Biodiversity is the material basis for human survival, sustainable development of agriculture and plant breeding, and the excellent germplasm resources provide a necessary foundation for breeding good varieties (Yang et al., 2004). Throughout the ages, a large number of innovative germplasms and new varieties of fruit trees have been created using conventional breeding methods, such as sexual hybridization and seedling selection, and this diversity plays an important role in the sustainable development of fruit industry (Wang et al., 2012). However, with the deepening of the breeding work, it is difficult to make a breakthrough in plant breeding by conventional methods due to the depletion of germplasm resources with good target traits in plant species, as well as genetic deficiencies in good or specific traits for some fruit trees (Yang et al., 2004). A large number of studies have shown that distant hybridization is a more effective method of enriching species and genetic diversity compared with close hybridization. This method combines the biological characteristics of distant species, which breaks species limits and amplifies genetic variations, resulting in creation of new variations or species (Chen et al., 2018; Li et al., 2016; Ma et al., 2015; Singh et al., 2011; Wang et al., 2012).
Distant hybridization refers to crosses between two different species, genera, or higher-ranking taxa (Chen et al., 2018; Li et al., 2016), and overcoming the prefertilization reproductive barrier is the key to its success. However, due to the reproductive isolation, distant hybridization tends to have a certain degree of incompatibility, which is an obstacle to the breeding process. Complications can include pollen not experiencing germination (Sui et al., 1999; Zhai et al., 2009), pollen tubes exhibiting lateral growth or twine and distortion (Luo et al., 1992; Rieseberg, 1997), pollen tubes bursting (Dutta, 2009), and pollen growth occurring slowly or briefly, preventing entrance to the ovary (Ma et al., 2005; Wu et al., 2006). Even if the pollen tube can enter the ovary, and reach the embryo sac, it may not be fertilized, or only the egg nucleus or polar nucleus produces single fertilization (Yang and Yu, 2004). Therefore, the distant hybridization incompatibility that has been a major concern for researchers may be overcome by several possible solutions, including pollination before anthesis (Chen et al., 2004), mixed pollen pollination (Zhao et al., 2008), repeated pollination (Xie et al., 2009), use of suitable parents (Yang et al., 2004), and obtaining the interspecific hybrids from Prunus avium × Prunus pseudocerasus, Myrica rubra × Myrica nana, Dimocarpus longgana × Litchi chinensis, and Prunus salicina × Prunus armeniaca. However, studies on how to overcome the distant hybridization incompatibility in the loquat have not been reported.
The loquat is an evergreen rosaceous fruit tree that is native to southeastern China, but is cultivated worldwide in more than 30 countries, where it grows predominantly in subtropical regions experiencing mild temperature (Yang et al., 2018). It flowers from late autumn to early winter, the fruit flesh is soft and succulent and rich in amino acids and microelements, and its taste makes it a popular choice among consumers (Yang et al., 2012). It also has ornamental significance and is grown for its use in the treatment of lung-related diseases, including cough, asthma, and chronic bronchitis, and as a result, it has been cultivated for more than 2000 years (Kikuchi et al., 2014; Yang et al., 2018; Zhang et al., 2015). Nevertheless, there are ≈30 species of loquats and only E. japonica was a cultivated species with narrow genetic diversity; when used as parents, several excellent varieties have been obtained by conventional cross breeding (Dong et al., 2008; Zheng, 2007). Through this process, some common flaws have been hard to overcome; for example, low edible percentage due to large seeds, easy lodging due to weak roots, low yield due to small, hydrophobic bud branches, and young fruits are susceptible to frost damage due to flowers from autumn to winter (Li et al., 2016).
In contrast, some wild species have many good traits, such as single seed, small seeds, high germination percentage, and strong branching ability, as well as spring flowers, but there are very few studies that have attempted to carry out distant hybridization with these wild species in loquats. For example, Fukuda et al. (2007) obtained three fruits and four seeds by distant hybridization of E. japonica ‘Mogi’ and Rhaphiolepis indica, among which three seedlings were true hybrid, but survived for only 4 months. It also was found that the opposite cross can also sit fruit but produce no seeds. Coombes and Robertson (2008) have obtained true hybrids from intergeneric hybridization of E. deflexa and R. indica. Li et al. (2016) tried breeding E. prinoides, E. japonica, and E. prinoides var. dadunensis as the male parent with E. deflexa and its variant that the pollen tube could not reach the ovule through the style after germination, which was determined by fluorescence microscopy. However, although these three aforementioned articles reported success with obtaining the distant hybrid, the problem of low fruit setting percentage also exists, and there have been no reports discussing how to overcome the incompatibility of distant hybridization among species of loquat.
The present study uses E. japonica ‘Dawuxing’ as the female parent and E. deflexa (blooms in winter) and E. bengalensis (blooms in spring) as the male parents, to investigate different cut-styles of treatment during pollination. The analysis of difference in pollen germination, pollen tube dynamics, fruit set, and average number of seeds per fruit after different pollination treatments was conducted, and the S-RNase gene AS-PCR amplification was used to identify the true hybrid of the obtained hybrid seedlings. The effect of cut-style on distant hybridization affinity of loquats was studied systematically to provide a scientific basis for overcoming the incompatibility of distant hybridization between loquat species, and make full use of the fine characters of wild resources to improve the varieties, which is of great significance to germplasm resource innovations in the loquat.
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