Isoetes ×atruensis (Isoetaceae – Lycopodiopsida), a new interspecific sterile hybrid from Rajasthan of Western India

Interspecific and intraspecific hybrid sterility is a typical and common phenomenon of postzygotic reproductive barrier in rice. This is an indicator of speciation involved in the formation of new species or subspecies, and it significantly hampers the utilization of favorable genes from distant parents for rice improvement. The Oryza genus includes eight species with the same AA genome and is a model plant for studying the nature of hybrid sterility and its relationship with speciation. Hybrid sterility in rice is mostly controlled by nuclear genes, with more than 50 sterility loci genetically identified to date, of which 10 hybrid sterility loci or pairs were cloned and characterized at the molecular level. Comparing the mapping results for all sterility loci reported indicated that some of these loci from different species should be allelic to each other. Further research revealed that interactions between the multiple alleles at the hybrid sterility locus caused various genetic effect. One hypothesis for this important phenomenon is that the hybrid sterility loci are orthologous loci, which existed in ancient ancestors of rice. When one or more ancestors drifted to different continents, genetic divergence occurred because of adaptation, selection, and isolation among them such that various alleles from orthologous loci emerged over evolutionary time; hence, interspecific hybrid sterility would be mainly controlled by a few orthologous loci with different alleles. This hypothesis was tested and supported by the molecular characterization of hybrid sterility loci from S1, S5, Sa, qHMS7, and S27. From this, we may further deduce that both allelic and non-allelic interactions among different loci are the major genetic basis for the interspecific hybrid sterility between O. sativa and its AA genome relatives, and the same is true for intraspecific hybrid sterility in O. sativa. Therefore, it is necessary to raise the near-isogenic lines with various alleles/haplotypes and pyramided different alleles/haplotypes from sterile loci in the same genetic background aiming to study allelic and non-allelic interaction among different hybrid sterility loci in the AA genome species. Furthermore, the pyramiding lines ought to be used as bridge parents to overcome hybrid sterility for rice breeding purposes.

We observed mapped individuals of three species of Botrychium Sw. at a single site in Waterton Lakes National Park, Alberta for six years. Our study showed that the interspecific sterile hybrid, B. xwatertonense W.H. Wagner and its putative parents, B. hesperium Maxon & R.T. Clauson and B. paradoxum W.H. Wagner, are stable, unique genotypic entities. For B. hes- perium and B. Xwatertonense, the degree of sporophore pinnation and the number of trophophore pinnae are plastic characters with much of the observed variation explained by the size of the plants. Although B. Xwatertonense is reported to be sterile, most sporophytes appeared more than 4 cm from other conspecifics, indicating that they are probably not produced vegetatively. Mean height of B. Xwatertonense was greater than that of either of its parents, indicating hybrid vigor. Many members of the Botrychium subg. Botrychium are considered rare (Lellinger, 1985). Nine species in the continental United States are can- didates for listing as threatened or endangered species under the Federal En- dangered Species Act of 1973 (USDI-Fish and Wildlife Service, 1993), and nine species are listed as rare in Canada (Argus and Pryer, 1990). Often rare species occur together with more common species in genus (Wagner and Wagner, 1983). However, these communities are natural common garden experiments and can be a tool to evaluate the discreteness of conge- neric species growing together. Species that maintain their morphological dif- ferences in communities are evidence that these differences are genet- ically fixed. The sterility of interspecific hybrids is additional evidence of ge- netic differentiation between the parent species (Wagner and Wagner, 1983). Members of Botrychium are often difficult to differentiate because the plants are simple and offer few characters for recognizing species (Cody and Britton, 1989). Furthermore, intraspecific variation in leaf shape within and among populations and among years is not well understood (Graham and Wagner, 1991). Keys to the taxa of Botrychium subg. Botrychium include sporophore and trophophore characters (Wagner and Wagner, 1986), but the range of vari- ation among species for these characters is often not known. The number of hybrids found in communities is usually very small (Wagner, 1991). One reason for the paucity of hybrids may be the low fre- quency of intergametophytic fertilizations (Soltis and Soltis, 1986). However, at the B. Xwatertonense type locality studied by Wagner et al. (1984), almost 25% of the plants were sterile hybrids. The reason for this high frequency of

The comparison of electrophoretic isozyme patterns of multimeric enzymes in leaf and pollen extracts from the same individual has been used to distinguish allozymes and isozymes. This is possible because in genetic heterozygotes leaf tissue exhibits both homomers and heteromers whereas pollen shows only homomers. I show that pollen of nearly sterile interspecific plant hybrids contains isozymes that can be examined in the same way. This permits a test to determine whether genes encoding particular isozymes in the parental species have remained allelic; i.e., located at corresponding positions on homologous chromosomes. Such a test is illustrated by examining cytosolic PGI in two classic hybrid combinations in Clarkia. The evidence shows that the coding PGI genes of the parental species are located on different chromosomes (or at different sites on the same chromosome), possibly resulting from reciprocal translocation as proposed by Lewis and Raven. The ability to determine whether genes in different species segregate as alleles or not will likely be useful in studying the extent of gene linkage conservation and as a supplement to RFLP and other DNA-directed maps. Formal genetic analysis is generally restricted to crosses between individuals of a single species because fertile hybrids are necessary to produce an F2 or other segregating progeny. However, for genes encoding multimeric enzymes expressed in pollen and detectable by electrophoretic assays, I show here that tests of allelism can be carried out between species even when their hybrids have very low fertility. This new test is an extention of previous results in which the electrophoretic isozyme pattern of extracts from leaves or other somatic tissues is compared with that of pollen from the same individual (Weeden and Gottlieb 1979). The test depends on the fact that following meiosis the haploid cells that develop into pollen receive one or the other allele at every locus, but not both. Thus if a particular locus is heterozygous for alleles encoding allozymes having different electrophoretic mobilities, leaf tissue exhibits both homomers and heteromers, but only homomers are synthesized in the pollen because any single grain receives only a single allele. For a dimeric enzyme in heterozygous individuals, extracts from a mass of pollen exhibit two homodimers but not the electrophoretically intermediate heterodimer. Its absence is apparent when patterns from the pollen and leaves are compared. In contrast, if the two homodimers are encoded by two different homozygous loci, then both leaf tissue and pollen show the same three-banded pattern. The pollen-leaf comparison has proved useful to test allelism of electrophoretic variants of numerous enzymes within plant species in which formal breeding studies cannot be readily carried out (Crawford 1990). The allelism of variants in different species can also be tested by the pollen-leaf comparison because inviable pollen often characteristic of interspecific hybrids contains many functional enzymes detectable by electrophoresis. In this report, I demonstrate the use of the pollen-leaf test to assess allelism of electrophoretic variants in two classical interspecific hybrids in Clarkia: C. franciscana x amoena and C. amoena x rubicunda (Lewis and Raven 1958a, 1958b). MATERIALS AND METHODS Plants. Interspecific hybrids were produced by crossing Clarkia amoena subsp. huntiana (Jepson) Lewis & Lewis x C. rubicunda (Lindley) Lewis & Lewis and x C. franciscana Lewis & Raven. Clarkia amoena subsp. huntiana was collected in Lake Co., near Manning Creek Bridge on Rt. 175, west of the junction with Rt. 29 (LDG 9025), and in Marin Co., on Lucas Valley Rd., 5.8 miles east of Nicasio Schoolhouse (LDG 714). Clarkia rubicunda was collected in Marin Co., at Ft. Barry (LDG BAR). Clarkia franciscana was represented by two populations, the type locality at The Presidio in San Francisco Co., and a newly discovered population in the foothills above

Interspecific and intergeneric hybridization within the Festuca-Lolium complex is frequently used in forage plant breeding. However, little is known about the natural occurrence and competitiveness of such hybrids. We collected naturally formed hybrids between Festuca apennina, Festuca pratensis, and Lolium perenne in different habitats of Switzerland and the British Isles and studied their origin, the ease of their spontaneous formation, and their competitiveness with parental species. A special attention was paid to the largely sterile triploid forms and their rare sexual progeny. The triploid hybrid F. apennina × F. pratensis proved to be widespread and often highly competitive in Swiss permanent pastures. The majority of these hybrids originated from F. apennina as the seed parent although little or no F. apennina grew nearby. In an experimental setting with ample F. pratensis pollen provided by neighbouring plants, up to 20 % of seeds from open pollinated F. apennina plants were interspecific hybrids; among seeds collected in natural habitats, only 0.35 % were hybrids. At an experimental site at 1 000 m altitude, these triploid hybrids grew much more vigorously than corresponding tetraploid pure F. apennina, confirming their great competitiveness at such altitudes in permanent grasslands. The triploid hybrids were only marginally fertile suggesting that vegetative propagation by rhizomes is the cause of their competitive success in grassland. Moreover, triploid progeny retained the chromosome constitution of their mother plants indicating the possibility of apomixis. Natural triploid F. pratensis × L. perenne hybrids were partially female fertile (a seed set of 0.1 % or less) whereas diploid hybrids did not produce any viable seeds. Progenies of these triploids showed considerable chromosome alterations, such as loss of a genome or recombination due to homoeologous pairing, and only rarely the chromosome constitution of the triploid mother plant was retained. It was concluded that natural triploid interspecific hybrids could expand the range of their progenitor species and might function as bridges transferring genes between them.

Fourteen interspecific hybrids in sexual diploid Allium senescens var. minor× apomictic tetraploid Allium nutans L. crosses, and eight interspecific hybrids in sexual diploid A. senescens var. minor× apomictic hexaploid A. senescens L. crosses were produced. The number of chromosomes was 2n= 24 in interspecific hybrids of diploid × tetraploid, and 2n= 32 in diploid × hexaploid crosses. Triploid and tetraploid interspecific hybrids showed intermediate parental morphological characteristics. Tetraploid interspecific hybrids of A. senescens var. minor×A. senescens crosses formed two groups based on leaf colour and leaf width. Seeds were formed in 11 out of 14 triploid interspecific hybrids under natural conditions. In cytological observations of parthenogenesis, three out of 12 triploid interspecific hybrids and five out of eight interspecific tetraploid hybrids were observed. Parthenogenesis ranged from 26.0% to 86.0% in five tetraploid interspecific hybrids. Non‐parthenogenesis to parthenogenesis segregated in a 3:5 ratio in A. senescens var. minor×A. senescens crosses.

Visualizing Meiotic Chromosome Pairing and Segregation in Interspecific Hybrids of Rice by Genomic in situ Hybridization

Common wheat is an allohexaploid species, derived through endoreduplication of an interspecific triploid hybrid produced from a cross between cultivated tetraploid wheat and the wild diploid relative Aegilops tauschii. Hybrid incompatibilities, including hybrid necrosis, have been observed in triploid wheat hybrids. A limited number of A. tauschii accessions show hybrid lethality in triploid hybrids crossed with tetraploid wheat as a result of developmental arrest at the early seedling stage, which is termed severe growth abortion (SGA). Despite the potential severity of this condition, the genetic mechanisms underlying SGA are not well understood. Here, we conducted comparative analyses of gene expression profiles in crown tissues to characterize developmental arrest in triploid hybrids displaying SGA. A number of defense-related genes were highly up-regulated, whereas many transcription factor genes, such as the KNOTTED1-type homeobox gene, which function in shoot apical meristem (SAM) and leaf primordia, were down-regulated in the crown tissues of SGA plants. Transcript accumulation levels of cell cycle-related genes were also markedly reduced in SGA plants, and no histone H4-expressing cells were observed in the SAM of SGA hybrid plants. Our findings demonstrate that SGA shows unique features among other types of abnormal growth phenotypes, such as type II and III necrosis.

Interspecific hybridization can be used to broaden the genetic base, generate novel species, postulate genetic relationships, and to introgress elite alien genes. However, interspecific hybridizations using wild parents outside the Ipomoea section Batatas are very difficult and have not been much studied. We used an improved hybridization technology to generate three novel interspecific hybrids by crossing Ipomoea batatas (L.) Lam. × I. hederacea Jacq., I. batatas (L.) Lam. × I. muricata (L.) Jacq., and I. batatas (L.) Lam. × I. lonchophylla J.M. Black. The ploidy level of the interspecific hybrids was determined by flow cytometry. The cross, I. batatas × I. hederacea, yielded the first artificial pentaploid Ipomoea hybrid ever. The other two hybrids, I. batatas × I. hederacea and I. batatas × I. muricata were tetraploid. The first two hybrids showed normal storage roots, a significant improvement in the storage roots of currently existing interspecific Ipomoea hybrids. AFLP (Amplified Fragment Length Polymorphism) molecular markers were used to explore the genetic relationship of these three novel interspecific hybrids with three other natural diploid, tetraploid, and hexaploid species of the Ipomoea section Batatas. Cluster analysis of AFLP bands showed that these three new interspecific hybrids were closely related to cultivated sweet potato (I. batatas/L./Lam.), which indicated that these novel hybrids can be used as an interspecific bridge to transfer alien genes from wild to cultivated species.

Background. The use of alien genetic material of bulbous barley Hordeum bulbosum L. to increase the diversity of cultivated barley Hordeum vulgare L. is an important task, since H. bulbosum is characterized by a number of valuable traits. One of the ways to use the genetic potential of bulbous barley is the interspecific hybridization and obtaining fertile introgressive lines of H. vulgare based on interspecific hybrids. The aim of the study was to obtain new introgressive forms of spring barley using interspecific triploid (HvHbHb) and tetraploid (HbHbHvHv) hybrids of cultivated barley with bulbous barley to expand the collection of introgressive lines of H. vulgare. Materials and methods. To create new introgressive forms, diploid barley H. vulgare (2x) cv. ‘Roland’, was crossed with interspecific hybrids H. vulgare cv. ‘Roland’(2x) × H. bulbosum W851 (4х) (HvHbHb), and H. bulbosum A17 (4x) × H. vulgare ‘Borwina’ (4x) (HbHbHvHv). Cultivated barley forms with introgression of the bulbous barley genetic material were selected from the offspring from these crosses; then the selection was continued in two progenies from self-pollination of selected BC1 plants with three alien introgressions. Identification and localization of introgressions was carried out using the method of fluorescent in situ hybridization (GISH and FISH with chromosome-specific markers). Results. The crossing of cultivated barley with triploid and tetraploid interspecific hybrids H. vulgare × H. bulbosum yielded new forms of cultivated barley with recombinant chromosomes, among which two plants with three terminal introgressions of the genetic material of bulbous barley were identified. The first plant, derived from a triploid hybrid, showed introgressions in chromosomes 5HL, 1HL and 3HS. When two homologues with the 5HL introgression of the initial size are combined in the karyotype, the lethality of seedlings is observed in the offspring plants. In the second plant obtained on the basis of a tetraploid hybrid introgression was revealed in chromosomes 5HL, 2HL, and 7HS. In the offspring from self-pollination of this form, the presence of the 2HL introgression of the original size in both homologues led to plant sterility. Forms with a change in size of the introgression in 5HL and 3HS in the offspring of the first plant and with a change in size of the introgression in 2HL in the offspring of the second plant were detected, which indicated that meiotic recombination had occurred in those chromosome regions in BC1 plants. Conclusions A barley plant with the introgression of bulbous barley chromatin into chromosomes 1HL, 5HL, and 3HS of cultivated barley was identified in the offspring from a cross of cultivated barley with a triploid interspecific hybrid H. vulgare × H. bulbosum. In crosses with a tetraploid interspecific hybrid, a barley plant with the introgression into chromosomes 2HL, 5HL, and 7HS was found. On the basis of ‘Roland’ spring barley cultivar, two series of new introgressive forms of H. vulgare with various combinations of recombinant chromosomes have been created.

Diploid (2n = 2x = 16), triploid (2n = 3x = 24), and tetraploid (2n = 4x = 32) interspecific hybrids between alfalfa (Medicago sativa L.) and M. papillosa Boiss. were recovered either from seed (the triploid hybrids) or from ovule–embryo culture (the diploid and tetraploid hybrids). Cytogenetic analysis of diploid interspecific hybrids (with one genome of M. sativa, designated S, and one genome of M. papillosa, designated P), indicated significant genomic affinity, with an average of 7.6 bivalents and 0.8 univalents per pollen mother cell. In contrast, cytogenetic analysis of the triploid interspecific hybrids (with one S genome and two P genomes) indicated little if any genomic affinity between M. sativa and M. papillosa. In 7 of 14 triploid hybrids analyzed no trivalent configurations were observed, and in the other hybrids, trivalent frequency ranged from 0.1 to 0.4 per pollen mother cell. Tetraploid interspecific hybrids with two S and two P genomes had predominantly bivalent pairing. Based on the lack of homology of S and P genomes, the tetraploid hybrids are basically allotetraploids (SSPP). Therefore, backcross progeny from crossing the tetraploid hybrids with tetraploid M. sativa have the genomic constitution SSSP. Univalents and trivalents were observed in first backcross (BC1) progeny, as expected, based on an allotetraploid interpretation. Most of the BC1 progeny were partially or completely male sterile, and female fertility was significantly reduced. Potential uses of homoeologous genomes such as M. papillosa in alfalfa genetic and breeding studies are discussed.Key words: cytogenetics, interspecific hybrids, ovule –embryo culture.

Hieracium s.str. (Asteraceae) is one of the largest angiosperm genera notorious for its taxonomic complexity caused by widespread interspecific hybridization. This process is tightly coupled with polyploidization and apomixis – asexual reproduction by seeds, which has ensured the persistence of otherwise sterile interspecific hybrids. As a result, apomictic polyploid taxa dominate the taxonomic diversity of the genus whilst sexual diploid species are extremely rare and mostly confined to southern Europe. As diploid taxa are assumed to be the parents of apomictic lineages, the discovery of any new diploid species is important for understanding evolutionary processes and diversity patterns in the genus. Here, we describe a new diploid species, Hieracium vranceae, a narrow endemic to the Munţii Vrancei (Vrancea Mountains, Eastern Carpathians, Romania). This taxon with a distinctive morphology and a strong affinity to relic rocky habitats was first collected in 2013. This means that, even in Europe, some regions, like the Eastern Carpathians, are botanically underexplored and might still preserve an undiscovered diversity of plants. Phylogenetic analyses with multiple molecular markers (low‐copy nuclear genes gsh1 and sqs, nuclear ribosomal ETS, four cpDNA loci) applied to all diploid Hieracium species support specific rank for H. vranceae. In contrast, molecular data suggest conspecifity of two pairs of species, H. lucidum/H. cophanense from Sicily and H. naegelianum/H. renatae from the Balkans, which are moreover only weakly differentiated morphologically. Molecular (cpDNA) and cytogenetic (GISH, FISH) analyses furthermore revealed that H. vranceae has been involved as a maternal parent in the origin of an allotriploid apomictic species, H. telekianum, to which H. vranceae contributed one haplome. The other putative parents of H. telekianum are the diploid narrow endemic H. pojoritense and probably H. sparsum s.l. Our data thus stress the importance of interspecific hybridization for an evolutionary shift from sexuality to apomixis in Hieracium. Our findings furthermore highlight the significance of that part of the Carpathians as one of the most important evolutionary/refugial centres of Hieracium and suggest dynamic species ranges at a regional scale allowing physical contact of taxa whose distributional ranges are nowadays completely allopatric.

Triticales (XTriticosecale Wittmack) at three ploidy levels (8x, 6x, 4x, x=7) were crossed with diploid rye (Secale cereale L.) to produce a solitary hypopentaploid hybrid (2n=32), and a number of tetraploid (2n=4x=28) and triploid (2n=3x=21) hybrids. The hybrids exhibited a morphology which was intermediate between the parents. The number of bivalents ranged from 1-7 (4.65 per cell) in hypopentaploid, from 2-12 (7.13 per cell) in tetraploid and from 4-9 (6.84 per cell) in triploid hybrids. In 4x and 3x hybrids, trivalents and quadrivalents were also observed at low frequencies (range 0-1; mean 0.01-0.03 per cell). Chiasmata frequency was highest in triploid hybrids (12.44 per cell), lowest in hypopentaploid (5.37 per cell) and intermediate in tetraploids (10.54 per cell). More than 7(11) were found in 39.7% pollen mother cells (PMC's) in the 4x hybrids and in 5.0% PMCs in 3x hybrids. It is concluded that an increase in the relative proportion of wheat chromosomes in the hybrids had a slight suppression effect on homologous as well as homoeologous pairing of rye chromosomes. Contrary to this, the relative increase in rye complement promoted homoeologous pairing between wheat chromosomes. In triploid hybrids, the chiasmata frequency as well as the 'c' value were the highest, suggesting that in tetraploid hybrids rye chromosomes had a reduced pairing (low frequency of ring bivalents).

Cytological mechanisms of interspecific incrossability and hybrid sterility between Oryza sativa L. and O. alta Swallen

Hybridization and polyploidy in Polystichum (Dryopteridaceae) have contributed to the morphological diversity and taxonomic complexity in this widespread genus. Patterns of reticulate evolution are particularly evident in the Polystichum complex from western North America (W. Wagner, 1973; D. Wagner, 1979), and several sterile interspecific hybrids have been reported (see P. Soltis et al., 1987, for review). As part of a comprehensive molecular analysis of polyploidy, its causes, and genetic consequences, we have studied two additional interspecific hybrids. In this paper we provide electrophoretic documentation of hybridization between P. andersonii and P. munitum and between P. lemmonii and P. munitum. Both hybrids were previously reported based on morphological and cytological evidence (W. Wagner, 1973). Polystichum andersonii occupies lowland coastal forests in British Columbia and southeastern Alaska, montane forests in western Washington and Oregon, and also occurs disjunctly in southeastern British Columbia, northern Idaho, and northwestern Montana. This species is tetraploid (2n= 164; W. Wagner, 1973; D. Wagner, 1979), but its ancestry is uncertain. Warren Wagner (1973) suggested that this species is of autopolyploid origin based on its distinctive leaf morphology and the presence of a vegetative bud on the rachis of the frond. However, cytological data (W. Wagner, 1973) show that P. andersonii contains a chromosome complement homologous with the common diploid P. munitum, suggesting instead an allopolyploid origin for P. andersonii. Anatomical, chromatographic, and electrophoretic data also support this hypothesis (D. Wagner, 1979; P. Soltis et al., unpubl. data). Although P. munitum is a likely candidate for one of the diploid progenitors of P. andersonii, the identity of the other parental species is currently unknown, although D. Wagner (1979) described a specimen from northern British Columbia that seems to fit the predicted morphology of the second progenitor. Polystichum lemmonii (2n = 82) occurs on open, serpentine, montane slopes in northern California, southwestern and central Oregon, and central Washington. Following D. Wagner (1979), P. lemmonii is herein considered distinct from the South American P. mohrioides.

A mixed model approach for evaluating yield improvements in interspecific hybrids of shrub willow, a dedicated bioenergy crop