Variability of pollen grains quality in oat amphiploids and their parental species

The reproductive potential of oat species and their hybrid progeny (amphiploids) was evaluated during three vegetation seasons. Morphotypes and viability of pollen grains were described by means of correlations and regression, while relationships between taxa were analysed with the use of numerical taxonomy methods. Size of pollen grains varied between the growing seasons, but the relations between the taxa appeared to be stable. Viability of pollen grains was environmentally modified and showed no correlation with pollen length. In an ordination space, amphiploids were discriminated from parental species. In both group of plants, a positive correlation between the pollen size and the level of ploidy was maintained; however, along a regression line, amphiploids were located among species with a high level of ploidy and were extreme units deviating from the regression line. Developmental anomalies of pollen grains had a low frequency, with the formation of micrograins being the most common event. Such a pattern of development can prove that some pollen grains were chromosomally unbalanced. Anomalous morphotypes of pollen were more common in hybrid types than in species, including pollens with many poruses, which were found only in amphiploids. Frequencies of multiporate grains and micropollens were strongly correlated. In an ordination space, monoporate types (species) were discriminated from multiporate types (amphiploids). In general, the high level of pollen viability in amphiploids can prove their genomic stabilisation through many generations of their reproduction.


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Research on the development and variability of pollen grains is important for understanding 3 9 the mating system in plants, including their common hybrid swarms. The variability of the 4 0 mating systems influences the variability and the differentiation processes observed in the 4 1 populations of wild and cultivated plants (Grant 1981;Richards 1986). In cereals, the most gametes. It may also be caused by disturbances in the formation of cell walls during 1 8 3 subsequent cytokineses in the microsporogenesis process, which was confirmed by the 1 8 4 irregular shape and depressions in the surface of some large pollen grains (Fig. 3c,g,h). However, the frequency of large pollen grains was low and did not exceed 1%. In some cases, 1 8 6 the irregular shape of the pollen grains suggests an asymmetric division that led to the 1 8 7 formation of micropollens (Fig. 3j). In the amphiploid e/sa, a single case of a huge, highly 1 8 8 elongated pollen was recorded (Fig. 3k). It was estimated to be about 4-5 times larger than a 1 8 9 normally developed grain, and had seven poruses, including two connected. Anomalous 1 9 0 pollen grains were observed in all amphiploids, but also in one hexaploid and three tetraploid 1 9 1 species (Table 4). However, they constituted a small percentage of the total pollen pool 1 9 2 analysed. The highest amount of pollen of irregular shape was observed in octoploids, but 1 9 3 their frequency was only 0.06% and 0.05% in e/sa and b/sn, respectively. Micrograins 1 9 4 appeared to be most common among other anomalies. Their average frequency was in 1 9 5 amphiploids 0.51%, while in the parental species it was distinctly lower 0.21%. As shown in Fig. 3, irregularly shaped pollen grains often had more than one porus. The  However, in all tested amphiploids, also in their normal pollen grains, two poruses were 2 0 1 observed sporadically. This phenomenon concerned only amphiploids and was absent in the 2 0 2 parental species. The position of the poruses in relation to each other was different. poruses. An extreme case of the multiporate grain was noted in this amphiploid (Fig. 3k).

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Pollen with three and four poruses were not observed in the remaining four amphiploids.  Table 5 shows that the frequency of pollen grains with an increased number of poruses was 2 1 0 low. b/sn showed the highest frequency of pollen grains with many poruses in comparison to 2 1 1 other amphiploids. Pearson's correlation coefficient matrix was calculated. Table 6 shows that the viability of 2 1 5 pollen grains was positively correlated only with pollen grains having one porus. There were 2 1 6 also negative correlation coefficients between pollen viability and all other characteristics.

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This means that taxa which produce micropollens, non-reduced pollen grains, irregular pollen 2 1 8 and multiporate grains will show a lower ability to germinate pollen grains. Disturbances in 2 1 9 pollen development significantly affect its viability. The viability of the pollen was also 2 2 0 negatively correlated with the length of the pollen grains. In plants with a higher level of 2 2 1 ploidy, and thus having larger pollen grains, a reduced pollen viability can be observed 2 2 2 comparing to diploid species producing haploid pollen grains. The presence of micrograins of the pollen grains, as well as between multiporate pollen grains. On the other hand, the 2 2 8 presence of irregular pollen was negatively correlated with one-porus grain. When analysing 2 2 9 the relationships between the characteristics of pollen grains related to the number of poruses, 2 3 0 a negative correlation was noted between one-porus pollen and pollen with two, three and 2 3 1 four poruses. The development of two-, three-and four-porus grains was positively correlated. All these data clearly show that the larger the pollen, and thus the higher the level of ploidy, 2 3 3 the more disturbances in microsporogenesis, leading to the formation of anomalous pollen 2 3 4 grains.

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The Pearson's correlation coefficient matrix of ten pollen grain characteristics ( Table   2  3  6 6) was used to distribute the fifteen studied oat accessions (OTUs) in the space of three 2 3 7 ordination axes using the Kruskal's non-metric multidimensional scaling (nmMDS). A  to the amphiploid b/sa. The phenotypic distance between these two OTUs was very small. The second extreme group in MST diagrams was created by octoploids. Their location in the 2 4 9 ordination space was due to the slightly reduced viability of pollen grains, as well as the 2 5 0 higher frequency of irregular pollen grains compared to the other studied accessions. OTUs could also be influenced by the differences in the percentage share of micropollens and 2 5 8 non-reduced pollen grains in the studied pools. The small distance between Ab and Astr 2 5 9 indicates their phenotypic similarity of the analysed traits. The viability of pollen grains did 2 6 0 not differentiate them from other studied accessions, but both species were characterised by 2 6 1 the production of pollen of a similar size. It is clear that mono-and multiporate taxa (species 2 6 2 versus hybrid units) were well discriminated in the diagram. The pollen variability (Dzyuba et. al. 2006;Karabournioti et. al. 2007;Costa et al. 2016) can 2 6 5 be evaluated by measuring pollen viability, assessing its morphology, and studying pollen- are important for assessing the ploidy of plants, in particular of newly formed hybrid units. The lengths of the pollen grains were provided for some species and amphiploids of oat  The differences may be related to the inter-cultivar variation, or the influence of inter-OTU differences was maintained (see Table 2, 3).

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The size of the pollen is determined by the ploidy level and genotypes of both the of pollen grains. According to Katsiotis and Forsberg (1995b), the size of pollen grains was 2 8 4 determined primarily by the level of ploidy, and only to a small extent by interspecific 2 8 5 variation at the same level of ploidy. They proved that there were no significant differences in 2 8 6 pollen size between diploid and tetraploid accessions of oats and between hexaploids and 2 8 7 octoploids. However, Southworth and Pfahler (1992) found that the size of pollen was  Pollen grains are considered viable when they are able to germinate on the stigma. There are 3 0 7 many different methods for determining pollen viability, and their advantages and 3 0 8 disadvantages have been discussed by Dafni and Firmage (2000). The authors suggested the 3 0 9 use of several tests simultaneously, but the results obtained by Platje (2003) Tables   3  1  6 2 and 3). Some amphiploids and parental species studied here showed a reduced viability of 3 1 7 pollen grains in one of the growing seasons, which was most likely caused by unfavorable 3 1 8 weather conditions, such as high temperature or air humidity. It is known that the early stages conditions, which resulted in a reduced viability of pollen grains in some growing seasons. Our studies showed that the percentage of viable pollen was slightly lower in oat between accessions, plants from one accession, and between flowers located in one spikelet.

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Significant information on the viability of pollen grains was provided by studies on the 3 2 7 reproductive system of other grass hybrids. The frequency of viable pollen was significantly   lead to the formation of gametes with a reduced or increased number of chromosomes.   Numerous examples of anomalous pollen grains can be found in the literature.

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Micropollens and ultra-micropollens seem to be quite common in oats (Kosina and Florek   Morphogenesis of poruses seem to be related to de novo synthesis of callose in a The number of poruses in the pollen wall, through which pollen tubes can germinate, grains can germinate through many pores. As a result, competition occurs between the tubes 4 0 7 during their growth, which significantly reduces their effectiveness in the fertilization process.  The increase number of poruses may be caused by the independent activity of genes 4 1 1 located in different genomes (Kalinowski et al. 2001(Kalinowski et al. , 2005. Therefore, plants formed by  impossible due to sterility of pollen, thus apomixis was an alternative to sexual reproduction.

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It was found that a shift from the generative reproduction to apomixis occurred in plant  The pollen of the examined oat species and the amphiploids obtained from their crosses pollen viability of amphiploids was several percent lower than in species. In addition, 4 3 7 anomalous pollens were produced at low percent by the amphiploid flowers. It was found that occurred only in amphiploids and was strongly correlated with the frequency of micropollens.

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Multiporate grains will be less efficient for reproductive success due to their germination with