‘l fi‘i'i"‘\"¢'\2“f'\ V,.{ .;' r. :1: -: V.~»u.-\“.-J.fiua.. s..'dr'i;-.. This is to certify that the thesis entitled A CYTOGENETIC INVESTIGATION OF X AGROHORDEUM PILOSILEMMA presented by Lynn Ellen Murry has been accepted towards fulfillment of the requirements for _Eh.Jl.___degree in.Bn1'.an;L_and_ Plant Pathology Datcziz/A’b” 1221/, /973~ 0-7639 .‘ LIBRARY BINDSRS ;; ? Quantum”: 3",. “4-" l . [a The cytol was investigat W Hitch plants studied M ilosile'" m, the and (4x), and scope observa stages of mic diakinesis-me for each plan- ability and (I Analysi 3%.“ . | tmnships 0*. 0f the ampm‘ ABSTRACT A CYTOGENETIC INVESTIGATION OF X AGROHORDEUM PILOSILEMMA By Lynn Ellen Murry The cytology of X Agrohordeum‘pilosilemma Mitchell & Hodgson *was investigated to determine the genome relationships of Agropyron sericeum Hitchc., Hordeum jubatum L., and Hordeum vulgare L. The plants studied were Agropyron sericeum, Hordeum jubatum, X Agrohor- deum pilosilemma, its amphiploid, the amphiploid x Agropxron sericeum, the amphiploid x Hordeum vulgare (4x), Hordeum vulgare (2x) and (4x), and Hordeum vulgare (2x) x Hordeum jubatum. Light micro- scope observations of chromosome behavior included examination of all stages of microsporogenesis and compilation of comparative data for diakinesis-metaphase I, anaphase I, telophase I, and the quartet stage for each plant. Plant fertility was estimated from pollen stain- ability and seed set. Analysis of microsporogenesis in Agropyron sericeum, Hordeum jubatum, X Agrohordeum pilosilemma, the amphiploid,and the backcross of the amphiploid to Agropyron sericeum elucidated the genome rela- tionships of Agropyron sericeum and Hordeum jubatum. The tetraploid eental specie artially horml evidenced in th ;'.aht§ are: I}; llcrcherdeum I the amphiploid tin-‘lguratiOns mm'gurations Pairing with de M v behavior that bliity, Thep mwas cc “L” lndic tIVely isolat. Pairing. The assigned to E Lynn Ellen Murry parental species, Agropyron sericeum and Hordeum jubatum, share a partially homologous genome which affects the pairing relationships evidenced in their hydrids. The genome formulae assigned to these plants are: Agropyron sericeum. A"A"BB; Hordeum jubatum, AAA'A'; X Agrohordeum pilosilemma, AA'A"B; the amphiploid, AAA'A'A"A"BB; and the amphiploid x Agropyron sericeum, AA'A"A"BB. Observed pairing configurations were compatible with the expected maximum pairing configurations predicted under the assumption of genetic control of pairing with dosage effects. Hordeum vulgare x Hordeum jubatum was found to display asynaptic behavior that is believed to represent a physiogenetic incompati- bility. The pairing configuration of the amphiploid x Hordeum vulgare was comparable to the pairing seen in X Agrohordeum pilo- silemma indicating that the genomes of Hordeum vulgare are effec- tively isolated from the genomes of both Agropyron sericeum and Hordeum jubatum either by homology or through genes controlling pairing. The genome formulae, AA'V and AA'A"BVV, were tentatively assigned to Hordeum vulgare x Hordeum jubatum and the amphiploid x Hordeum vulgare, respectively. A CYTOGENETIC INVESTIGATION OF X AGROHORDEUM PILOSILEMMA By Lynn Ellen Murry A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Botany and Plant Pathology 1975 DEDICATION To my Mother and Father ii I would l Tai, my major and Stephen N. assistance, a for their con 1 would Gilbert Start and suggesti: friends trul The fin Jones Predoc New York. i s ACKNOWLEDGEMENTS I would like to express my sincere appreciation to Dr. William Tai, my major professor, and Drs. William G. Fields, John E. Grafius, and Stephen N. Stephenson, my committee members, for their guidance, assistance, and encouragement during the course of this research and for their constructive criticism of the manuscript. I would also like to thank Michael Christianson, Margaret Mead, Gilbert Starks, Robert Steidl, and Joanne Whallon for their interest and suggestions. The moral support given by my family and many friends truly sustained me through the completion of this work. The financial support for my final year of study, a Donald F. Jones Predoctoral Scholarship granted by the Research Corporation of New York, is gratefully acknowledged. iii LIST OF TABLES LIST OF FIGUR! Imaoucnou . MTERIALS AND RESULTS ,,,,, uscussmh _, 5mm ,,,,, l LITERATURE C TABLE OF CONTENTS Page LIST OF TABLES ............................................. v LIST OF FIGURES ............................................ vi INTRODUCTION ............................................... 1 MATERIALS AND METHODS ...................................... 6 RESULTS .................................................... 13 DISCUSSION ................................................. 68 SUMMARY .................................................... 82 LITERATURE CITED ........................................... 84 iv Origin 1 Diakine X Agroh Anaphas pilosil Diakine the An; Anapha Diakin Anapha Diakir Horde; Anaph. M93 Diaki HV x ~ Diak: SUWru Asso Freq Free Full Table 10. ll. 12. l3. I4. 15. LIST OF TABLES Origin and Designations of the Research Materials ...... Diakinesis-Metaphase I Chromosome Association in X Agrohordeum pilosilemma ............................ Anaphase I Chromosome Distribution in X Agrohordeum pilosilemma .......................................... Diakinesis-Metaphase I Chromosome Association in the Amphiploid ....................................... Anaphase I Chromosome Distribution in the Amphiploid.. Diakinesis-Metaphase I Chromosome Association in AHPA. Anaphase I Chromosome Distribution in AHPA ........... Diakinesis-Metaphase I Chromosome Association in Hordeum vulgare (4x) ................................. Anaphase I Chromosome Distribution in Hordeum vulgare (4x) ......................................... Diakinesis-Metaphase I Chromosome Association in HV x HJ .............................................. Diakinesis-Metaphase I Chromosome Association in AHPV. Summary of Diakinesis-Metaphase I Chromosome Association ......................................... Frequency of Micronuclei in Telophase I Cells ........ Frequency of Micronuclei in Quartets ................. Pollen Stainability and Seed Set ..................... Page 8 27 29 33 34 39 4O 48 53 53 58 65 66 67 LIST OF FIGURES Figure Page l. X Agrohordeum pilosilemma Breeding Program ,,,,,,,,,,,, g 2. Spike Morphology ...................................... l4 3. Stages of Microsporogenesis in Agropyron sericeum (2n=28) ............................................... 16 4. Stages of Microsporogenesis in Hordeum jubatum (2n=28). 20 5. Stages of Microsporogenesis in X Agrohordeum pilosilemma (2n=28) ................................... 23 6. Stages of Microsporogenesis in the Amphiploid (2n=56).. 3O 7. Stages of Microsporogenesis in AHPA (2n=42) ........... 36 8. Stages of Microsporogenesis in Hordeum vulgare (2n=l4). 42 9. Stages of Microsporogenesis in Hordeum vulgare (2n=28). 45 lO. Stages of Microsporogenesis in HV x HJ (2n=21) ........ 50 ll. Stages of Microsporogenesis in AHPV (2n=42) ........... 55 l2. Irregular Meiotic Behavior in HV x HJ and AHPV ........ 59 13. Possible Pairing Relationships among the Genomes of A ro ron sericeum, Hordeum jubatum, the Amphiploid.. and AHPA, Assuming Genetic Control of Pairing ......... 70 vi Investig CEZ’US hybrids :ytclogical a natural, Paci were Agrogvrc Based on thei U946) sugges sented collec have been adl Keller between Sien fO'xtail barl this same CC both the pa ”filmed a £0khlclne. :Grah parentage INTRODUCTION Investigations of North American X Agrohordeum G. Camus ex A. Camus hybrids began with the work of Stebbins, et 21, (l946). This cytological and morphological study revealed that the parents of a natural, Pacific Coast hybrid classified as Elymus macounii Vasey were Agropyron pauciflorum (Schwein) Hitchc. and Hordeum nodosum L. Based on their examination of herbarium specimens, Stebbins, gt_gl, (1946) suggested that materials classified as Elymus macounii repre- sented collections of several different, sterile hybrids between Hordeum jubatum L. and various Agrogyron species. Subsequently there have been additional investigations of the Elymus macounii complex. Keller (l948) stated that Elymus macounii was a natural hybrid between slender wheatgrass, Agropyron trachycaulum (Link) Malte, and foxtail barley, Hordeum jubatum. Booher and Tryon (1948) arrived at this same conclusion through their study of herbarium specimens of both the parents and sterile hybrid from Minnesota. A publication on forage crops by Forsberg (1953) indicated the same parentage and reported a fertile amphiploid of this hybrid had been obtained by colchicine-doubling. Lepage (1952, 1953) renamed the taxon, X Agrohordeum macounii (Vasey) Lepage, on the basis of its presumed parentage and on a comparison of the morphological characters of cf ACID ‘v’ron 1 In l955. investigatior and morpholog ieiosis in tl normal; four‘ artificial h; The present a 15.45 I, 5.8. attributed b aim sterility of ComPletely c cial hybrid Indistinguj of Agropyron, X Agrohordeum, and Hordeum. In 1955, Boyle and Holmgren published the first cytogenetic investigation of X Agrohordeum macounii. They studied the cytology and morphology of the parental species, Agropyron trachycaulum and Hordeum jubatum; the natural;and the reciprocal, artificial hybrids. Meiosis in the parental species, assumed to be allotetraploids, was normal; fourteen bivalents consistently formed. The natural and artificial hybrids (2n=28) displayed similar chromosome associations. The present author's calculations from their data show averages of 15.45 I, 5.88 II, 0.07 III, and 0.14 IV per cell. Boyle and Holmgren attributed bivalent formation to allosyndesis between the chromosomes of Agropyron trachycaulum and Hordeum jubatum and interpreted the sterility of the hybrids as failure of the two complements to synapse completely during meiosis. Morphologically, the natural and artifi- cial hybrids were more or less intermediate between the parents and indistinguishable from one another. They suggested AABB and AACC as genome formulae for Agropyron trachycaulum and Hordeum jubatum, respectively. Ashman and Boyle (1955) continued the previous investigation and reported on the meiotic behavior of the fertile, colchicine- doubled amphiploid, X Agrohordeum macounii. They found an average of 1.3 I, 24.6 II, 0.15 III, and 0.7 IV per cell at metaphase I; laggards and precocious dyad division at anaphase I; laggards and, rarely, bridges at anaphase II; an average of 5.8 micronuclei per quartet; 56 % pollen fertility; and 30 % seed set. Ashman and Boyle rerarked ti amphiploid the genome Bowde' northern g and the pa by Boyle a their loca wherever tl typified E (Vasey) Le; collected l ciarified 1 assume the garded as r The d‘ by Gross (1 D'ifllarily 1 to result . to salinit I CYCOiOgiCa Studies b Grogr remarked that the only morphological character distinguishing the amphiploid from the F1 was the presence of caryopses. They advanced the genome formula, AAAABBCC, for the amphiploid. Bowden (1959) discussed the intergeneric hybrid, X Agrohordeum nacounii in his paper on the chromosome numbers and taxonomy of northern grasses. He corroborated the chromosome number of 2n=28, and the parentage, Agropyron trachycaulum x Hordeum jubatum, reported by Boyle and Holmgren (1955); listed several voucher specimens and their localities; and stated that the hybrid may be expected to occur wherever the two parental species are sympatric. In 1960, Bowden typified Elymus macounii Vasey, the basonym of X Agrohordeum macounii (Vasey) Lepage. The type specimens were selected from materials collected by J. Macoun in Saskatchewan in 1879. Bowden (1960) also clarified the status of the fertile, artificial amphiploids; they assume the same binary name as the natural hybrid and should be re- garded as cultivars. The distribution and cytology of Elymus macounii was restudied by Gross (1960). The hybrids' widespread distribution was determined primarily from examination of herbarium specimens and hypothesized to result from a combination of frequent hybridization between Agro- pyron trachycaulum and Hordeum jubatum and of the hybrids' tolerance to salinity and flooding. Some differences are evident between the cytological data presented by Gross (1960) and that from the earlier studies by Boyle and Holmgren (1955) and Ashman and Boyle (1955). Gross recorded an average of 20.24 I, 3.87 II, and 0.004 IV (this author njgrld but 1" tiralentS- Further Gdtby Mitch differences the hybrid had glumes E type specmé Hodgson (19! first spike lenra pubes and the spe Acropyron s with Horde! servaticns dam piles d5 Agmgxr tion of X about 66° (Mitchell include I' Mitchell .i Ltd. (1 genome tc (this author's calculations from the published data) for the sterile hybrid but reported only those amphiploid cells which displayed 28 bivalents. Further investigations of X Agrohordeum macounii were carried out by Mitchell and Hodgson (1965a) in an attempt to explain the differences in lemma pubescence apparent in Alaskan collections of the hybrid. In 1942, Hulten had mentioned that Alaskan specimens had glumes and lemmas that were pilose whereas the lemmas of the type specimen of Elymus macounii were glabrate. Mitchell and Hodgson (1965a) studied the comparative morphology (length of the first spike internode, glume length, glume epidermal pattern, and lemma pubescence) of the "Alaskan hybrid", X Agrohordeum macounii, and the species, Agropyron latiglume (Scribn. & Smith) Rydb., Agropyron sericeum Hitchc., and Agropyron trachycaulum, which coexist with Hordeum jubatum in Alaska. Their morphological and field ob- servations resulted in the establishment of a new taxon, X Agrohor- deum pilosilemma Mitchell & Hodgson, whose parents were identified as Agropyron sericeum and Hordeum jubatum. The original distribu- tion of X Agrohordeum pilosilemma, "from south of the Brooks range, about 66° N. latitude, to southcentral Alaska, about 61° N. latitude“ (Mitchell and Hodgson, 1965a) has been extended by Bowden (1967) to include the Yukon and the District of Mackenzie. The work of Mitchell and Hodgson (1965a) reconfirms the contention of Stebbins, gt_gl, (1946) that more than one Agropyron species contributed its genome to the hybrids comprising the Elymus macounii complex. The MP of the specie ’- n-‘rare L. t. u a snrrogenesi s The purpose of the present cytogenetic investigation of X Agrohordeum pilosilemma was to determine the genome relationships of the species, Agropyron sericeum, Hordeum jubatum, and Hordeum vulgare L. through observations on chromosome behavior during micro- sporogenesis of these species and their hybrids. The pl obtained fr Michigan St search mate appears as The re field, grep are naintai of Botany a Plant Scier conditions cuddled, o Eel 37-14 buelve 25 and temper The r F Nth colc" enCeg, M; 300 treat'l MATERIALS AND METHODS The plants, Agropyron sericeum, Hordeum jubatum, X Agrohordeum . pilosilemma, Hordeum vulgare (2X), and Hordeum vulgare (4X), were .obtained from Dr. John Grafius, Department of Crop and Soil Sciences, Michigan State University. The origins and designations of the re- search materials are given in Table l, and the breeding program appears as Figure l. The research materials were cultivated under a combination of field, greenhouse, and growth chamber conditions. Perennial stocks are maintained year-round in the barley nursery at the Department of Botany and Plant Pathology farm. Duplicates are kept in the Plant Science greenhouse under 14 hour light and 21 C temperature conditions from early October to early May. Plants being crossed, coddled, or forced to bloom are grown in a Scherer-Gillett Model Cel 37-14 growth chamber in which sixteen 6' fluorescent tubes and twelve 25 N incandescent bulbs provide a 14 hour-a-day light regime and temperature settings are 21 C days and 13 C nights. The natural hybrid, X Agrohordeum pilosilemma, was treated vvith colchicine by Robert Steidl, Department of Crop and Soil Sci- eences, Michigan State University, in March, 1973. Approximately 300'treated culms were planted in the barley nUrsery in early June aimatlear iigust, cold caperature pats when th in the grout seed set wer ins‘estigatit The prr asfollows: 1. Aw 2. Up ca 3. Ti“ DC The ”Ore rent 6-8 daily the; 1aAse (Br In ~ of that year. Seeds produced by those plants were harvested in early August, cold-treated at 7 C for five days, and germinated at room temperature in a dark cabinet. Plantlets were transferred to clay pots when the coleoptyle was 1 cm. in length and grown to anthesis in the growth chamber. Both meiotic chromosome counts and relative seed set were considered in selecting the amphiploid used in this investigation. The procedures employed for crossing the research plants are as follows: 1. Awns of the female spike were clipped with cuticle scissors. 2. Upper and lower immature florets of the spike were removed carefully to avoid injuring the flag leaf. 3. The remaining florets were Opened and emasculated withfine- pointed tweezers; anthers were discarded. 4. Florets with feathery, receptive stigmas were individually hand-pollinated by breaking dehiscing anthers from selected male spikes over them. This process was repeated on the following day. 5. The pollinated spike was covered loosely with an aluminum foil envelope and supported by an iron rod. The florets of the covered spike were examined for ovary develop- lnent 6~8 days after pollination. Developing seeds were checked (daily thereafter for yellowing, an initial sign of endosperm col- Tapse (Brink, gt_al,, 1944). In the case of both interspecific (Davies, 1960; Konzak, gt_gl,, 3.3mm mafiw:= mpmum epoxeo gucoz .cmpoocum .m.< seem mvwmm zuwmcm>wc3 mpmum cemwguwz .mmocmwum pwom use aocu Go pcmEpcmqmo .inmpm .m >2 emczppzo oxcnem new nmmmoco cocuze >5 vwczppzo.ox;nem use vmmmogu ‘ zuwmgm>wc3 mpmpm cemwcuwz .mmocmwum pmom ccm aoco co Gamaycwaao ._ewmom .m »n umpamcp macaweupoo zHPmcm>Pcz mpmpm cmmwcuwz .mmocmwum Pwom can aogo mo pcmspceqmo .mswwmcu .m.c Eocm mummm zuwmcm>wcz mpmpm cmmwcowz .mmucmmom Peom use aocu mo newspceamo .chmgm .m an cmczupsu:oxcpsm use commocu exmap< .aaspma serm56SCZ .3.3 50cc memam exme_< .coe_ea .P_m;upez .3.3 50cc acopu exmmp< cmepma .coP>MH .m use Ppmcupwz .3.3 Eocm mummm szHmo >az< 2 onh Ezmuco: Axwv mgmmpa> an x neoFchaE< Eamopgmm am x uPoFchge< ewo_a+;ga< Axmv mgmwpz> Esmucoz Ezumnzw an x Axmv meme3> Eamuco: Eapmnzm Ezmugo: mEEmPPmo—wawszmucozocm< x Ezmowsmm coLAQOLm< Hz<4a mpmwcmpmz cugmmmmm esp mo mcowpmcmvmwo use cwmwco p m4maz< L Ammucmv mcmmpz> Ezmuco: 1|. APNnCNV a: x >: IL Ammncmv aesa_emopwme:mcco;ocm< x 7 #7 fir: Ammucmv uwopawcae< + m:..n=ou mcwow;u_oo _ Amencmv Ezmuco: iiiii.AwNncmv Ezpmnzw Esmvco: Ammucmv EsmUwcmm coce Emcmosa.mcwcmmsm ease meopmn Ezmugosocm< x p mmzwmm aogm< 10 1951) and intergeneric (Cooper and Brink, 1944) barley hybrids, endosperm collapse, interpreted as a nutritional incompatibility between the embryo and endosperm, necessitates embryo culture. The method outlined by Morrison, gt_al, (1959) was adapted for the pre- sent study. Embryo culture was carried out in a small darkroom lacking obvious air currents. The immediate transfer area was swabbed with 95 % alcohol and ringed with burning alcohol lamps. Tools, tweezers and needles, were flame-sterilized before each use. The caryopsis was surface-sterilized in 5 % "Clorox“ and placed in a petri dish containing sterile distilled water. Excision, complete removal of the endosperm and ovary wall from the embryo, was done under a dissecting microscope (15X-45X). The embryo was placed on the surface of 50 ml. of sterile culture medium (Norstog, 1973) con; tained in a foam-stoppered, 125 ml. Erlenmeyer flask. The flasks were stored in a room-termperature (21 C), dark cabinet until the embryos germinated. As soon as the embryos showed well-developed roots, the flasks were removed to a light table. After emergence *of the second leaf, the plantlets were potted and placed in the growth chamber. Spikes for cytological studies were fixed (1973-1975) in bottles of Newcomer's solution (Newcomer, 1953) from the field, be- tween 6 and 10 AM in early June; from the greenhouse, between 8 and 11 AM depending on the season; and from the growth chamber, be- tween 9 and 12 AM. These collection times were established to allow harvest during periods of maximum meiotic activity. After 11 24 hours at room temperature, the fixed materials were stored in a refrigerator until used. All cytological observations made in this investigation were from microspore mother cells. Temporary slides were prepared according to the technique described by Tai (1967). All stages of meiosis were examined for each plant, and comparative data from at least five spikes was compiled for a minimum total of 30 euploid cells at diakinesis-metaphase I and anaphase I. Minima of 60 telo- phase I cells (henceforth designated T-I cells) and 200 quartets were scored for micronuclei. Phase contrast microscopy was accomplished using either a Zeiss Standard WL Research Microscope with an external light source or a Zeiss Photomicroscope II with a built-in light source. Photomicro- graphs were recorded on Panatomic X film using the planapochromatic, oil immersion, objective lenses (40x/1.0 and 63x/l.4) and the built- in 35 mm. camera on the Photomicroscope II. Fertility of the parental plants and hybrids was eStimated from observations of pollen stainability and seed set. Pollen stainability was tested using IZKI (Johansen, 1940) on a minimum total of 1000 grains from at least five spikes per plant. Seed set was determined by counting the number of florets which developed seed on a minimum of five mature spikes. It was originally proposed that the plants included in this research program be karyoptyped using giemsa chromosomal banding techniques. Banding would allow the specific identification of 12 homologous chromosomes (or chromosomal segments) of the variods genomes. Despite almost seven months of experimental effort, attempts to obtain reproducible giemsa bands and karyotype barley chromosomes proved futile. Various techniques available in the literature (Gill and Kimber, 1974; Merritt and Burns, 1974; Verma and Rees, 1974; Doebel, g g” 1973; Schweizer, 1973; Stack and Clark, 1973; and Vosa and Marchi, 1972) have been tried, but faint heterochromatic bands are obtained less than 10 % of the time in Hordeum vulgare Larker seedling, root tip chromosomes. The vari- ables of giemsa banding continue to be elusive since two slides of the same material treated in the same way at the same time may re- sult in only one slide exhibiting chromosomal banding. Attempts at banding with leuco-basic-fuchsin (Feulgen Stain) and with aniline blue also failed. 13 RESULTS ‘Agropyron sericeum (Figure 2 A) is a self-fertile (Hodgson, 1964), perennial tetraploid endemic to Alaska, the Yukon, and the District of Mackenzie (Bowden, 1965). Hodgson (1956) first reported its chromosome number, 2n=28, and later published the only account of its cytology (Hodgson, 1964). Microsporogenesis in Agropyron sericeum (Figure 3) was normal with 14 bivalents formed at metaphase I (Figure 3 D). No univalents or multivalents were observed in 92 cells, but the bivalents were often difficult to separate and ap- peared to have tenuous connections one to another. This character- istic, previously noted by Hodgson (1964), was present regardless of fixative used, growth conditions, or the time of harvest. Chro- mosome segregation was regular, 14-14 (Figure 3 E) with 27.9% of the 43 anaphase I cells showing chromosome bridges. The T-I cells and quartets reflected this bridge formation and associated frag- mentation in that 11.8% of the 187 T-I cells and 7.2 % of the 807 quartets contained micronuclei. Pollen stainability was 84.0% under field conditions, and seed set was 88.6% in the growth cham- ber. The latter percentage falls within the range of 83-100% seed. set reported for Agropyron sericeum by Mitchell and Hodgson (1965 b). Hordeum jubatum (Figure 2 C) is a highly fertile (Mitchell and 14 Figure 2. Spike Murphology OD (BTW Agropyron sericggm_(0 6x) X Agrohordeum pilosilemma (0-8x) Hordeum jubatum (1.lx) Amphiploid (008x) AHPA (0.8x) Hordeum vulgare (0.6x) HV x HJ (1.0X) AHPV (0.8x) . Linden he...“ ...~ .d. IlfiJW/lzi/Jrufljss 1r. sic-"I... 1141i . . . InihCVs‘ 15 Figure 2 16 Figure 3. Stages of Microsporogenesis in Agropyron sericeum (2n=28) Zygotene (1450x) Pachytene (ll75x) Diplotene (l375x) Metaphase I with 14 bivalents (1975x) Anaphase I with a 14 - 14 distribution and a double bridge involving three dyad chromosomes (1550x) Two daughter cells from the first meiotic division (1550x) Metaphase II (2240x) Telophase II (1060x) Figure 3 18 3 (cont'd.) Figure 19 Wilton, 1964; Smith, 1944), perennial, segmental allotetraploid (Starks and Tai, 1974; Redmann and Bergaonkar, 1966; Rajhathy, gt 21,,1964; and Wagenaar, 1959, 1960) with arctic-alpine-temperate distribution in both the Old and New World (Bowden, 1962; Hitchcock and Chase, 1950; Covas, 1948; and Nevski, 1934). From meiotic counts of microspore mother cells, Aase and Powers (1926) published the first determination of chromosome number, 2n=28, for Hordeum jgbatgm, Microsporogenesis (Figure 4) was completely normal with consistent formation of 14 bivalents in 40 diakinesis-metaphase I cells (Figure 4 B-D). Quadrivalents, observed in Hordeum_jubatum by Schooler, gt_al, (1966) and Rajhathy and Morrison (1961), have never been seen in the Alaskan material used in this investigation (cf. Huang, 1975; Starks and Tai, 1974). Anaphase I segregation (Figure 4 E) was an orderly 14-14 with chromosome bridges found in 10.0 % of the 30 cells. Micronuclei were present in 17.7% of the 96 T-I cells (Figure 4 F) and 11.1% of the 207 quartets (Figure 4 H) counted. Under field conditions, pollen stainability was 84.8% (Starks, 1975), and seed set was 93.7%, higher than the 72% reported by Smith (1944) for greenhouse-grown plants. Mitchell and Hodgson (1965 a) established the chromosome number, 2n=28, for X Agrohordeum pilosilemma (Figure 2 B), but its cytology has not been studied previous to this investigation. Microsporo- genesis of the spontaneous hybrid is represented in Figure 5. Meta- phase I configurations (Figure 5 C) averaged 13.32 I, 5.89 II, 0.08 III, and 0.05 IV for 38 cells (Table 2). Secondary association of 20 Figure 4. Stages of Microsporogenesis in Hordeum jubatum (2n=28) A. Zygotene (1375x) B. Diakinesis with 14 bivalents (1880x) C. Prometaphase I with 14 bivalents (2750x) D. Metaphase I with 14 bivalents (2075x) E. Anaphase I with a 14-14 distribution, one fragment (arrow), and one bridge (1625x) F. Telophase I (1350x) G. Metaphase II (ll75x) H. Quartet (1075x) 21 22 .7. 1 led ‘81:? {a o I . a 33' Xi." r .44 Figure 4 (cont'd.) S?- b. \ .---"‘l 23 Figure 5. Stages of Microsporogenesis in X Agrohordeum pilosilemma KC; (2n=28) Zygotene (1600x) Pachytene (l625x) Metaphase I with 111, 411, and (arrows) 3III (1625x) Anaphase I with an 8-9 distribution, 11 laggards, and one bridge (1325x) Telophase I with two micronuclei (1075x) Late telophase I with trailing laggards (1175x) Late telophase I with tripolar segregation (1125x) Prophase II (l450x) Metaphase II (1175x) Anaphase II with a 14-14 distribution and two laggards (2015x) Quartet with six nuclei and six micronuclei (l450x) Linear quartet with eight micronuclei (1200x) 24 . 409...! .«Vtrwwtufi Figure 5 25 c 33%; 1. ‘ ';- (NI-{4. .\ .- . . . ‘ . “’~n;--;_. ’.‘.l “ n' r .. -.' ‘ 'v r E 1- s ."" :5: 3-"; ~. .- :. \’:"~ ,-. 35...... ,s rs.” I" Ix.-. - l ‘ i. I. ~_. '7- s . . .3.- , 31,}. 4",“. - _ ‘9‘ -. J" ’1 " . 110‘ T ' H . 1‘. ‘ , ‘. l' s e 0 v “-” xii..." ' ‘ 4.; ' .t g: . 44FVDEE. ti _ ., U C Figure 5 (cont'd.) 26 ca '82". .. . . “:3- p. . ‘ r? v, . '3‘.“ 9. 3-. ° . I {D (‘5 Tl}. o\".__§ . C ‘T “:35 . ' Figure 5 (cont'd.) 27 mo.o mm.o om.m mm.m~ mmacm>< mm N cm ¢NN mam pouch mo.N F m my mo.N p m o_ m~.mp m p m mp ~m.mN o? m up 0N.m N F m «P 9N.m N N e .ep Nm.op ¢ F m mp mmaN F m m mp mm.~ m w Np Nm.op ¢ N m NF mo.N F — n —_ mN.m N m c FF mo.N P N o o_ mo.N P F m m & mP—mu mo * >H HHH HH H cowomwuomm< mEomosocsu mEEmpwmopwa samucococmm.x cw cowumwoomm< mEomoEocgu H mmmcnmpmzumwmmcpxewo N m4m< NN NON NNN NNN NNNON NN.N N NN e NN NN.N N NN N oN NN.N N NN N NN NN.N N N oN N NN.N N NN N N NN.N N NN N N NN.N N NN N N NN.N N N NN N NN.NN N N NN N NN.N N ON ON N NN.N N NN N N NN.N N N «N N NN.N N N NN N NN.N N oN NN N NN.N N N NN N NN.N N N NN N .1NN1. mNNNN N0 N «NON NNNNN co NNoa , NuNNNNNN mmcwmaocw Nesmpwmopwa Esmucogocm< x :N corpanNpmNo msomosocgu N mmmzamc< m m4m Pachytene (1225x) B. Metaphase I with 21, 2111, and (arrows) 31V (l650x) C. Anaphase I with a 26-26 distribution and 4 laggards undergoing precocious centromere division (1050x) 0. Late telophase I with five micronuclei (1075x) E. Metaphase II (1075x) F. Anaphase II w1th a 263E§ddfifiggibfiffign and two m1cronucle1 (1175x) G. Quartet with eight micronuclei (1350x) H. T-shaped quartet with two micronuclei (1075x) 31 1‘9 W 6 Figure 32 Q.. 6 (cont'd.) Figure 33 TABLE 4 Diakinesis-Metaphase I Chromosome Association in the Amphiploid Chromosome Association I. II 111 IV # of Cells __3;__ 1 18 1 4 3 10.00 1 22 1 2 3 10.00 2 l8 2 3 1 3.33 2 19 4 2 6.67 2 21 3 2 6.67 3 15 1 5 1 3.33 3 17 1 4 1 3.33 3 19 1 3 2 6.67 3 21 1 2 1 3.33 4 20 3 1 3.33 4 22 2 1 3.33 5 l8 1 3 3 10.00 5 19 3 1 1 3.33 6 18 2 2 1 3.33 8 18 3 1 3.33 20 4 2 6.67 22 3 1 3.33 24 2 3 10.00 Total 73 594 21 89 30 Average 2.43 19.80 0.70 2.97 34 NN.NN NN.N NN.NN NNNNN>< NN NNN NNN NNN NNNoN NN.N N NN NN NN.N N NN NN NN.NN N NN N NN NN.NN N NN N NN NN.NN N NN N NN NN.NN N NN N NN NN.N N NN N NN NN.N N NN N NN NN.N N NN N NN NN.N N NN N NN NN.N N NN N 4N NN.N N NN N 3N NN.N N NN N NN NN.N N NN N NN NN.N N NN N NN N NNNNN N6 N NNN; NNNNN co NNoN chmmmNN wchasocw uNoNqNgae< mgu cw coNu=NNNuNNo meoNoEoczu N mmmcqmc< m m4mN NNN NH H :oNpNNuoNN< mEomoEocgu :Nmz coNNNNUONN< mEoNoeocgu H mNNcaNNmzimNchNxNNo mo Nemessm NN m4m Esmuco: >NI< Esmucoz a: x >1 EspNnnw.Ezchoz NEENNNNoNNm Ezmucozocm< x EzmuNNmN cocxmmgmm NNNNN 65 NN.N N-N N.NN NNN a NN.N N-N N.NN NNN NN.N NN-N N.NN NNN NN.N N-N N.N NNN N NN.N N-N N.NN NN NN.N N-N N.NN NN NN.N N-N N.NN NNN NNN: NNNNN zz-NNNN N-N N .mmummwrw Ppmu H1H\Nmpu:cocuwz NNNmo N mmmcaonN :N NmNuzcocoNz mo Nucmzcmcm mp m4m<~ .pxmp mom .NNNonE NNNammNNNN Nxcv mcnmpa> Ezmccoz >mz< «NNN NNNNNNNNEN AxNv mNNmN3> Ezmvcoz a: x >1 Eaamnmw,szmccoz NEENNNNonm Ezmcco;ocm< x Eamuwcmm cocxaosm< pampm 66 mN.N NN-N N.om «N.N NN-N N.Nm o¢.N 0N1N N.Nm NN.N N-N N.N mm.N N-N N.NN NN.N NN-N .ooN NN.N oNiN N.N cNmz mmcmm zziumpcmzo N umpNNNdVNmNuacocoNz NNN wPoP Nmm mom a NON Num now umcoom N .pxmp mom .NNNoNoE NNNammNNH N Axev «NNmN:> ancco: >mx< E N.N NNoNNNNNe< NXNV wcmmNz> Ezmuco: a: x >1 Enumnzn Ezmuco: NEENNNNonm EzmucogonN x EzmuNcmN coczmoxm< vamp; Npmucmao :N NmNozcocoNz mo Nucmzcmcu «P m4mwcn mumpm :wacomz .NmoNospma chNm ucm Newpom mo “cospgmawo .Nxcmpm .w Eocm Name a N.NN NNNN Nxev 88N2, Eameao: o NNNN >N=< N.NN NNNN 1 N.NN « NNNN ENNNNNNNENNNNOI o 000—. mEEmZmoPE Ezmtgocosm< x oéw 000—. Ezmotmm cotaegq mNaNcNNNm N cmcoom NcNNNo madam NNNNNNNNNNNN NNNNoN NNN NNNN NNN NNNNNNNNNNNN NNNNNN NN NNNNN 68 DISCUSSION - Cytological analysis of microsporogenesis in Agropyron sericeum revealed that 14 bivalents were formed at metaphase I (Table 12). The complete absence of multivalents evidenced in both this investigation and that of Hodgson (1964) suggests that only homologous pairing occurs and that Agropyron sericeum is an allotetraploid. Hordeum jubatum displays identical metaphase I behavior (Table 12) but is believed to be a segmental allotetra- ploid. This designation was proposed by Wagenaar (1959, 1960) after a thorough study of the chromosome behavior of hybrids between Hordeum jubatum and Secale cereale L. In Wagenaar's hybrids, the smaller chromosomes of Hordeum jubatum usually paired autosyndetically rather than with the chromosomes of the Secale cereale complement. Whether Hordeum jubatum displayed autosyndetic pairing with chiasma in that cross with Secale cereale or some phenomenon analogous to distributive pairing (Grell, 1967) based on chromosomal size differences reamins open to queStion. Starks and Tai (1974), in an article on Hordeum jubatum x Hordeum com- pressum Griseb. hybrids, agreed with Wagenaar's interpretation of Hordeum jubatum as a segmental allotetraploid. In addition they proposed that homologous versus homeologous chromosome association 69 in Hordeum jubatum is genetically controlled and suggested that the genome formula, AAA'A', be assigned to Hordeum jubatum. In order to detennine the genome formula for Agropyron sericeum, the chromo- some associations of X Agrohordeum pilosilemma were analyzed. The average chromosome association for X Agrohordeum pilosilemma (Table 2) was 13.32 I, 5.89 II, 0.89 III, and 0.05 IV, which approaches a maximum pairing configuration of 14 I + 7 II. If auto- syndetic pairing occurred in Hordeum jubatum, it may be assumed that the genomes of Agropyron sericeum did not pair with one another, the behavior expected of genomes from a strict allotetraploid. The single quadrivalent recorded for X Agrohordeum pilosilemma is be- lieved to represent two loosely associated bivalents, a pseudoquad- rivalent (Walters, 1954). The presence of 1-3 III in 55% of the hybrid cells suggests that one Agropyron sericeum genome is partially homologous with one of the genomes of Hordeum jubatum. From the results of this study, the genome formula, A"A"BB, is assigned to Agropyron sericeum, and, AA'A"B, to X Agrohordeum pilosilemma. Possible pairing relationships of the parents and hybrid are pre- sented in Figure 13. Subscripts S and J delineate the genomes of Agropyron sericeum and Hordeum jubatum, respectively. , Indirect support for the supposition that Agropyron sericeum is an allotetraploid with a genome formula partially homologous to a genome of Hordeum jubatum was found in the cytotaxonomic literature. Taxonomically Agropyron sericeum is closely related to two other northern, slender wheatgrasses, Agropyron latiglume ".."~'—.a.2_-"~"1.""v&". . " "smfiifil 70 FIGURE 13 Possible Pairing Relationships among the Genomes of Agropyron sericeum, Hordeum jubatum, X Agrohordeum pilosilemma, the Amphiploid, and AHPA, Assuming Genetic Control of Pairing A; A; BS BS AJ AJ AS A; 711 711 711 711 A. 4:. 441:. III 71 611+61 AMPHIPLOID Au A3~A3-AJ Ag Ag BS 33 3IV+3II 7II III+I 311 311 311 AHPA A: A: A; A; 3.3 “”3IV ‘ 7II “III+I ‘ 3II+3I 31 71 and Agropyron trachycaulum (Bowden, 1965; Mitchell and Hodgson, 1965a). Dewey (1966) noted close similarities between the genomes of Agropyron latiglume and Agropyron trachycaulum, both allotetraploids which carry the basic Agropyron spicatum (Pursh) Scribn. & Smith genome (Stebbins and Snyder, 1956). In a later paper, Dewey (1971) wrote, "Although Hordeum species do not contain a genome drived from Agropyron,.a . modified Hordeum genome apparently occurs in Agropyron...". Inia recent study of X Agrohordeum.macounii, Huang (1975) stated that one of the genomes of allotetraploid Agropyron trachycaulum was homoelo- gous to a Hordeum jubatum genome. Future cytogenetic investigations will answer the obvious question: what are the chromosome associa- tions among Agropyron latiglume, Agropyron sericeum, and Agropyron trachycaulum and between Agropyron latiglume and Hordeum jubatum. Since the optimum pairing configuration for X Agrohordeum pilo- silemma would be 7 I + 7 III and the data (Table 2) show limited tri- valent formation, it is suggested that the extent of multivalent formation in the amphiploid and the backcross of the amphiploid to Agropyron sericeum, AHPA, may offer more accurate indices of chromosome homology. Chromosome association in the amphi- ploid averaged 3.67 multivalents per cell (Table 4) with 1-3 III in 57% of the cells and 1-5 IV in 100%. Multivalent formation in AHPA averaged 4.26 (Table 6) with 1-4 III in 61% of the cells and 1-6 IV in 100%. Considering that multivalent formation is governed by the size and number of chromosomes per cell, chiasma frequency and dis- tribution, environment, and genetic control of pairing (Thomas and 72 Kaltsikes, 1972; Morrison and Rajhathy, 1960 b; Hovin, 1958; Grun, 1952; Sears, 1941; and Myers and Hill, 1940), the amphiploid with a maximum association of 31, 1511, III, and 5 IV, and AHPA with 41, 711, and 61V approach their predicted maximum pairing configurations, 14 II + 7 IV and 7 II + 7 IV, respectively. Figure 13 shows the possible pairing relationshipsand genome formula, AAA'A'A"A"BB, of the amphiploid. As the amphiploid was created by colchicine treatment, its genomes appear in duplicate and were interpreted to pair as follows. The BB genome from Agropryon sericeum paired homologously. The segmentally homologous AA, A'A' and ABA? genomes from Agropyron sericeum and Hordeum jubatum paired either autosyndetically as bivalents or autoallosyndetically as multivalents. Since at least one chiasma per chromosome pair is re- quired for multivalent formation (Darlington, 1929), segmentally homologous parts must have been exchanged between A and A' of Hordeum jubatum and A or A' and A“ of Hordeum jubatum and Agropyron sericeum approximately 57% of the time. Consequently, chromosomes with an Agropyron centromere may carry a Hordeum telomere; the converse situation would also exist. For the purpose of demonstrating pair- ing relationships in Figure 13, those genomes bearing exchanged chromosomes are designated A*. The exchange chromosomes may be assumed to assort independently and to pass into the gametes pro- duced by the amphiploid. The constitution of these gametes is reflected in the genome formula of the subsequent hybrid, AHPA (Figure 13) and in its pairing 73 configuration. Chromosome pairing in some plants is believed to depend on telomere recognition. The literature on this subject be- gan with the work of Cleland and Blakeslee (1931) who proposed that chromosome pairing was initiated at or near the ends of chromosomes. The fusion of homologous, telocentric heterochromatin during pachy- tene was reported by Kostoff (1938) in Triticum, by Thomas and Revell (1942) in Ciggr, and by Kasha and Burnham (1965) in barley. Recent articles include theoretical papers by Wagenaar (1969), Comings (1968), Jones (1968), Sved (1966), and Walters (1954) as well as papers by Godin and Stack (1975), Ashley and Wagenaar (1972), Wagenaar and Sadasivaiah (1969), Brown and Stack (1968), Kumar and Natarajan (1966), Wagenaar (1960), Riley and Chapman (1957), and Ostergren and Vigfusson (1953), which contain supporting data. In AHPA, the BB genomes from Agropyron sericeum paired homol- ogously and the four A genomes from Agropyron sericeum and Hordeum jubatum paired as fully as telomere recognition in a hexaploid cell allows. The average 6.58 univalents observed in the AHPA cells may have resulted from mechanical obstruction to complete pairing (Sears, 1941), the failure of chiasma to form between A* chromosomes with one or two matched telomeres but different centromeric regions (Swanson, 1940) or the occurence of multivalents in all of the cells (Sears, 1941). Genetic influence on chromosome pairing has been reported by Starks and Tai (1974), Gottschalk (1973), Ellis, 22.9.1; (1973)”, Driscoll (1972), Douglas and Brown (1971), Harlan, 93.213 (1970), 74 Feldman (1966), Rajhathy, §t_gl, (1964), and Riley and Chapman (1958). Starks and Tai (1974) proposed that control of pairing in Hordeum jubatum x Hordeum compressum hybrids is governed by a gene or genes on the Hordeum jubatum A genome and that the type of pair- ing results from a dosage effect, i.e., a single dose allows homeolo- gous pairing whereas a double dose promotes homologous pairing. This hypothesis was employed to construct the maximum pairing con- figurations for X Agrohordeum pilosilemma, the amphiploid. and AHPA. and can be used to explain the possible pairing relationships seen in Figure 13. Pairing in X Agrohordeum pilosilemma and in AHPA with one dose of the A genes is generally homeologous, and in the amphi- ploid with two doses is primarily homologous. The frequency of Agropyron sericeum (27.9%), Hordeum jubatum (10.0%), and diploid Hordeum vulgare (38.0%) anaphase I cells dis- playing bridges was rather high. Hodgson (1964) reported a 10% frequency in Agropyron sericeum, and Redmann and Borgaonkar (1966), 2-5%, in Hordeum jubatum. It seems probable that late separating bivalents, especially in the case of Hordeum vulgare (2x), were in- terpreted as bridges, thus inflating the frequencies reported in this investigation. X Agrohordeum pilosilemma showed asynchrony, similar to that reported in Triticale (Thomas and Kaltsikes, 1922),_during anaphase I with 4-14 chromosomes, univalents and bivalents, lagging on the metaphase plate. This behavior may be attributable to a differential duration of meiotic stages (Bennett, 1971; Bennett, gt 31. 1971; 75 and Riley, 1968) or lack of homology between the kinetochores and spindle organizers (Tai, 1970) of Agropyron sericeum and Hordeum jubatum; the meiotic times of the parental species have not been determined. The late-aligning univalents (cf. Wagenaar and Bray, 1973) of X Agrohordeum pilosilemma, the amphiploid, and AHPA commonly underwent precocious centromere division, a meiotic phe- nomenon generally correlated with the presence of unpaired chromo- somes in hybrids, haploids, polyploids, or asynaptics (Clayberg, 1959). Precocious centromere division involves the separation of chromosomes into "chromatids" during anaphase I and the fragmenta- tion of some of these "chromatids" during anaphase II. If either the "chromatids" or fragments are not included in their respective daughter nuclei, they become micronuclei scorable in either T-I cells or quartets. Precocious centromere division was described in haploid wheat by Gaines and Aase (1926), in triploid maize by McClintock (1929), in Aegilotritricum by Kihara (1931), and in an asynaptic wheat hybrid by Smith (1936). The occurence of this phe- nomenon has been widely reported (Dewey, 1972; Sadasivaiah and Kasha, 1971; Lange, 1971 a; Tai and Dewey, 1966; Rajhathy and Morrison, 1959; Wagenaar, 1959; Lima-de-Faria, 1956; Dowrick, 1953; Walters, 1950; Elliott and Love, 1948; and Stebbins, et_al, 1946). The precocious centromere division observed in univalents and bi- ‘ valents of X Agrohordeum pilosilemma, and in univalents of the am- phiploid and AHPA is believed to have added significantly to the number of micronuclei found in their T-I cells and quartets. 76 Whether precocious centromere division results from lack of comple- ment balance, asynchrony or late alignment of the chromosomes on the metaphase plate, or is an inheritable character in these plants re- mains unknown. The frequency of micronuclei in the T~I cells and quartets of Agropyron sericeum, Hordeum jubatum, X Agrohordeum pilosilemma, the amphiploid, and AHPA (Tables 13, 14) may be related to cytological irregularities such as univalent frequency, bridge-fragment formation, precocious centromere division, laggards, and chromosomes excluded in the various meiotic stages. Likewise, pollen stainability and seed set (Table 15) may be related, although in polyploids, not necessarily correlated with the forementioned cytological irregularities. Overall fertility, as predicted from frequencies of pollen stainability and seed set, is determined by a combination of genetic, environmental, physiological, and cytological factors (Hsam and Larter, 1973; Weimarck, 1973; Merker, 1971; Rommel, 1961; Stebbins, 1950; and Muntzing, 1939). Irregular meiotic behavior was observed in both HordeUm vulgare (2X) x Hordeum jubatum, HV x HJ, and the amphiploid x Hordeum vulgare (4X), AHPV, despite the relatively stable constitutions and regular meiosis of their respective parents (Table 12). The euploid chromo- some association of HV_x HJ which averaged 19.85 I, 0.54 11, and 0.02 111 illustrates essentially asynaptic behavior. The rod bivalents and trivalent recorded in this study and the bivalents and trivalents reported for the reciprocal hybrid (Kerber, cited in Wagenaar, 1960; 77 Rajhathy and Morrison, 1959) may represent persistent secondary associations, pseudochiasma resulting from heterochromatic fusion of paired telomeres (see references, discussion AHPA). Obvious secondary associations were noted in approximately 90.0% of the HV x HJ metaphase cells, and 98% of these associations were end-to- end. Since genome suppression of chromosome pairing has been re- ported in other interspecific crosses involving Hordeum vulgare; Hordeum bulbosum bu, vulgare (Lange, 1971 a), (Hordeum compressum x H, stenostachys)2 x H, vulgare (Rajhathy, §t_§l, 1964), (Hordeum ‘a pusillum x.H. californicum) x H,.vuLgare.(Rajhathy,.et.al, 1964),. (Hordeum jubatum x H, brachyantherum) x H, vulgare (Rajhathy and Morrison, 1959), and Hordeum depressum x H, vulgare (Morrison and Rajhathy, 1959); it is suggested that genome interaction is respon- sible for the asynapsis displayed in HV x HJ. The aneuploidy (2N = 16-22) revealed in microspore mother cells of HV x HJ is attributed to premeiotic loss of chromosomes by chromosome elimination. Chromosome elimination subsequent to fertilization in interspecific hybrids was first suggested by Schooler (1963, cited in Subrahmanyam and Kasha, 1973) and has been reported for Hordeum lechleri x H, vulgare (Rajhathy, gt_gl, 1964) and Hordeum bulbosum x H, vulgare (Lange, 1971 a,b; Kao and Kasha, 1971; Kasha and Sadasivaiah, 1971; and Subrahmanyam and Kasha, 1973). In these investigations of Hordeum bulbosum x H, vulgare, it was pro- posed that the balance between genetic factors of the two parents regulated the stability or elimination of chromosomes. Whereas a 78 1:2 genome ratio (Hordeum vulgare : H, bulbosum) is relatively stable, a 1:1 hybrid may lose chromosomes until it becomes a pure haploid or dihaploid Hordeum vulgare. Chromosome elimination in the 1:1 hybrid may begin in the embryo and continue during plant maturation. The exact mechanism of chromosome elimination or timing of chromosome loss in HV x HJ has not been determined; however, lagging chromosomes at premeiotic telophase (Figure 10 A) and uncondensed or disintegrating univalents at diakinesis (Figure 10 C) were noted in the material analyzed here. A series of Hordeum jubatum x H, vulgare hybrids, similar to those studied in Hordeum bulbosum x H, vulgare (Lange, 1971 a, b: Kasha and Sadasivaiah, 1971) would be necessary to deter- mine which chromosomes are being eliminated, the mechanism through which this occurs, and which genome genetically controls this process. The average chromosome association for AHPV, 16.47 I, 10.1 11, 0.73 111, and 0.73 IV, was comparable to the pairing observed in X Agrohordeum pilosilemma (Table 12) which seems to indicate that the genomes of Hordeum vulgare are isolated from the other genomes by homology or through genes controlling pairing. As secondary asso- ciations were quite prevalent among obvious univalents and the modal class was 14 I + 14 II, most of the AHPV multivalents are believed to be loosely-associated I + II, II + II, and I + III combinations. Interchromosomal synapsis was particularly difficult to distinguish from secondary association in HV x HJ and AHPV, .The metaphase I chromosomescfiiHV x HJ always assumed an appearance more typical of mitosis and never assembled on the metaphase plate (Figure 10 D): 79 metaphase I in AHPV was defined by the presence of a few bivalents on the metaphase plate and numerous univalents scattered throughout the cytoplasm (Figure 11 C). Since the putative influence of the Hordeum vulgare genome on pairing was not evidenced in AHPV, in the manner previously described for other hybrids (see HV x HJ discussion), the genome formula, AA'A"BVV, is tentatively assigned to AHPV. Then, by extrapolation, the genome formula for HV x HJ becomes AA'V. Centromere misdivision, a term coined by Darlington (1939), occurs in the anaphase I cells of both HV x HJ and AHPV. Apparently the telomeres of a univalent acquire centromeric activity (neocentro- mere, Rhoades, 1952) and bring about the transverse division of the chromosome; the products of this division are telocentrics, isochromo- somes, acentrics and accessory chromosomes (Rieger, et 31, 1968; Sayed, _t._l, 1973). Centromere misdivision has been studied in 859mg; (Walters, 1952), maize (Rhoades and Vilkomerson, 1942), rye (Ostergren and Prakken, 1946) and wheat (Sears, 1952; Sanchez-Monge, 1950). The irregularity of meiosis II in HV x HJ.and AHPV precluded both identi- fication of the products of centromere misdivision and analysis of the disposition of these products in the T-I cells and quartets. Multipolar cell division is a spontaneous or induced spindle apparatus abnormality resulting in genome separation during either mitosis or meiosis (Chen, 1975). This inheritable phenomenon, which occurs in both plant and animal tissues, has been described in several grasses (Chen, 1975; Huang, 1975; Dewey, 1974; Maguire, 1974; Sosniklina, 1973; Tai, 1970; Kabarity, 1966; Nielson and Nath, 1961; 80 and Walters, 1958, 1960). In an article on Agropyron cristatum (L.) Gaertn., Tai (1970) proposed that multipolar cell division occurs via genome specific spindle organizers, cell organelles which govern chromosome migration and cytokinesis, and provides an evolutionary mechanism for haploidization in higher plant polyploids. In this investigation, multipolar spindles were first detected in late ana- phase I cells of the hybrids, X Agrohordeum pilosilemma, HV x HJ, and AHPV. Figures 5-G and lO-G show tripolar and quadripolar spindles, respectively. Genome separation is manifested in the quartets of X Agrohordeum pilosilemma (Figure 5 K) by binucleate microspores and of HV x HJ and AHPV by supernumerary cytokinesis producing extra microcells (Figures lO-H, ll-H). The irregular meiotic behavior (Figure 12) originally observed in HV x HJ and AHPV (spindle suppression, reformed nuclei, erratic cytokinesis, and budding) may be explained as the effect of tempera- ture stress on an unbalanced genome incurred under summer field and greenhOuse conditions. Gene-independent asynapsis provoked by high temperatures has been reported in cotton (Douglas and Brown, 1971), wheat (Riley, 1968), Tradescantia (Dowrick, 1957; Sax, 1937) and Uvularia (Dowrick, 1957). Riley (1968) views such asynapsis as a result of altered meiotic timing and states that high-temperature shortens prophase I preventing stable, homologous, zygotene pairing which usually leads to synapsis. Sax (1937) reported that temperature shock in Tradescantia induced asynapsis and budding, conditions that lasted up to several months after the experiment. In wheat and rye 81 grown at 25 C, Bennett, gt al. (1972) observed the termination of meiosis after the first division, dyads with greatly thickened wall and germ pores, and abnormal, persistent tapetal cells. They attributed the meiotic irregularity to temperature—mediated phys- iological failure. Wagenaar (1959) reported spindle suppression during second meiotic division in Hordeum jubatum x Secale cereale hybrids. Irregular nuclear formation, budding, and cell degenera- tion were ascribed to abnormal physiologic conditions created by combining the genomes of these two genera. It is proposed that genome interaction between Hordeum jubatum and Hordeum vulgare in HV x HJ and AHPV presents a similar physio-genetic incompatibility. Temperature stress accentuated the meiotic failure in these plants, but even under ideal growth conditions, asynapsis in HV x HJ and multipolar cell division, centromere misdivision, and the low level of synapsis in AHPV assures sterility, the absence of pollen stain- ability and seed set (Table 15). 82 SUMMARY Cytogenetic investigation of microsporogenesis in Agropyron sericeum, Hordeum jubatum, their spontaneous hybrid, X Agrohordeum pilosilemma, its amphiploid, and the backcross of the amphiploid to Agropyron sericeum elucidated the genome relationships of Agropyron sericeum and Hordeum jubatum. The tetraploid parental species, Agropyron sericeum and Hordeum jgbatum, share a partially homologous genome which affects the pairing relationships evidenced in their hybrids. The genome formulae assigned to these plants are: Agropyron sericeum, A"A”BB; Hordeum jubatum, AAA'A'; X Agrohordeum pilosilemma, AA'A"B; the amphiploid, AAA'A'A”A“BB; and the amphiploid x Agropyron sericeum, AA'A"A”BB. Observed pairing configurations were compatible with the expected maximum pairing configurations predicted under the“ assumption of genetic control of pairing with dosage effects. This is interpreted as further support for the hypothesis that pairing in the hybrids of Hordeum jubatum is controlled by its A genome; one dose of A allows homeologous pairing and two doses of A promotes homolo- gous association. Microsporogenesis in the hybrids, Hordeum vulgare (2X) x Hordeum jubatum and the amphiploid x Hordeum vulgare (4X) was also investigated. Hordeum vulgare x Hordeum jubatum was found to display asynaptic 83 behavior that is believed to represent a physiogenetic incompati- bility. 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