Development of Specific Thinopyrum Cytogenetic Markers for Wheat-Wheatgrass Hybrids Using Sequencing and qPCR Data

The cytogenetic study of wide hybrids of wheat has both practical and fundamental values. Partial wheat-wheatgrass hybrids (WWGHs) are interesting as a breeding bridge to confer valuable genes to wheat genome, as well as a model object that contains related genomes of Triticeae. The development of cytogenetic markers is a process that requires long and laborious fluorescence in situ hybridization (FISH) testing of various probes before a suitable probe is found. In this study, we aimed to find an approach that allows to facilitate this process. Based on the data sequencing of Thinopyrum ponticum, we selected six tandem repeat (TR) clusters using RepeatExplorer2 pipeline and designed primers for each of them. We estimated the found TRs’ abundance in the genomes of Triticum aestivum, Thinopyrum ponticum, Thinopyrum intermedium and four different WWGH accessions using real-time qPCR, and localized them on the chromosomes of the studied WWGHs using fluorescence in situ hybridization. As a result, we obtained three tandem repeat cytogenetic markers that specifically labeled wheatgrass chromosomes in the presence of bread wheat chromosomes. Moreover, we designed and tested primers for these repeats, and demonstrated that they can be used as qPCR markers for quick and cheap monitoring of the presence of certain chromosomes of wheatgrass in breeding programs.

Diversity of Productivity Traits in Hybrid Lines of Aegilops L. with Triticum aestivum L.

Variation in productivity traits and their correlations were studied in 16 hybrid lines of <em>Aegilops kotschyi</em> Boiss. and <em>A. variabilis</em> Eig. with <em>Triticum aestivum</em> L. and their parental components. In comparison to the wheat cultivars, most of the hybrid lines exhibited higher tillering, whereas the length of the main tiller and spike rachis were similar to the values for the wheat cultivars. The values of other traits, such as basal stem diameter, main spike density, fertility, and a 1,000-grain weight in hybrid lines were varied. In some lines, the value of these traits was higher or similar to that of wheat cultivars, whereas in others it was lower. Among the hybrids, [(<em>A. kotschyi</em> × ‘Rusałka’) × ‘Begra’] × ‘Turnia’ was distinguished in having the greatest basal stem diameter, longest spikes, highest fertility, and greatest 1,000-grain weight. The hybrid lines exhibited greater variation in the analyzed traits than did the wheat cultivars. In the hybrid lines, the main tiller length and basal stem diameter were positively correlated with fertility and 1,000-grain weight, whereas in wheat cultivars there were negative correlations for these traits. The results confirmed that wide hybrids can be used to eliminate the negative correlations between productivity traits in wheat. Analysis of clusters in terms of productivity traits provided information on the similarity and diversity of hybrid lines, which may prove useful in their further selection.

Reproductive Behavior of Diploid and Allotetraploid Rhododendron L. ‘Fragrant Affinity’

Wide hybridization can lead to recombination of diverse traits and creation of unique phenotypes, but the resultant hybrids are often sterile as is the case with the intersubgeneric hybrid Rhododendron L. ‘Fragrant Affinity’. Sterility in wide hybrids can either be genic or chromosomal; the latter may be overcome by induction of polyploidy, which can restore chromosome homology and fertility. Cytologic studies of ‘Fragrant Affinity’ appear to support the presence of bridges between bivalents in metaphase I and laggard chromosomes in anaphase I. In the current study, an allotetraploid form of R. ‘Fragrant Affinity’ was developed using oryzalin (4-(dipropylamino)-3, 5-dinitro-benzenesulfonamide) as a mitotic inhibitor and chromosomal doubling agent. Genome sizes (2C) were determined using flow cytometry and found to be ≈1.6 and 3.2 pg for the diploid and allotetraploid, respectively. Pollen viability, determined by staining and germination tests, was 4% and 0%, respectively, for the diploid and 68% and 45%, respectively, for the allotetraploid. No seeds were produced when the diploid R. ‘Fragrant Affinity’ was used as a pistillate parent when pollinated with pollen from viable diploid and tetraploid parents. The allotetraploid produced viable seeds and seedlings when pollinated with pollen from either diploid or tetraploid parents, including self-pollination, demonstrating restored male and female fertility.

Genetic Diversity Estimates for the Genus Hydrangea and Development of a Molecular Key Based on SSR

Using 14 codominant microsatellite markers that amplify loci across 14 different Hydrangea L. species, we analyzed gene diversity and genetic similarity within Hydrangea. Samples also included Dichroa Lour., Platycrater Sieb. and Zucc., and Schizophragma Sieb. and Zucc. genera to establish their relatedness to Hydrangea species since previous work suggests they may be closely related. Our results support the close affiliation between Macrophyllae E.M. McClint. and Petalanthe (Maxim.) Rehder subsections and their separation from the other Hydrangea species. Most of the Hydrangea species analyzed cluster within their designated sections and subsections; however, genetic distance between species within each subsection varied considerably. Our data suggest that morphological analyses which labeled H. serrata (Thunb.) Ser. as a subspecies of H. macrophylla (Thunb. Ex J.A. Murr.) Ser. are probably more accurate than recent genome size data suggesting H. macrophylla ssp. macrophylla (Thunb.) Ser. and H. macrophylla ssp. serrata (Thunb.) Makino are separate species. Gene diversity estimates indicate that 64.7% of the total diversity is due to differences between species and 49.7% of the overall variation is due to differences between subsections. Low diversity suggests a lack of gene flow between species and subsections and underscores the difficulty in making wide hybrids. Since only 35.3% of the genetic variation is common to all species, unique alleles were used to develop a molecular key for unambiguous species identification and interspecific hybrid verification. Genetic similarity estimates for all 85 samples suggests a roadmap for introgressing horticulturally important traits from different Hydrangea species.

REPRODUCTIVE BEHAVIOR OF DIPLOID AND ALLOTETRAPLOID AZALEODENDRONS

Wide hybridization can potentially lead to the combination of diverse traits, but these hybrids are often sterile as is the case with the inter-subgeneric hybrid Rhododendron `Fragrant Affinity'. Induction of polyploidy can restore chromosome homology and fertility in wide hybrids. In this study we successfully developed an allopolyploid form of R. `Fragrant Affinity' using oryzalin as a mitotic inhibitor and chromosome doubling agent. Approximate genome size (2C), determined using flow cytometry, was 1.6 pg for the diploid and 3.2 pg for the allotetraploid. Pollen viability, determined by staining and germination tests, was 4% and 0%, respectively for the diploid and 68% and 45%, respectively for the allotetraploid. No seeds were produced when the diploid R. `Fragrant Affinity' was crossed with pollen from viable diploid and tetraploid parents. The allotetraploid produced viable seeds and seedlings when crossed with viable pollen from either diploid or tetraploid parents, including self pollination, demonstrating restored fertility. Additional crosses were successfully completed using the allotetraploid as part of an ongoing breeding program to develop new fragrant, cold hardy, evergreen rhododendron.