Meiotic behaviour and pollen fertility of F₁, F₂ and BC₁ progenies of Iris dichotoma and I. domestica

Iris dichotoma and I. domestica are herbaceous perennials belonging to the family Iridaceae, with a diploid chromosome number of 2n=2x=32 native to China, and have a long flowering period in summer and high resistance to cold, drought and barren conditions, which make them excellent materials for iris breeding (Zheng et al., 2017). Therefore, I. dichotoma and I. domestica have been attracting the attention of plant breeders (Bi et al., 2012). In nature, I. dichotoma is found with violet, yellow and white flowers, but no red or orange flowers. Its individual flowers stay open for about 4 h in form the late afternoon to the early evening (Luo et al., 2016). To improve its flower colour and flowering time, interspecific crosses were conducted to introgress useful genes from I. domestica to I. dichotoma and this has resulted in some novel varieties (Yang et al., 2013; Lian et al., 2016; Ruan et al., 2017; Xu et al., 2017; Gao et al., 2020). Previous studies concentrated on violet and white-flowered I. dichotoma plants (Chimphamba, 1973; Ruan et al., 2017), whereas I. dichotoma with yellow flowers has not been comprehensively investigated. The F₂ generation of I. dichotoma (violet-flowered) and I. domestica shows great variation in flower colours, but not with yellow (Lenz, 1972). Therefore, creating new germplasm with yellow-flowered I. dichotoma is a significant goal of Iris-breeding programs.

Pollen fertility is very important for plant development and reproduction (Consolaro et al., 1996; Conterato et al., 2006). Variations in pollen fertility are usually attributed to abnormal chromosome pairing and abnormal meiotic division (Yabuya, 1991). Abnormal meiosis, such as early chromosome migration, lagging chromosomes, bridge, unequal separation, micronuclei, multiple spindles, nonsynchronous division, unequal separation and so on, was observed in many plants, especially in interspecific hybrids (Wang et al., 2010; Hu et al., 2021). Compared with their parents, interspecific hybrids of Dendranthema had a higher frequency of chromosome bridges and lagging chromosomes (Cui et al., 2007). Approximately 56.5% of the cells of allotriploid lily ‘Cocossa’ demonstrated meiotic abnormalities at telophase II (Zhang et al., 2017). Sometimes, the high frequency of abnormal meiosis was also found in intraspecific hybrids. The hybrids of Hydrangea aspera subsp. aspera Kawakami group × subsp. sargentiana showed a higher frequency of meiotic abnormalities (48.2%) compared with the two parents and resulted in reduced pollen fertility (Crespel and Morel, 2014). Generally, the fertility of interspecific/intraspecific hybrid could be restored by successive backcrosses. So far, there is no information on pollen viability and meiosis of I. dichotoma, I. domestica and their hybrids. Therefore, meiotic behaviour and pollen fertility of yellow-flowered I. dichotoma, I. domestica and their F₁, F₂ and BC₁ hybrid progenies were investigated in this study. Furthermore, the relationships between meiotic abnormalities and crossbreeding were discussed. The knowledge acquired will contribute to the rational use of these genetic resources.

The most striking form of the diffuse stage that we observed in these hybrids was the diffuse diplotene stage. To our knowledge, this phenomenon was found in the Iris species for the first time. The same phenomenon has also been reported in Rosa and large genome cereals (Colas et al., 2017) as well as in Larix (Kołowerzo-Lubnau et al., 2015). Especially in interspecific hybrid plants, the diffuse stage has been proposed to be widespread in plant meiosis, affecting restitution from diffuse to fully contracted chromosomes, with chromosomes often entering metaphase before fully contracting (Klášterská and Natarajan, 1974). In this study, it occurred in summer with high temperatures and drought stress. In Larix species, meiosis calmed during the autumn and pollen mother cells remained in the diffuse stage for several months to resist the cold (Ekberg et al., 1968). This possibly reflects that the diffuse stage is influenced by some extraordinary environmental conditions.

The high frequency of meiosis abnormalities was observed in BC₁-S-1 (58.7%), BC₁-S-2 (54.5%), BC₁-S-3 (52.7%), BC₁-S-4(47.7%) and F₂-1(57.3%), with low pollen-germination rates (0, 1.6%, 4.8%, 7.2% and 0, respectively). Pollen fertility analysis of different cross combinations in this study indicated that meiotic abnormalities, such as non-congression, lagging chromosomes, unequal separation and abnormal spindle fibres orientation, led to low pollen fertility. As previously reported, pollen viability is closely associated with meiosis of pollen mother cells, and meiotic abnormalities affect pollen development (Boff and Chifino-wittmann, 2002; Zha et al., 2007; Farco and Dematteis, 2014; Usandizaga et al., 2020). The relationship between meiotic abnormalities and fertility has been studied in many plant species, including Musa and Brassica (Adeleke et al., 2004; Yang et al., 2020). Meiotic abnormalities are shown to be negatively correlated with pollen fertility (Devi and Borua, 1997).

The pollen fertility of F₁-5 (10.1%) was much lower than that of its parents I. dichotoma (26.1%) and I. domestica (35.1%). Backcrossing F₁-5 to I. dichotoma resulted in rapid restoration of fertility, and the pollen fertility of BC₁-Y-1 and BC₁-Y-2 was higher than that of F₁-5. However, when backcrossing F₁-5 to I. domestica, the BC₁ hybrids had a high total meiosis abnormality rate (exceeding 50.0%) and produced no or shrunken pollen with relatively low pollen fertility (0 to 4.8%) compared with F₁-5 (10.1%). Low fertility of BC_l plants was also reported in Agropyron junceum (Charpentier et al., 1986) although backcrossing can restore the fertility of interspecific hybrids (Yushkina et al., 2009). F₁-5 was male fertile and presented only 1–4 unpaired chromosomes. Partial fertility of the interspecific hybrid could be associated with relatively high chromosome association frequency, indicating partial genomic homology between the parental species (Asano, 1984). The abnormal segregation of chromosomes in meiosis I and II can lead to the formation of micronuclei, which either may remain in microspore tetrads or may be eliminated in the form of microcytes for additional cytokinesis. In this case, the formed tetrads will be genetically unbalanced, and the resulting pollen will become dysfunctional or shrunken. Although tetrads were found in F₂-1 and BC₁-Y-1, they did not grow cell walls and form pollen grains. Therefore, the frequency of 2n pollen was low in I. dichotoma, I. domestica and their hybrids which exhibited a high frequency of dyad and triad. Additionally, microspores in tetrads could originate from an unequal chromosome division, usually leading to sterile pollen (Do Nascimento et al., 2014).

In this study, the lagging chromosomes, unequal separation and nonsynchronous division led to chromosomes slowly moving to cell poles, forming the micronucleus or disappearing in the cytoplasm, and finally led to the production of unbalanced tetrads or polyad and abnormal pollen grains. Similar meiotic aberrations have been reported in Brachiaria (Gallo et al., 2007), Begonia (Dewitte et al., 2010), Hydrangea aspera (Crespel and Morel, 2014) and allotriploid lily ‘Cocossa’ (Zhang et al., 2017). They can be contributed to chromosome pairing problems due to their lack of homology and are the expression of the typical behaviour of univalent or non-paired chromosomes. In some rare cases, unequal and incomplete nuclei separation resulted in monads, such as asymmetric cytokinesis in pollen mother cells in which the entire nucleus was included in one of the cells (Barba-Gonzalez et al., 2005). In this study, the presence of dyads and/or triads at the tetrad stage imply the production of 2n pollen grains. Abnormal spindle fibres orientation is associated with the production of 2n gametes. The formation of disoriented spindle fibres led to a dyad (composed of two 2n microspores) in the case of fused or parallel spindle fibres, and to a triad (composed of one 2n microspore and two n microspores) in the case of tripolar spindle fibres. The production of 2n gametes was observed in interspecific hybrids of potato (Conicella, 1991), Populus (Dong et al., 2015) and strawberry (Luo et al., 2018).

The spontaneous occurrence of unreduced gametes can produce unexpected polyploids such as triploids and tetraploids in both natural and artificial crosses in various plants such as Lilium (Van Tuyl et al., 2005), Tulip (Marasek-Ciolakowska, 2012) and Primula (Kato et al., 2008). When they occur in interspecific hybrids with some extent of viability, 2n gametes can be used to produce sexual progeny through either crossing or selfing (Ramanna and Jacobsen, 2003; Van Tuyl and Lim, 2003). In the case of meiotic polyploidisation, polyploid progenies are raised by crossing with 2n gamete-producing genotypes. The most important advantage of meiotic polyploidisation is that homologous recombination occurs at a high frequency between parental chromosomes during meiosis. Therefore, a large amount of heterozygosity can be transmitted to the polyploid offspring. Because of the function of unreduced gametes, a considerable number of Iris garden varieties with large flowers, improved form and plant habit, and fully fertile were produced through intercrossing by meiotic polyploidisation (Bee et al., 1986). In this study, the selection of individuals producing 2n gametes allowed manipulating the ploidy level of the offspring. Because unreduced pollen is not always viable, as previously demonstrated in Begonia (Dewitte et al., 2010), screening for viable unreduced gametes should be undertaken in the future, as these cells present an interesting tool for polyploidisation and can be used as pollen donors to produce polyploid iris individuals exhibiting larger flowers as well as higher pollen and seed fertility (Barba-Gonzalez et al., 2004; Nikoloudakis et al., 2018).

In summary, this study analysed the relationship between meiotic abnormalities and pollen fertility in 12 Iris individuals comprising parents and hybrids resulting from their crosses and backcrosses. Meiotic abnormalities negatively affected pollen fertility. The diffuse diplotene stage was observed for the first time in the genus Iris. Poor pollen fertility of BC₁-S presented difficulties for further breeding. As all BC₁-Y individuals displayed relatively similar flower colour (yellow) and higher pollen fertility than other cross combinations, they are excellent plant material for yellow-flowered iris breeding. In addition, BC₁-Y-2, BC₁-Y-1, BC₁-S-2, BC₁-S-3 and BC₁-S-4 have potential in polyploid breeding.