Exploration of natural allelic variations from AA genome species
Genetic diversity and allele were lost during the domestication from the wild species of rice to the cultivated rice (Sun et al. 2002), whereas narrow genetic basis led to the yield bottleneck of Asian cultivated rice, thus, exploration and utilization of favorable allelic genes in AA genome species is an accessible approach to improve rice breeding. Recently years, mining and utilization of useful allele genes have made great progress in rice breeding. For example, the allelic variation in the Wx gene and SSSI were proved to contribute greatly to the differences in ECQs in the two subspecies (Li et al. 2018). Allelic variation at the E1/Ghd7 locus allowed expansion of the rice cultivation area through adjusting heading date (Saito et al. 2019). The allelotypes of BPH9 confer varying levels of resistance to different biotypes of BPH and enable rice to combat planthopper variation (Zhao et al. 2016). The allelic variation at the rice blast resistance (R) Pid3 locus were analyzed based on the 3K RGP sequencing data, and different strategies were developed to apply the functional Pid3 alleles to indica and japonica rice breeding (Lv et al. 2017). Thus, exploration of natural allelic variation and artificial shuffling within useful genes may allow breeding to be tailored to control emerging traits.
Though wild accessions from O. nivara, O. rufipogon, O. meridionalis, O. barthii, O. glumaepatula, O. longistaminata, O. barthii and Africa cultivated rice O. glaberrima were used as donor parents in the backcross breeding program (Chen et al. 2006; Tian et al. 2006; McCouch et al. 2007; Rangel et al. 2008; Hao et al. 2009; Gutierrez et al. 2010; Ramos et al. 2016; Bhatia et al. 2017), most of introgression lines were derived from a single accession of AA genome species in a single background leading to the loss of some valuable allelic information. In this study, introgression library with multiple donors from different relatives of Asian cultivated rice is a powerful resource platform to discover novel and functional allele QTLs/genes. One locus for grain length and one locus for grain width were explored from the six and five different donor species, respectively. Two loci for grain length, three loci for grain width and one locus for the ratio of grain length to grain width were detected from the donors of four species respectively (Figure 4-5), mining the natural functional variations in the useful genes derived from the multiple donors and combing the different alleles through diversification could be useful for accurate rice breeding program.
By now, more than 20 sets of introgression lines were raised, derived from the cross between the indica varieties and japonica varieties (Kubo et al. 2002; Ebitani et al. 2005; Chen et al. 2007; Ando et al. 2008; Hao et al. 2009; Bian et al. 2010; Xu et al. 2010a; Lin et al. 2011; Zhang et al. 2011; Kanjoo et al. 2012; Pinson et al. 2012; Uga et al. 2015; Ookawa et al. 2016; Ujiie et al. 2016), 2 sets of ILs from indica x indica (Chen et al. 2014; Liu et al. 2016), 2 sets of ILs from japonica x japonica (Hori et al. 2010), 6 sets of introgression lines were developed from the cross between O. sativa and O. glaberrima (Doi et al. 1997; Ghesquière et al. 1997; Li et al. 2004; Gutierrez et al. 2010; Shim et al. 2010), 5 sets of introgression lines were raised from O. sativa and O. rufipogon (Chen et al. 2006; Hao et al. 2006; Tian et al. 2006; Cheema et al. 2008; Furuta et al. 2014), 2 sets of introgression lines from cross the between the O. sativa and O. glumaepatula (Sobrizal et al. 1999; Rangel et al. 2008). One set of introgression line from the cross between O. sativa and Zhangpu wild rice (Yang et al. 2016), 1 sets of introgression lines from the cross between O. sativa and O. minuta (Guo et al. 2013), 1 sets of introgression lines from the cross between O. sativa and weedy rice (Subudhi et al. 2015). Above CSSL library were derived from the cross between single donor or few donors and single recurrent parent. And some CSSL library from multiple donors and single recurrent were also built for rice improvement and gene identification in rice (He et al. 2005a; He et al. 2005b; He et al. 2005c; Xi et al. 2006; Yasui et al. 2010; Arbelaez et al. 2015). These CSSL library were developed based on the genotype selection so that the introgression segments could cover the donor genome completely and the donor information were not lost, but some of introgression lines could not show the significant phenotype, compared to the recurrent parent. Thus, it could not be used for the further study and breeding program. Previous reports indicated the IL library was generated by crossing 70 accessions of six AA genome species with two elite cultivars of O. sativa (Bhatia et al. 2017). In this study, 334 accessions of AA genome species as the donor parents were transferred into three different cultivars of O. sativa. These IL libraries were raised from a large number of the donors in the multiple recurrent parents based on the phenotype selection, which exhibited the desirable agronomic traits and provided abundant information of favorable genes or QTLs and allelic variations. This approach could save a couple of times and cost and obtained more interesting information. We could select the desirable traits from the different donors using this approach, yield component traits and disease resistance were paid more attention during selection (Bhatia et al. 2017) , whereas we emphasized on the selection of plant and panicle architecture, grain size and aerobic adaptation. IL libraries derived from the multiple donors have some advantages: 1) An abundant genetic variations were introgressed into the cultivated rice genome; 2) Target genes/QTLs for the same phenotype could be validated by the different donors, and it will provide the information that these target genes/QTLs could be the same haplotype; 3) The genes or QTLs responsible for the opposite phenotypes, for example, long grain size and short grain size, could also be confirmed using the different populations from multiple donors, and it could be the different haplotype; 4) Introduction of new genetic variation, selection of favorable alleles in the wild relatives could speed up to fix the useful genes. Therefore, Constructing IL library is not only the breeding method, but also the domestication power. Thus, these IL libraries will help us improving rice breeding and interesting genes discovery and utilization, as well as the development of rice domestication.
Introgression library for QTL mapping and cloning
Introgression lines harboring one or more donor chromosome segments exhibited distinguish traits, compared to the recurrent parent. Since the background of introgression lines is similar to the recurrent parent and it is easier to correlate a particular chromosomal region to phenotypic variation, they were used for mapping and cloning QTLs or genes for complex traits. Many QTLs or genes have been mapped based on introgression lines, such as GS2 for grain size and weight (Hu et al. 2015), GL7 for grain size (Wang et al. 2015), DEP1 for dense and erect panicle (Huang et al. 2009), GS3 for grain length and weight (Fan et al. 2006), qPGWC-7 for grain chalkiness (Zhou et al. 2009), QTLs for heterosis (Tao et al. 2016), QTLs for seed dormancy (Marzougui et al. 2012), qGRH9 for green rice leafhopper (Fujita et al. 2010), Pi54rh for rice blast (Das et al. 2012), qSPP2.2 controlling spikelet per panicle (Kaur and Singh, 2018).
Previous reports indicated that some genes or QTLs for agronomic traits were identified based on this introgression library, such as Spr5(t) for spreading panicle from O. glaberrima (Xu et al. 2010b), qph1 for plant height from O. longistaminata (Chen et al. 2009), EP4 for erect panicle from O. glumaepatula (Zhang et al. 2015), Er1 for erect panicle from O. glaberrima (Zhou et al. 2008), GS3 for grain length from O. nivara, O. glumaepatula, O. longistaminata and O. glaberrima (Zhang et al. 2014), HS1 for hybrid seed shattering between O. barthii and O. sativa (Zhang et al. 2019). In this study, 41 loci for grain length, 44 loci for grain width on 12 chromosomes, 42 loci for the ratio of grain length to grain width were explored on 12 chromosomes (Figure 4-6) , and some loci were identified in the same location with published genes, such as GW2 (Song et al. 2007), GL2 (Hu et al. 2015), PGL2 (Heang and Sassa, 2012b), PGL1 (Heang and Sassa, 2012a), GL3.2 (Xu et al. 2015), GS3 (Fan et al. 2006; Takano-Kai et al. 2009; Mao et al. 2010), qGL3-1 (Qi et al. 2012), qGL3.3 (Hu et al. 2018), GS6 (Sun et al. 2013), TGW6 (Ishimaru et al. 2013), GL7 (Wang et al. 2015), OsSPL13 (Si et al. 2016). In addition, 29 loci might be new QTLs or genes controlled for grain size from the different AA genome donors, and most of published genes for grain size were found based on the introgression library. Moreover, a novel QTL for grain width, qGW6.1, was identified from the O glumaepatula based on BC4F2 and BC4F3 populations. It suggested that this library is an effective tool to systematically discover and map novel genes and allelic variations. Moreover, introgression lines showing superior agronomic traits could accelerate the improvement of rice breeding. The ILs with the desirable traits could be used to improve rice breeding program through marker-assisted selection (Ashikari and Matsuoka, 2006).
Hybrid sterility is the major barrier to develop interspecific introgression library, which is also an ideal model for studying the relationship between the reproductive isolation and speciation
The major barrier is hybrid sterility exhibiting complete or partial pollen sterility and/or spikelet sterility in the crosses between AA genome species and O. sativa. We observed that pollen fertility of F1 varied from 1.92% to 93.19 % dependent on the different accessions of O. nivara and O. rufipogon, whereas all the crosses with the accessions of O. barthii, O. glumaepatula and O. meridionalis showed complete pollen sterility in the F1 combinations（data not shown）. For the accessions of O. longistaminata, the crosses using the japonica varieties Dianjingyou 1 and Yundao 1 as the recurrent parents were failed, only the crosses using an indica variety RD23 as the recurrent parent was obtained by embryo rescue. Thus, hybrid sterility between O. sativa and AA genome species is the main difficulty to transfer favorable genes between them. Fortunately, the female gametes from the interspecific hybrids were partially fertile, and some backcross seeds in the different combinations could be obtained by backcrossing F1 as the female parent with O. sativa as the male parent.
Genus Oryza probably originated at least 130 million years ago and spread eventually in Asia, Africa, Americas, Australia and Antarctica, which contains twenty-four wild rice species and two cultivated rice species representing 11 genomes (Khush, 1997). The AA genome includes six wild rice species (O. nivara, O. rufipogon, O. barthii, O. glumaepatula, O. longistaminata, O. meridionalis) and two cultivated species (O. sativa and O. glaberrima). Though reproductive isolation, especially hybrid sterility, was existed in the hybrids between Asian cultivated rice and AA genome species, direct cross and backcross could be made for raising introgression library. Thus, introgression library from AA genome species is an ideal model to study the relationship between reproductive isolation and AA genome species divergence. Using this resource platform, we identified a series of QTLs or genes for interspecific hybrid sterility, including S1, S29(t), S37(t), S38(t), S39(t), S40, S44(t), S51(t), S52(t), S53(t), S54(t)、S55(t)、S56(t) and qHMS7(Hu et al. 2006; Zhao et al. 2012; Xu, et al. 2014; Chen et al. 2017; Xie et al. 2017; Li, et al. 2018; Zhang, et al. 2018; Xie et al. 2019). Moreover, we found some orthologous genes for hybrid sterility across the species and populations. For examples, S29 (t) from O. glaberrima, S53 (t) from O. meridionalis, and S22B from O. glumaepatula had good co-linear relationship on chromosome 2 (Hu et al. 2006; Sakata et al. 2014; Li et al. 2018). S56(t) locus from O. glumaepatula was mapped around S20 and qSS-7 region identified in the cross between O. sativa and O. glaberrima (Doi et al. 1999; Li et al. 2011). In addition, It was observed that S23 from O. glumaepatula, S21 from O. glaberrima and O. rufipogon, qHMS7 from O. meridionalis were located into the similar region on chromosome 7 (Doi et al. 1999; Sobrizal. et al. 2000; Miyazaki et al. 2007; Yu et al. 2018). These results suggested that alleles of S29 (t) / S53(t) / S22B were required for the divergence among O. sativa and O. glaberrima, O. meridionalis, O. glumaepatula, S56(t) /S20 /qSS-7 played an important role in the species formation of O. glumaepatula, O. glaberrima and O. sativa, S23/S21/qHMS7 are necessary for the speciation of O. glaberrima, O. rufipogon, O. glumaepatula and O. meridionalis. Thus, introgression library with the multiple AA genome donors is an excellent resource for studying the reproductive isolation and speciation in rice.
Interspecific hybridization is an important driving force for evolutional process
The process of crop domestication is driven by artificial selection, cultivation practices, as well as agricultural environments (Petr et al. 2018). Large-scale chromosomal structural changes, polyploidy, copy-number variation and changes in transposable-element content exhibited distinguished difference between wild and cultivated plants, which are the important mechanism for crop evolution (Yang et al. 2012; Wang et al. 2015; Salman-Minkov et al. 2016). In addition, Hybridization is major power in crop domestication, increases crop diversification and arises new crop species (Michael, 2019). Archaeological evidence and genomic analysis supported that indica, a subspecies of rice evolved from the hybridization between japonica and O. nivara (Fuller et al. 2010; Fuller, 2011; Silva et al. 2018). Interspecific hybridization can lead to novel allelic variations, gene combinations and/or novel patterns of gene expression, which in turn provide the variation on which natural selection can act. The decrease in diversity caused by artificial selection and bottlenecks could be counteracted by interspecific hybridization, interspecific introgression may provide for novelty, superior quality gene resources which could increase crop yield, quality or adaptive ability. In this study, multiple donors were introgressed into Asian cultivate variety, Dianjingyou 1. ILs library exhibited abundant and diverse traits in plant architecture, seed characteristics, biotic or abiotic resistance (Table S3-S5). Thus, interspecific hybridization could be an important mechanism for rice species diversity and increase adaptive ability in the different environment, which is valuable resource for meeting the demand of breed challenge in rice.