likely represent pollen contamination or mutations. Hewever, the occur- rence of chNA.mutations in 8.3%.of the progeny of intraspecific hybrids of Douglas-fir, or in 33.3% of interspecific larch hybrids as reported by Neale et al. (1988) and Szmidt et al. (1988) respectively, would not fit the current model of the chNA genome as slowly evolving (Zurawski and Clegg 1987). The mechanism for paternal inheritance in Picea is still unclear, though microscopy studies of fertilization in the Pinaceae indicate the pollen tube plastids enter the egg cell, and the female plastids are 43 excluded. Studies by Maheshwari and Konar (1971) and Willemse (1974) on fertilization in Pinus, have shown that plastids from the pollen tube enter the egg cytoplasm during fertilization, and Willemse (1974) also demonstrated that probably all plastids from the female parent are ex- cluded from the neocytoplasm. Owens ard Simpson (1988) have shown that during fertilization of Douglas-fir (Pseudotsuga menziesii), mle organ- elles enter the egg cell, ard a small portion of the pollen tube cyto- plasm migrates along with the male gamete to the female nucleus. In Biota, a member of the Cupressaceae, Chesnoy (1967) demonstrated by light microscopy, the exclusion of female plastids from the neocytoplasm. Thus, in the Coniferales, the mechanism of paternal inheritance of plas- tids appears to involve plastids of paternal origin entering the archae- gonium during fertilization, coupled with the exclusion of Internal plas- tids from the neocytoplasm. The evidence for paternal inheritance of plastids in gymosperms is increasing, with reports showing genetic evidence of paternal inheritance for two genera of the Taxoideacae, Crytaneria (Ohba et al 1971), Sequoia (Neale ard Sederoff 1988); ard four genera of the Pinaceae, Pinus (Neale et al. 1988, Wagner et al. 1987 ), Picea (Stine and Keathley 1988, ard this report), Pseudotsuga (Neale ard Sederoff 1988) ard Larix ( Szmidt et al. 1987). There are still 10 other families of gymosperms for which there have been no reports. The further study of plastid inheritance in gymnosperms may elucidate whether paternal inheritance is a derived char- acter or represents the primitive state. It may also lead to a further understarding of why uniparental inheritance of plastids (either maternal or paternal) is much more cannon than biparental inheritance. 44 LITERATURE CITED Bongarten BC, Hanover JW (1982) Hybridization among white, red, blue and white x blue spruces. For Sci 28:129-134 Bowman ()4, Bonnard G, Dyer TA (1983) Chloroplast DNA variation between species of Triticum and Aegilops. location of the variation on the chloroplast genome and its relevance to the inheritance of the cytoplasm. Theor Appl Genet 65:247-262 Chesnoy L (1969) Sur la participation du gamete male a la constitution du cytoplasme de l’embryon chez le Biota orientalis Erdl. Rev Cytol Biol veg 32:273-294 Chiu WL, Sears BB (1985) Recombination between chloroplasts DNAs does not occur in sexual crosses of Oenothera. Mol Gen Genet 198:525-528. Fox TD, Leaver CJ (1981) The Zea mays mitochordrial gene coding cytochrome oxidase subunit II has an intervening sequence ard does not contain T'GA codons. Cell 26:315-323 Hanover JW, Wilkinson RC (1969) A new hybrid between blue spruce ard white spruce. Can J Bot 47:1693-1700 Liu TS (1982) A new proposal for the classification of the genus Picea. Acta Phytotax Geobot 33:227-244 Maheswari P, Konar RN (1971) Pinus Council of Scientific ard Irdustrial Research, New Dehli, 107p Maniatis T, Fritch EF, Sambrook J (1982) Molecular cloning: A laboratory manual. Cold Spring Harbor Lab, Cold Spring Harbor, New York. Metzlaff M, Borner T, Hagemann R (1981) Variations of chloroplast DNAs in the genus Pelargoniun ard their biparental inheritance. Theor Appl Genet 60:37-41 Neale DB, Sederoff RR (1988) Inheritance and evolution of conifer organelle genoms. In: Hanover JW, Keathley DE (eds) Genetic Manipulation of woody plants. Plenun Press, New York, pp 251-264 Neale DB, Wheeler NC, Allard RW (1986) Paternal inheritance of chloroplast DNA in Douglas-fir. Can J For Res 16:1152-1154 Ohba K, Iwakawa M, Okada Y, Murai M (1971) Paternal transmission of a ' plastid anoualy in some reciprocal crosses of sugi, Cryptaneria japonica D. Don. Silvae Genet 20:101-107 Owens JN, Simpson SJ (1988) (Abstract) An ultrastructural study of fertilization and cytoplasmic inheritance in Douglas-fir. In: Hanover JW, Keathley DE (eds) Genetic Manipulation of woody plants. Plenun Press, New York, p 483 45 Palmer JD (1985) Isolation and structural analysis of chloroplast DNA. Methods Enzymol 118:167-186 Sears BB ( 1980) Elimination of plastids during spermatogenesis and fertilization in the plant kingdom. Plasmid 4:233-255 Sederoff, RR (1987) Molecular mechanism of mitochondrial-genome evolution in higher plants. Amer Nat 130(3) :30-45 Stine M, Sears BB, Keathley DE (1988) Inheritance of plastids in interspecific hybrids of blue spruce and white spruce. In preparat ion . Sytsma KJ , Gottlieb ID (1986) Chloroplast DNA evolution and phylogenetic relationships in Clarkia sect. Peripetasrm (Quagraceae). Evolution 40: 1248-1261 Szmidt AE, Alden T, Hallgren J-E (1987) Paternal inheritance of chloroplast DNA in Larix. Plant Molec Biol 9:59-64 Wagner DB, Furnier GR, Saghai-Phroof MA, Williams m, Dancik BP, Allard RW (1987 ) Chloroplast DNA polymorphism in lodgepole and jack pines and their hybrids. Proc Natl Acad Sci USA 84:2097-2100 Whatley JM (1982) Ultrastructure of plastid inheritance: green algae to angiosperms. Biol Rev 57:527-569 Willemse MM (1974) Megagametogenesis and formation of neocytoplasm in Pinus sylvestris L. In: Linskens HF (ed) Fertilization in higher plants. North-Holland, Amsterdam. pp 97-102 Wilson MF, Burley N (1983) Mate choice in plants: Tactics, mechanisms, and consequences. Princeton University Press, Princeton, New Jersey. 251 p. Wright JW (1955) Species crossibility in spruce in relation to distribution and taxonouw. For Sci 1:319-349 Zurawski G, Clegg MP (1987) Evolution of higher-plant chloroplast DNA- encoded genes: implicatoms for structure-function ard phylogenetic studies. Ann Rev Plant Physiol 38:391-418 CHAPTER III Paternal inheritance of plastids in Engelmann spruce X blue spruce hybrids. ABSTRACT Chloroplast DNA (chNA) was purified from blue spruce (Picea pungens Engelm.) and Engelmann spruce (It engelmannii Parry ex Engelmm), and was digested with several restriction endonucleases. Restriction fragment length polymorphisms (RFLPs) were identified that differentiated.both species. Intraspecific conservation of the RFLPs that distinguished.each species was confirmed.by examining eight trees from the natural range of blue spruce and five of Engelmann spruce. Fbur Engelmann spruce x blue spruce Fl-hybrids were examined, and the chNA from each showed the band- ing pattern of blue spruce. Three naturally occurring, putative hybrids showed typical Engelmann spruce banding patterns, which is inconsistent with the reported unilateral crossing incompatibility (crosses work only with Engelmann spruce as the female parent), and the apparent paternal inheritance of plastids in Fficea. These results suggest that these trees are not hybrids. Cloned Petunia.chNA containing part of the rbcL gene hybridized to the polymorphic bands, while a cloned.maize mtDNA prObe of the coxII gene, failed to hybridize to any bands in the gels. 46 47 INTRODUCTION The classic method for studying the inheritance of plastids is to utilize plastid mutants (either natural or induced) in reciprocal crosses. In such analyses angiosperms exhibit either maternal inheri- tance or biparental inheritance of the plastids (Sears 1980, Whatley 1982). Among gymnosperms, evidence suggests that the inheritance of the plastids may be either strictly or largely paternal. Ohba et al. (1971) followed the inheritance of induced chloroplast mutants in sugi (crypto- .meria japonica D. Don) and determined that the plastids were inherited paternally approximately 90 to 99% of the time, providing the first genetic evidence for predominantly paternal transmission of plastids in the gymnospermae. These genetic data have been supplemented by the application of molecular techniques in the restriction analysis of chloroplast DNA (opDNA) and mitochondrial DNA (mtDNA). It has been possible by comparing restriction fragment length polymorphisms (RFLPs) of either chNA or mtDNA to ascertain the inheritance of the organelles with a high degree of certainty in Pelargoniun (Metzlaff et al. 1981) ard in Triticun ard Aegilops (Bowman et al. 1983). The advantages of this technique are that there is no need to find or induce chloroplast or mitochondrial mutants, there is no question whether or not the mutation affects the inheritance of the organelles, and there is no ambiguity as to whether differential sorting out of the organelles occurs following fertilization. Several recent reports have utilized RFLP analysis of chNA to determine the inheritance pattern of chloroplasts in members of the Coniferales. Neale et al. (1986) reported predominantly paternal trans- mission of plastids in intraspecific crosses of Douglas-fir (Pbeudotsuga 48 menziesii (Mirb) Franco). Szmidt et al. (1987) also have reported find- ing the paternal pattern of chNA in interspecific Fl-hybrid progeny of Larix. Paternal inheritance of chNA was demonstrated in Fl-hybrids of lodgepole pine (Pinus contorta Dougl. ex. Loud.) x jack pine (P. bank- siana Lamb.) (Wagner et al. 1987 ), ard in interspecific hybrids of Picea pungens (Engelm.) ard P. glauca (Moench) Voss (Stine et al.) ard hybrids of P. glauca (Moench) Voss ard P. anorika (Panic) Purkyne (Stine ard Keathley 1988) . Neale atd Sederoff (1988) have also reported paternal chNA transmission in redwood (Sequoia sempervirens D. Don Erdl. ) . We present here an additional demonstration of paternal inheritance of chNA in Picea. Interspecific Fl-hybrids of P. engelmannii x P. pungens were examined, as were several putative hybrids from Colorado, in an area sympatric to the two species. MATERIALS ANDW Plant Material The blue spruce trees used in this study are located in the Kellogg Experimental Forest, 7060 N. 42rd St., Augusta, MI 49012. Accession number, plantation nunber, atd the row ard colunn nunbers for each tree are listed in Table A1, ard the parentage of the hybrids is listed in Table 1. The blue spruce are located in plantation 70.22, which is a rangewide provenance test of blue spruce established in 1970. Detailed records on the trees used in this study can be obtained from the Michigan Cooperative Tree Improvement Program (MICHCDI‘IP) , Michigan State University, East Lansing, MI 48824. 49 Table 1 . Parentage of Engelmann x blue spruce hybrids . Female Parent Pollen Parent Hybrid Species Accession Species Accession Number! Number Nmber 610083 P. engelmannii 170509 P. pungens 310915 610113 P. engelmannii 170517 P. pumgens 310905 610114 P. engelmnnii 170517 P. pungens 310903 610111 P. engelmannii 170516 P. pungens 310915 x Add 67,000,000 to obtain the complete MICHCXII'IP accession nunbers. The Engelmnn spruce, ard putative Engelmann ard blue spruce hy- brids, are located in the Dolores River drainage in southwest Colorado, described by Ernst et al. (1988). The Engelmann spruce x blue spruce hybrids are located at the Tree Research Center, Michigan State Univer- sity, East Lansing, MI 48824. The perimeter of the crown of each tree was sampled at least two points, and between 75 ard 100g of the current ard previom season's needles were sampled. The procedures for chloroplast DNA (0%) prep- aration ard analysis were described previously (Stine et al. 1988) . Digestion of the DNA samples with restriction erdonucleases followed the instructions of the various manufacturers. Stardard procedures for agar- ose gel electrophoresis, and Southern transfer, as described in Maniatis et al. (1982) were used. Petunia chNA clones, described by Systm ard Gottlieb (1986), were provided by J. Palmer, c/o D. Neale, Pacific South- west Forest ard Ihnge Experiment Station, 1960 Addison St. , Berkeley, CA 94701. A mize mtDNA probe (pZmEl) , described by Fox ard Leaver (1981) and containing the gene for cytochrome oxidase subunit II (coxII) , was 50 provided by T. Fox. The probes were labeled by random primed labeling according to the manufacturer (Boehring Mannheim Biochemicals), and were hybridized to the filters according to the procedures of Maniatis et al. 1982. The filters were washed as described in Stine et al. (1988). mm 1911) DISCUSSION RFLPs that differentiated Engelmann spruce from blue spruce were identified using several enzymes. Figure 1A shows the RFLPs that are produced by the enzymes Cla I and Ava I. Diagnostic bands are labeled with arrows, and are also listed in Table 2. The broad band of fluores- cence near the top of each lane is most probably nuclear DNA. Those bands with a "3" represent Cla I fragments of 2.9 and 4.45-Rb in size from blue spruce and Engelmann spruce respectively, and are used to demonstrate paternal transmission by autoradiography. Table 2. Diagnostic chNA RFLPs shown in Figure 1. Fragment Blue Engelnnnn (kb) spruce spruce C18. I 4045 - + Cla I 2.9 H + + = present - = absent H = Stoichiometrically double instensity Figure 13 shows hybridization of a 4.1 kb Retmia chNA probe (P16) to a Southern filter produced from the gel shown in Fig 1A. P16 contains part of the large subunit of ribulose bi sphosphate carboxylase-oxygenase 51 A. B. Cla I Aval Cla I Ava I 1 2 3 4 1 2 3 4 '~ Ma Fig. 1. Identification of RFLPs that differentiate Engelmann spruce and blue spruce. A. Restriction patterns of chNA from Engelmann spruce (lanes 1 and 3) and blue spruce (lanes 2 and 4). RFLPs that differenti— ate blue spruce and white spruce are identified by arrows. For RFLPs labeled "X" see text. Fragments were electrophoretically separated in 0.8% agarose, TBE buffer, 1.0 V/cm, for 8 hours. B. Hybridization of P16 to a Southern filter from the gel in A. 52 (rbcL). Another probe (P3), which contains the rest of the rbcL gene on a fragment of 21.0 kb in size, also hybridizes to these RFLPs, in addi- tion to several other non-polymorphic bands (data not shown). There are many reports of DNA sequences of putative chloroplast origin being located in the mitochondrial genome (Sederoff 1987). To determine whether our chNA preparations contain mtDNA, the southern filter from.Fig. 1A was also prdbed with a 2.1 kb mtDNA prObe containing the gene for ooxII from maize. The probe failed to hybridize to any'band visible in Fig. 1A (not shown), but did.hybridize to total DNA prepara- tions from blue spruce (David, personal communication) under identical hybridization and washing conditions. It has been previously demonstrated that the Cla I chNA restriction pattern is conserved in blue spruce throughout its range (Stine et al. 1988). The conservation of the Cla I chNA restriction pattern in Engel- mann was checked in 5 trees from Colorado, either in or near the Delores river drainage (Fig. 2), and one tree of unknown origin, on the Michigan State university campus (not shown). No variation of the diagnostic Cla I chNA RFLPs was Observed among these six trees, although the tree shown in lane 1 has a unique hand of approximately 2.6—kb in size (lower arrowhead). Figure 3A shows the Cla I restriction patterns of four Fl-hybrids from controlled crosses, and three putative hybrids, compared to the parental species. All four hybrids are from different controlled crosses. The four Fl-hybrids show the restriction pattern of blue spruce, the paternal species. The putative hybrids show banding patterns characteristic of Engelmann spruce. A Southern filter from this gel was probed with P16 (same probe as used in fig. 18), and is shown in Fig. BB. 53 A. B. 1234512345 Fig. 2. Conservation of diagnostic chNA RFLPs in Engelmann spruce. A. Cla I restriction patterns of Engelmann spruce from within the Delores river drainage (lanes 1, 2 and 4), but outside the sympatric zone. Lanes 3 and 5, Engelmann spruce from outside the Delores river drainage. Fragments were electrophoretically separated in 0.8% agarose, TBE buffer, 1.5 v/cm, 7 hours. B. Hybridization of P16 to the Southern filter from the gel shown in A. 54 A. B. E PH ExB B E PH ExB B 72345678372345678? Fig. 3. Comparison of chNA from Fl-hybrids and putative hybrids to parental species. A. Cla I restriction patterns of chNA from Engelmann spruce (lane 1), putative Engelmann and blue spruce hybrids (lanes 2-4), Engelmann spruce x blue spruce Fl—hybrids 610083, 610113, 610114 and 610111 (lanes 5-8 respectively), blue spruce (lane 9). The restriction fragments were electrophoretically separated in 0.8% agarose, TBE buffer, 1.5 V/cm, 7 hours. B. Hybridization of P16 to a Southern filter of the ‘ gel shown in A. 55 The bands to which P16 hybridized represent the paternal chNA type. The filter was then over exposed, to check for possible heteroplasmy or chi- meric Fl-hybrids, and no evidence of maternal patterns was Observed (data not shown). Ernst et al. (1988), Fechner and Clark (1969) reported.that viable progeny resulted from.interspecific hybridization of blue spruce and Engelmann spruce, only when Engelmann spruce was used as the female parent. The three putative hybrids examined were identified as likely hybrids based on discriminant analysis of morphological and.nonoterpene composition data (Schaefer and.nanover 1985). RFLP analysis of chNA from.these trees showed typical Engelmann ch A patterns. If the unidi- rectional hybridization pattern found by Ernst et al. (1988) and Fechner and Clark (1988) is correct, and the paternal transmission of chNA found in the Fl-hybrids examined also is correct, then we would expect to find blue spruce chNA restriction patterns in the putative hybrids. Thus, the putative hybrids are most likely not Fl-hybrids. POssibly these trees represent Fz-hybrids (or later generations) that have backcrossed to Engelmann spruce. In this study we have confirmed that the bands produced by the di- gestion of our DNA samples with Cla I or Aya I, are ch A‘by the fact they are produced.by methylation sensitive enzymes, cloned.chNA.prObes from FEtunia.hybridize to the RFLPs, while a.mtDNA cloned.probe from maize failed to hybridize to the same Southern filters, and the RFLPs do not assort independently as would.be expected for nuclear DNA. As wdth other interspecific hybrids in spruce, hybrids of white spruce and blue spruce (Stine et al. 1988) and those of Serbian spruce and.white spruce (Stine and Keathley 1988), we found the paternal chNA restriction 56 pattern in the Fl-hybrids. Proposed mechanisms for paternal inheritance of plastids have been discussed previously (Stine and Keathley 1988, Stine et al. 1988) 57 1.1%ng CITE_D Bongarten BC, Hanover JW (1982) Hybridization among white, red, blue and white x blue spruces. For Sci 28:129-134 Bowman CM, Bonnard G, Dyer TA (1983) Chloroplast DNA variation between species of Triticun and Aegilops. Location of the variation on the chloroplast genome and its relevance to the inheritance of the cytoplasm. Theor Appl Genet 65:247-262 Ernst SG, Keathley DE, Hanover JW (1987) Inheritance of isozymes in seed and bud tissues of blue and Engelmann spruce. Genome 29:239-246 Fechner GH, Clark RW (1969) Preliminary observation on the hybridizarion of Rocky Mountain spruces. In: Proc Com For Tree Breeding in Canada 11:237-247 ~ Fox TD, Leaver CJ (1981) The zea mys mitochondrial gene coding cytochrome oxidase subunit II has an intervening sequence and does not contain T'GA codons. Cell 26:315-323 Hanover JW, Wilkinson RC (1969) A new hybrid between blue spruce and white spruce. Can J Bot 47 :1693-1700 Maniatis T, Fritch EF, Sambrook J (1982) Molecular cloning: A laboratory manual. Cold Spring Harbor Lab, Cold Spring Harbor, New York. Metzlaff M, Borner T, Hagemann R (1981) Variations of chloroplast DNAs in the genus Pelargoniwz and their biparental inheritance. Theor Appl Genet 60:37-41 Neale DB, Sederoff RR (1988) Inheritance and evolution of conifer organelle genomes. In: Hanover JW, Keathley DE (eds) Genetic Manipulation of woody plants. Plenun Press, New York, pp 251-264 Neale DB, Wheeler NC, Allard RW (1986) Paternal inheritance of ‘ chloroplast DNA in Douglas-fir. Can J For Res 16:1152-1154 Ohba K, Iwakawa M, Okada Y, Murai M (1971) Paternal transmission of a plastid anomaly in some reciprocal crosses of sugi , Cryptameria japonica D. Don. Silvae Genet 20:101-107 Sears BB (1980) Elimination of plastids during spermatogenesis and fertilization in the plant kingdom. Plasmid 4:233—255 Sederoff RR (1987) Molecular mechani-I of mitochondrial-genome evolution in higher plants. Amer Nat 130(8) :30-45 Stine M, Keathley DE (1988) Inheritance of plastids in reciprocal crosses of white spruce and Serbian spruce. In preperation. 58 Stine M, Sears BB, Keathley DE (1988) Inheritance of plastids in interspecific hybrids of blue spruce and white spruce. In preparat ion . Sytsma KJ, Gottlieb LD (1986) Chloroplast DNA evolution and phylogenetic relationships in Clarkia sect. Peripetasma (Onagraceae). Evolution 40: 1248-1261 Szmidt AE, Alden T, Hallgren J-E (1987) Paternal inheritance of chloroplast DNA in Larix. Plant Molec Biol 9:59-64 Wagner DB, Furnier GR, Saghai-Maroof MA, Williams SM, Dancik BP, Allard RW (1987) Chloroplast DNA polymorphisms in lodgepole and jack pines and their hybrids. Proc Natl Acad Sci USA 84:2097—2100 Whatley, JM (1982) Ultrastructure of plastid inheritance: green algae to angiosperms. Biol Rev 57:527-569 Willemse M'IM ( 1974) Megagametogenesis and formation of neocytoplasm in Pinus sylvestris L. In: Linskens HF (ed) Fertilization in higher plants. North-Holland, Amterdam. pp 97-102 Wilson MF, Burley N (1983) Mate choice in plants: Tactics, mechanism and consequences. Princeton University Press, Princeton, New Jersey. 251 p. Wright JW (1955) Species crossibility in spruce in relation to distribution and taxonouw. For Sci 1:319-349 SUMMARY AND CONCLUSIONS In these studies, I have been able to identify chNA.RFLPs that differentiate the four species of spruce under study. By examining the ch A from either the actual parents of Fl-hybrids, or from.representa- tives of the natural range of each species, I have been able to identify species-specific plastome types in the Fl-hybrids. In the examination of Fl-hybrids, I have found only the chNA restriction pattern of either the actual pollen parent, or the species used as the pollen parent. The results of all three hybrid systems examined are listed in Table 1, and the summary of the pure species examined to assign the species-specific chNA restriction pattern are listed in Table 2. The only trees that did not show the expected chNA genotypes were three putative hybrids of blue and Engelmann spruce. The hybridization of blue and Engelmann spruces has been successful only when Engelmann spruce has been used as the female parent. The putative hybrids were expected to show a blue spruce chNA restriction pattern. Since the opposite result was Obtained.the hybrid nature of these three trees is left in question, but these find- ings do not rule out that these trees are Fz-hybrids (or later genera- tions) that resulted from.backcrosses with Engelmann spruce as the pollen parent. The evidence for paternal inheritance of plastids in gymnosperms is increasing, with reports showing paternal inheritance for two genera of the Taxoideacae, Ckytameria.(0hba et a1 1971), sequoia (Neale and 59 60 Table 1 . Hybrid spruces examined for plastid inheritance . Hybrid Nunber of Total Number Plastome Crosses of Trees Type White X Serbian 3 10 Serbian Serbian X White 2 7 White Blue X White 4 4 White White X Blue 6 6 Blue Engelmann X Blue 4 4 Blue Putative Hybrids 3 Engelmnn Totals 19 34 Paternal Table 2. Conservation of intraspecific RFLPs in "pure" species. Species Nunber Variation of trees in RFLPs White spruce 9 No Blue spruce 13 No Serbian spruce 4 No Engelmann spruce 6 No Totals 32 No and Sederoff 1988); and four genera of the Pinaceae, Pinus (Neale et al. 1988, Wagner et al. 1987 ), Picea (Stine and Keathley 1988a,b; Stine et a1. 1988) , Pseudotsuga (Neale and Sederoff 1988) and Larix ( Szmidt et al. 1987). There are still 10 other families of gymnosperms for which there have been no reports . The further study of plastid inheritance in gymnosperms may elucidate whether paternal inheritance is a derived char- acter or represents the primitive state. It may also lead to a further understanding of why uniparental inheritance of plastids (either maternal or paternal) is much more cannon than biparental inheritance. 61 Another example of paternal inheritance in the Pinaceae was reported by Allen (1976), who showed that hybrids of either longleaf pine (Pdnus palustris Mill.) or slash pine (P. elliottii Engelm.) with loblolly pine (P. taeda L.) or shortleaf pine (P. echinata Mill.) showed rates of 02 evolution in the dark, and stomate closure patterns that were similar to the paternal parent in reciprocal crosses. While there is still very limited information on the chloroplast genome of gymnosperms, there is even less known about the mitochondrial genome of gymnosperms. Neale and Sederoff (1988) demonstrated the mater— nal inheritance of mtDNA in loblolly pine (Pinus taeda) by the RFLP analysis of mtDNA, which is to the author’s knowledge the only report on the analysis of mtDNA in a gymnosperm. If paternal inheritance of plas- tids, and.naternal inheritance of mitochondria, prove the rule, then several very interesting questions can be addressed. First, it will be possible to do phylogenetic analysis of the gym- nospermae through the analysis of two relatively simple genomes, and to simutaneously develop both maternal and paternal lineages. Theoreti- cally, if the molecular clock hypothesis is correct, then both phylo- genies should be similar. If rates of molecular evolution are not cons- tant within both genomes, but vary independently, then very different phylogenies may be obtained. It will also be possible to follow gene flow in pOpulations for both maternally and paternally inherited traits. This will allow differences in dispersal mechanisms to be studied, ie. seed verses pollen. Analysis of chNA could also be of use in confirming the hybrid nature of Fl-hybrids, and for identifying the male parent species of progeny resulting from open pollination of Fl-hybrids. 62 LITERATTLRE CITED Allen RM (1976) Gas exchange of detached needles of southern pines and certain hybrids. USDA For Serv Res Pap SO—125 Neale DB, Sederoff RR (1988) Inheritance and evolution of conifer organelle genomes. In: Hanover JW, Keathley DE (eds) Genetic Manipulation of woody plants. Plenun Press, New York, pp 251-264 Neale DB, Wheeler NC, Allard RW (1986) Paternal inheritance of chloroplast DNA in Douglas-fir. Can J For Res 16:1152-1154 Ohba K, Iwakawa M, Okada Y, Murai M (1971) Paternal transmission of a plastid anomaly in some reciprocal crosses of sugi, Crwptaneria japonica D. Don. Silvae Genet 20:101-107 Stine M, Keathley DE (1988a) Inheritance of plastids in reciprocal crosses of white spruce and Serbian spruce. In preparation. Stine M, Keathley DE (1988b) Paternal inheritance of plastids in Engelmann spruce x blue spruce hybrids. In preparation. Stine M, Sears BB, Keathley DE (1988) Inheritance of plastids in interspecific hybrids of blue spruce and white spruce. In preparation. Szmidt AE, Alden T, Hallgren J-E (1987) Paternal inheritance of chloroplast DNA in Larix. Plant Molec Biol 9:59-64 Wagner DB, Furnier GR, Saghai-Maroof MA, Williams SM, Dancik BP, Allard RW (1987) Chloroplast DNA polymorphisms in lodgepole and jack pines and their hybrids. Proc Natl Acad Sci USA 84:2097-2100 APPENDIX 63 Table A1. location of blue spruce, white spruce, and hybrid spruce trees used. Species Origin Accession Plantation Row Colunn Nunbert Nunber P. pungans Utah 310070 70.22 24 CG P. pungans N. Mexico 310045 70.22 24 FF P. pungans Utah 310068 70.22 24 E P. pungans Colorado 310106 70.22 24 CC P. pungans Wyoming 310128 70.22 24 BB P. pungans Arizona 310192 70.22 23 DD P. pungans Colorado 310246 70.22 24 DD P. pungans Arizona 310193 70.22 23 AA P. glam (htario 191687 63.05 B 2 P. glauca New York 191644 63.05 B 4 P. glauca Manitoba 191631 63.05 B 5 P. glauca S. Dakota 191628 63.05 B 6 P. glauca Labrador 191657 63.05 B 7 P. glauca N. Hampshire 191649 63.05 B 8 P. glauca B. Colunbia 191672 63.05 B 9 P. glauca Saskacthewan 191665 63.05 B 10 P. glauca x pungens 720007 70.21 8 27 P. glauca x pungens 720008 70.21 7 36 P. glauca x pungans 720009 70.21 8 36 P. glauca x ptmgans 720010 70.21 8 39 P. glauca x pungans 720011 70.21 8 41 P. glauca x pungans 720017 70.21 7 29 P. pungans x glauca 720058 70.21 21 21 P. mans x glauca 720059 70.21 25 23 P. pwzgans x glauca 720060 70.21 23 33 P. pungans x glauca 720061 70.21 25 33 I! Add 67,000,000 to obtain the complete MICHtflI‘IP accession nunbers. 64 Table AZ. Location of white spruce, serbian spruce, and hybrid spruce trees used. Species Accession Plantation Row Column NunberllI Nunber Number Nunber Hybrid 710003 78 . 01 4 22 Hybrid 710003 78 . 01 25 20 Hybrid 710003 78 . 01 14 22 Hybrid 710003 78 . 01 57 22 Hybrid 710004 78.01 11 21 Hybrid 710004 78.01 ‘ 32 21 Hybrid 710004 78.01 13 21 Hybrid 710004 78 . 01 12 20 Hybrid 710004 78.01 30 21 Hybrid 710005 78 . 01 25 20 Hybrid 710005 78.01 8 21 Hybrid 710005 78 . 01 58 22 Hybrid 710005 78 . 01 40 22 Hybrid 710006 78 . 01 7 22 Hybrid 710006 78 . 01 44 22 Hybrid 710006 78 . 01 41 22 Hybrid 710006 78 . 01 39 22 Hybrid 710007 78 . 01 38 22 P. amorika 270002 u xx n P. amorika 270003 it an n P. glauca 190423 -- -- -- P. glauca 190424 -- -- -- I Add 67 ,000,000 to obtain the complete Michigan cooperative tree improvement program (MICHGII‘IP) accession nunbers. n See text for locations. HICHIG STA TE UN ll IIUIJIILIHIII IWHlWlll“HIIWIHIIIHIWMllHl