CROSSING RELATIONSHIPS AMONG DIPLOID SPECIES OF THE SOLANUM NIGRUM COMPLEX IN NORTH AMERICA

Crosses were made in all combinations of the six diploid species of the Solarium nigrum complex that occur in North America. Some interspecific pollinations failed to yield viable seed; successful crosses gave rise to moderately to highly sterile F₁ hybrids. Results of interspecific crosses suggested phylogenetic relationships that were not completely in accord with those suggested by morphology. Interspecific crosses also gave varied results. All interpopulational crosses within S. interius and S. sarachoides produced fully fertile hybrids. In contrast, hybrids within S. americanum and S. douglasii varied from fully fertile to almost completely sterile. Populations of S. pseudogracile could be divided into two groups which are geographically separated but not morphologically differentiated. Fully fertile hybrids resulted from crosses within a group, whereas crosses between groups gave hybrids with reduced fertility. Four crossing groups were observed within 5. nodiflorum; three of the parental groups are sympatric and are morphologically differentiated. Although hybrid sterility in interspecific crosses is sometimes used to support delimitation of species, the presence of sterility in intraspecific crosses suggests that such an interpretation is unwarranted for the S. nigrum complex. Hybrid sterility, therefore, is not considered to have special taxonomic significance in this complex.     

Synthesis of Ogura male sterile rapeseed (Brassica napus L.) with cold tolerance by protoplast fusion and effects of atrazine resistance on seed yield

Twenty-one cold-tolerant, male sterile Brassica napus somatic hybrids were produced by protoplast fusion. The fusion partners were a coldsensitive, Ogura cytoplasmic male sterile cauliflower inbred (B. oleracea var. botrytis inbred NY7642A) and a cold-tolerant, fertile canola-type B. rapa cv. Candle. Hybridity was confirmed by morphology, isozyme expression, flow cytometry, and DNA hybridization. Organellar analyses revealed a very strong bias for Brassica over Raphanus chloroplasts. Cold tolerance was confirmed by cold chamber studies and chloroplast DNA analyses. Good female fertility with 21.4 ± 3.1 seeds/pod was observed in the field using natural pollination vectors. Total seed yield was significantly greater for the atrazine-sensitive somatic hybrids produced in this study than for atrazine-resistant isolines.Abbreviations CMS cytoplasmic male sterility - IA iodoacetate - cpDNA chloroplast DNA     

Developmental and cytogenetic analyses of pollen sterility in Valeriana scandens L.

Valeriana scandens presents perfect and pistillate flowers, the latter with sterile anthers. The species is composed of two varieties with different ploidy; V. scandens var. scandens (2n = 28) and V. scandens var. candolleana (2n = 56), both of which occur in RS, Brazil. Crosses between these varieties may give rise to hybrids with pollen sterility. In this study, we analyzed the microsporogenesis and microgametogenesis of sterile and fertile anthers, and also investigate whether pollen sterility is caused by an irregular meiotic process. Developmental analysis using light microscopy and scanning electron microscopy showed that sterile anthers develop similarly to fertile anthers until the end of meiosis. After this stage, sterile tetrads do not separate as a consequence of exine fusion between adjacent microspores, which is similar to sterile pollen of Brassica ms-cdl1 mutants. In addition, vacuolated immature pollen grains degenerate after separation. The cytogenetic analysis of the microspore mother cell (MMC) showed that the diploid population of V. scandens var. scandens (2n = 28) has pollen sterility that is not caused by a cytogenetic disturbance. The MMCs analyzed from prophase I to tetrad stage showed a regular meiotic process, indicating the phenotype of V. scandens sterile pollen is a postmeiotic process formed by fusion of exine between opposite microspores.     

Rapid Male-Specific Regulatory Divergence and Down Regulation of Spermatogenesis Genes in Drosophila Species Hybrids

In most crosses between closely related species of Drosophila, the male hybrids are sterile and show postmeiotic abnormalities. A series of gene expression studies using genomic approaches have found significant down regulation of postmeiotic spermatogenesis genes in sterile male hybrids. These results have led some to suggest a direct relationship between down regulation in gene expression and hybrid sterility. An alternative explanation to a cause-and-effect relationship between misregulation of gene expression and male sterility is rapid divergence of male sex regulatory elements leading to incompatible interactions in an interspecies hybrid genome. To test the effect of regulatory divergence in spermatogenesis gene expression, we isolated 35 fertile D. simulans strains with D. mauritiana introgressions in either the X, second or third chromosome. We analyzed gene expression in these fertile hybrid strains for a subset of spermatogenesis genes previously reported as significantly under expressed in sterile hybrids relative to D. simulans. We found that fertile autosomal introgressions can cause levels of gene down regulation similar to that of sterile hybrids. We also found that X chromosome heterospecific introgressions cause significantly less gene down regulation than autosomal introgressions. Our results provide evidence that rapid male sex gene regulatory divergence can explain misexpression of spermatogenesis genes in hybrids.     

Chromosomal location of a pollen fertility-restoring gene, Rf, for CMS in Japanese bunching onion (Allium fistulosum L.) possessing the cytoplasm of A. galanthum Kar. et Kir. revealed by genomic in situ hybridization

In a previous study, we developed cytoplasmic male sterile lines of Allium fistulosum possessing the cytoplasm of A. galanthum, a wild species, by continuous backcrossing. Furthermore, we reported the presence of a pollen fertility-restoring gene (Rf) for cytoplasmic male sterility (CMS) in A. fistulosum from segregation of pollen fertility of backcross progenies. In the present study, genomic in situ hybridization (GISH), using genomic DNA of A. galanthum as the probe DNA and that of A. fistulosum as the blocking DNA, was applied to F₁ hybrids between both species and backcross progenies to determine the chromosomal location of the Rf locus. By means of GISH, eight chromosomes from A. galanthum were clearly discriminated from those of A. fistulosum in the F₁ hybrids, and chromosome substitution process through continuous backcrossing was visualized. Interestingly, the chromosome region from A. galanthum, specific to male fertile plants, was detected in one chromosome of BC₄ to BC₇ generations. Based on the karyotype analysis of the male fertile plants, the chromosome was identified as the 5F chromosome. Our results confirm that the Rf locus is located on the 5F chromosome of the male fertile plants. This is the first report that identified the chromosomal location of the pollen fertility-restoring gene in A. fistulosum.     

A study of morphology,cytology and sterility in interspecific hybrids and amphidiploids of Nicotiana knightiana X N. umbratica

Summary Interspecific hybrids and amphidiploids of Nicotiana knightiana Goodspeed (n= 12)x N. umbratica Burbidge (n = 23) resembled either parent in some characters and were intermediate in other characters. The F₁ hybrids (2n = 35) showed mostly univalents during meiosis, while the amphidiploids (2n = 70) formed bivalents almost regularly. The former were completely sterile and the latter fully male fertile but predominantly female sterile. This female sterility was due to disintegration of the embryo sacs leading to collapsed ovules. The few fertile ovules, however, showed normal development of embryo sac and embryo. The occurrence of fertile and sterile ovules was believed to be due to segregation of the genes governing sterility.     

SPERM PRODUCTION AND STERILITY IN HYBRIDS BETWEEN TWO SUBSPECIES OF DROSOPHILA PSEUDOOBSCURA

Subspecies of Drosophila pseudoobscura, one occurring in the United States and the other in Bogota, Columbia, exhibit Haldane's Rule in one direction of the cross. Additionally, D. pseudoobscura produces two sperm types: short, sterile sperm and long, fertile, sperm. Here I examine the relationship between the production of short and long sperm and hybrid sterility. Fertile and sterile hybrid males produce a greater proportion of short sperm compared to parental males with sterile hybrids producing mainly short, immotile sperm. Sperm transfer and storage patterns were similar between fertile hybrid and parental strains; and unexpectedly, short, immotile sperm from sterile hybrids were stored. These findings raise the question of whether different genetic mechanisms disrupt both sperm heteromorphic production and sperm motility and whether this indicates that females exert some control over sperm storage.     

SYNTHETIC AGROPYRON-ELYMUS HYBRIDS. I. ELYMUS CANADENSIS × AGROPYRON SUBSECUNDUM

Twenty-four seeds were formed in 34 hand-emasculated E. canadensis florets exposed to A. subsecundum pollen. Fifteen of the 24 seeds germinated, and 12 plants were raised to maturity. Each of the 12 plants proved to be a hybrid. The hybrids were morphologically intermediate between the parents, although they favored A. subsecundum in general appearance. The parent plants were meiotically regular and behaved cytologically as strict allotetraploids, 2n = 28. Fourteen bivalents were formed in 12.6% of the hybrid cells interpreted at metaphase I. All other hybrid cells contained various combinations of univalents, bivalents, and multivalents. Mean chromosome associations of 0.22 univalents, 11.70 bivalents, 0.11 trivalente, 0.23 quadrivalents, 0.01 pentavalents, and 0.55 hexavalents were observed in 135 hybrid cells. All hybrid pollen examined was shriveled and non-staining. Five hybrids produced eight seeds, and seven hybrids were completely sterile. The ready cross-compatibility of E. canadensis and A. subsecundum and the relatively good chromosome pairing in their hybrids suggest a much closer relationship between the parent species than is implied by the prevailing taxonomic treatment. Structural rearrangements appear to be responsible for the relatively few differences between E. canadensis and A. subsecundum genomes and for the sterility of the hybrids. Cytological data from this and other investigations indicate a close relationship between many self-fertilizing Agropyron, Elymus, and Sitanion species. It is postulated that this composite of self-fertilizing Agropyron, Elymus, and Sitanion species originated by hybridization between A. spicatum and an unidentified Hordeum species.     

Independent segregation of the plastid genome and cytoplasmic male sterility in Petunia somatic hybrids

Summary The chloroplast genomes of three sets of Petunia somatic hybrids were analyzed to examine the relationship between chloroplast DNA (cpDNA) composition and cytoplasmic male sterility (CMS). Chloroplast genomes of somatic hybrid plants were identified either by restriction and electrophoresis of purified cpDNAs or by hybridization of total DNA digests with cloned cpDNA probes that distinguish the parental genomes.The chloroplast genomes of a set of seven somatic hybrids derived from the fusion of Petunia CMS line 2423 and fertile line 3699 were analyzed. All seven plants were fertile, and all exhibited the cpDNA restriction pattern of the sterile cytoplasm. Similarly, four fertile somatic hybrids derived from the fusion of CMS line 3688 and fertile line 3677 were found to contain the CMS chloroplast genome. The cpDNA compositions of four fertile and two sterile somatic hybrids derived from the fusion of CMS line 3688 and fertile line 3704 were determined by restriction analysis of purified cpDNAs; all six plants exhibited the cpDNA restriction pattern of line 3704. Thus the CMS phenotype segregates independently of the chloroplast genome in Petunia somatic hybrids, indicating that CMS in Petunia is not specified by the chloroplast genome.     

HYBRIDIZATION IN THE GENUS GLYCINE SUBGENUS GLYCINE WILLD. (LEGUMINOSAE,PAPILIONOIDEAE)

The genus Glycine is composed of two subgenera, Glycine and Soja. Soja includes the cultivated soybean, G. max, and its wild annual counterpart G. soja, while Glycine includes seven wild perennial species. Hybridization was carried out within and between wild perennial species of the subgenus Glycine. The success rate (pods set/flowers crossed) was 11% for intraspecific and 8% for interspecific crosses. A total of 220 F₁ hybrids was examined morphologically and cytologically where possible. Hybrids within G. canescens (2n = 40) and G. latifolia (2n = 40) were fertile as expected. Glycine clandestina (2n = 40) was morphologically separable into at least three groups, which produced fertile hybrids within each group. One cross between two groups gave vegetatively vigorous but sterile hybrids. The majority of crosses within G. tabacina (2n = 80) were fertile, except that extremely narrow-leaved forms gave sterile hybrids in combination with more usual forms. Sterility was also encountered in G. tomentella when aneuploids (2n = 78) from New South Wales, Australia, were crossed with tetraploids (2n = 80) from either Queensland, Australia, or Taiwan; crosses between the latter two populations resulted in seedling lethality. Cytological behavior of sterile hybrids followed a similar pattern, whether at the diploid or tetraploid level. The frequency of chromosome pairing was approximately half that expected if genomes showed full pairing homology. Bivalent disjunction at anaphase I was usually followed by precocious division of the majority of univalents. Telophase I and II were characterized by lagging chromosomes and micronuclei, so that resulting pollen was misshapen and sterile. Chromosome pairing data from sterile intraspecific hybrids at the tetraploid level may indicate a polyphyletic origin of tetraploids, whereby different diploid populations were involved in their formation. Similarly, chromosome pairing in sterile intraspecific diploid hybrids may indicate that the various diploid groups arose independently of one another. Both 40- and 80-chromosome forms are fully diploidized, however, and if they are of ancient origin, divergence since that time could have resulted in the chromosomal differentiation which becomes apparent when intraspecific hybridization is effected. Diploid (2n = 40) interspecific hybrids G. falcata × G. canescens, and G. falcata × G. tomentella grew poorly and did not reach flowering stage. Diploid (2n = 40) crosses between G. latifolia and G. tomentella produced inviable seedlings. Tetraploid (2n = 80) hybrids between G. tomentella and G. tabacina were vegetatively vigorous but sterile owing to low chromosome pairing at meiosis, indicating little pairing homology between the two species. Diploid hybrids between G. canescens and G. clandestina, however, showed almost complete chromosome pairing at diakinesis and partial fertility. Although morphologically distinct, these two species have not diverged sufficiently to prevent hybridization and possible gene exchange through recombination. Self compatibility, perennial growth habit, and geographic isolation have favored divergence among Glycine populations to the point that gene exchange appears no longer possible in many cases. Internal isolating mechanisms have been shown to operate at various levels of plant development from hybrid lethality at seedling stage, to failure of seed-set in sterile but vegetatively vigorous hybrids.     

Several nuclear genes control both male sterility and mitochondrial protein synthesis in Nicotiana sylvestris protoclones

Summary Male sterile plants appeared in the progeny of three fertile plants obtained after one cycle of protoplast culture from a fertile botanical line and two androgenetic lines ofNicotiana sylvestris. These plants showed the same foliar and floral abnormalities as the cytoplasmic male sterile (cms) mitochondrial variants obtained after two cycles of culture. We show that male sterility in these plants is controlled by three independent nuclear genes,ms1, ms2 andms3, while no changes can be seen in the mitochondrial genome. However, differences were found between thein organello mitochondrial protein synthesis patterns of male sterile and parent plants. Two reproducible changes were observed: the presence of a new 20 kDa polypeptide and the absence of a 40 kDa one. Such variations were described previously in mitochondrial protein synthesis patterns of the cms lines. Fertile hybrids of male sterile plants showed normal synthesis patterns. The male sterile plants are thus mutated in nuclear genes involved in changes observed in mitochondrial protein synthesis patterns.     

New chromosome counts in the genus Centaurea (Asteraceae) from Turkey

Twenty‐two chromosome counts are reported in 16 species, four subspecies and two varieties of the genus Centaurea. These are mostly Turkish local endemics of section Cheirolepis, a complicated group from the Eastern clade of the Jacea group. Twenty‐one reports are new. Prevalence of the basic chromosome number x = 9 among the eastern sections of the Jacea group is confirmed. © 2009 The Linnean Society of London, Botanical Journal of the Linnean Society, 2009, 159 , 280–286.     

Cytological and Reproductive Studies of Japanese Diplazium (Woodsiaceae; Pteridophyta). III. The Cytological Complexity of Species Groups with Simply Pinnate to Bipinnatifid Leaves

Diplazium with simply pinnate or bipinnatifid leaves. Diplazium wichurae var. wichurae, D. wichurae var. amabile, D. okudairae, and D. pin-faense are sexual diploids (2n=82; n=41II); D.× kidoi and D. × okudairaeoides are sterile diploids (2n= 82; meiosis irregular); D. donianum var. donianum is an apomictic triploid (2n=123; n=123II); D. donianum var. aphanoneuron is a sterile triploid (2n=123; meiosis irregular); D. crassiusculum, D. cavalerianum, D. incomptum, D. longicarpum, and D. pullingeri are sexual tetraploids (2n= 164; n=82II); and D. lobatum is an apomictic tetraploid (2n=164; n=164II). This is the first report of the chromosome numbers of D. lobatum, D. crassiusculum, D. incomptum, D. longicarpum, D. pullingeri, and D. × okudairaeoides, as well as the mitotic chromosome numbers of D. wichurae var. amabile, D. okudairae, D. pinfaense, and D. ×kidoi. The mitotic chromosome number, meiotic behavior, sterility, and allozyme analysis confirm that D. × kidoi and D. × okudairaeoides are hybrids between D. pin-faense and D. wichurae var. wichurae and D. okudairae and D. wichurae var. wichurae, respectively. Diplazium with simply pinnate to bipinnatifid leaves displayed an extraordinary cytological and reproductive complexity: a polyploidal series with diploids to hexaploids, sexual and apomictic reproduction, and natural hybridization. Received 14 August 2001/ Accepted in revised form 1 October 2001     

Contribution to the cytotaxonomy of Oryzopsis (Poaceae)

Meiotic studies were performed in twelve populations of four Oryzopsis species (O. pubiflora, O. lateralis, O. holciformis var. longiglomis and O. barbellata) to obtain data on the ploidy level and cytological evolution of the genus. The chromosome number 2n=2x=24 was revealed in all the species and populations studied. The present and other studies show the occurrence of two basic chromosome numbers in the genus, i.e. x=11 and x=12. Although Oryzopsis species and populations studied are diploid and are expected to form only bivalents in metaphase of meiosis‐I, quadrivalents were observed in O. pubiflora and O. lateralis, possibly due to the occurrence of heterozygote translocations. B‐chromosomes of 0–2 were observed in all species and populations studied. This is the first report of the occurrence of B‐chromosomes in the genus Oryzopsis. Several meiocytes showed the presence of double chromosome number in O. lateralis, and multipolar cells were observed in populations of O. barbellata, O. lateralis and O. holciformis var. longiglomis. The occurrence of large pollen grains (possibly unreduced) was observed along with smaller (normal) pollen grains in these species. Significant differences observed in chiasma frequency and distribution among studied species may be of use in species delimitation. The Kakan population differed significantly from the other populations of O. lateralis in meiotic characteristics. If such cytological differences are accompanied by morphological variation (under investigation), we may consider this population as a new variety or subspecies.     

Synthesis of male sterile,triazine-resistant Brassica napus by somatic hybridization between cytoplasmic male sterile B. oleracea and atrazine-resistant B. campestris

Summary Fusion of leaf protoplasts from an inbred line of Brassica oleracea ssp. botrytis (cauliflower, n=9) carrying the Ogura (R1) male sterile cytoplasm with hypocotyl protoplasts of B. campestris ssp. oleifera (cv Candle, n=10) carrying an atrazine-resistant (ATR) cytoplasm resulted in the production of synthetic B. napus (n=19). Thirty-four somatic hybrids were produced; they were characterized for morphology, phosphoglucose isomerase isoenzymes, ribosomal DNA hybridization patterns, chromosome numbers, and organelle composition. All somatic hybrids carried atrazine-resistant chloroplasts derived from B. campestris. The mitochondrial genomes in 19 hybrids were examined by restriction endonuclease and Southern blot analyses. Twelve of the 19 hybrids contained mitochondria showing novel DNA restriction patterns; of these 12 hybrids, 5 were male sterile and 7 were male fertile. The remaining hybrids contained mitochondrial DNA that was identical to that of the ATR parent and all were male fertile.     

Allelic interaction of F1 pollen sterility loci and abnormal chromosome behaviour caused pollen sterility in intersubspecific autotetraploid rice hybrids

The intersubspecific hybrids of autotetraploid rice has many features that increase rice yield, but lower seed set is a major hindrance in its utilization. Pollen sterility is one of the most important factors which cause intersubspecific hybrid sterility. The hybrids with greater variation in seed set were used to study how the F(1) pollen sterile loci (S-a, S-b, and S-c) interact with each other and how abnormal chromosome behaviour and allelic interaction of F(1) sterility loci affect pollen fertility and seed set of intersubspecific autotetraploid rice hybrids. The results showed that interaction between pollen sterility loci have significant effects on the pollen fertility of autotetraploid hybrids, and pollen fertility further decreased with an increase in the allelic interaction of F(1) pollen sterility loci. Abnormal ultra-structure and microtubule distribution patterns during pollen mother cell (PMC) meiosis were found in the hybrids with low pollen fertility in interphase and leptotene, suggesting that the effect-time of pollen sterility loci interaction was very early. There were highly significant differences in the number of quadrivalents and bivalents, and in chromosome configuration among all the hybrids, and quadrivalents decreased with an increase in the seed set of autotetraploid hybrids. Many different kinds of chromosomal abnormalities, such as chromosome straggling, chromosome lagging, asynchrony of chromosome disjunction, and tri-fission were found during the various developmental stages of PMC meiosis. All these abnormalities were significantly higher in sterile hybrids than in fertile hybrids, suggesting that pollen sterility gene interactions tend to increase the chromosomal abnormalities which cause the partial abortion of male gametes and leads to the decline in the seed set of the autotetraploid rice hybrids.     

Bisexual Hybrid Sterility in DROSOPHILA MELANOGASTER

A new type of hybrid sterility was investigated in D. melanogaster . Matings between strain 27 males from Para Wirra, South Australia, and Canton-S females produce 70–80% fully sterile male and female progeny. Strain 27 males produce sterile progeny when crossed to females of other geographic origins, but produce fertile progeny when crossed to a second sympatric strain. The sterility is avoided by lower rearing temperatures. Heat shock and tetracycline produce no improvement in the fertility of the hybrids. Normal flies produce sterile progeny when injected with, or fed, homogenates of sterile flies. A combination of maternal and paternal factors may interact to produce sterile hybrids by inhibiting gonad development.     

Cytoplasmic male sterility in hybrids of sterile wild beet (Beta vulgaris ssp. maritima) and O-type fertile sugar beet (Beta vulgaris L.): molecular analysis of mitochondrial and nuclear genomes

The organization of the mitochondrial genome of B3, B4 and B5generations of hybrids created by backcrossing sterile wild beet Betamaritima with a fertile O-type sugar beet line was studied usingrestriction fragment length polymorphism (RFLP) analysis. Random amplifiedpolymorphic DNA (RAPD) analysis was used to study restoration of the fertile(O-type) sugar beet genotype in hybrids after multiple backcrossings.Restriction of mtDNAs from the cytoplasm of B. maritimaandhybrids revealed BamHI, EcoRI andXhoI restriction patterns different from those for sterileand fertile sugar beet lines. The most conspicuous feature of our accession ofsterile wild beet mtDNA was the absence of the 10.7-kbEcoRI fragment detected in the cytoplasm of S-type sterileB. maritima and sugar beet. The hybridization of digestedmtDNAs with coxII, atpA andatp6 homologous probes revealed alterations within thesegene loci that distinguished wild beet and hybrids from sugar beets.Characteristic hybridization profiles for the wild beet and B3, B4 and B5hybrids were observed for all probes regardless of the restrictase used todigest mtDNA. Notable changes in atpA andatp6 genes resulted when probes that comprised the5flanking sequences of these genes and a small part of the coding sequences wereused. RFLP analysis of the sterile B. maritimamitochondrial genome further supported the unique character of this source ofwild beet sterility. The genotypic differences between hybrids and parentalaccessions were determined by scoring PCR-RAPD reaction products for nineselected primers. The diversity of the B. maritimagenotyperesulted in a lower genetic similarity index in comparison with hybrids,sterileand fertile lines of sugar beet. The dendrogram obtained after cluster analysisdistinguished hybrids as a group that differed from wild beet and themaintainersugar beet line used for backcrossing. These results may indicate incompleterestoration of the fertile sugar beet genotype in hybrids.     

Polyploidy and sterility in relation to sex in Dioscorea alata L. (Dioscoreaceae)

Chromosome numbers and fertility studies of 73 male and 30 female flowering germplasm accessions of Dioscorea alata L. were carried out. All males were tetraploids showing the same chromosome number (n=20 or 2n=40) and were pollen fertile (10.9–96.2%), most of them being highly fertile. Among the female the majority were higher ploids (hexa-and octoploids; 2n=60 and 80) and sexually completely sterile. There were only two tetraploid female accessions which were sexually fertile. Pollination studies revealed that seed sterility in D. alata was due to female sterility associated with the occurrence of higher levels of ploidy. The female sex-limited occurrence of higher polyploidy and sterility observed in D. alata is a curious situation among dioecious higher plants.