Two Tightly Linked Genes at the hsal Locus Cause Both F1 and F2 Hybrid Sterility in Rice

Molecular mechanisms of hybrid breakdown associated with sterility (F2 sterility) are poorly understood as compared with those of F1 hybrid sterility. Previously, we characterized three unlinked epistatic loci, hybrid sterility-a1 (hsa1), hsa2, and hsa3, responsible for the F2 sterility in a cross between Oryza sativa ssp. indica and japonica. In this study, we identified that the hsa1 locus contains two interacting genes, HSA1a and HSA1b, within a 30-kb region. HSA1a-j (japonica allele) encodes a highly conserved plant-specific domain of unknown function protein (DUF1618), whereasthe indica allele (HSA1a-is) has two deletion mutations that cause disruption of domain structure. The second gene, HSA1b-is, encodes an uncharacterized proteinwith some similarity to a nucleotide-binding protein. Homozygous introgression of indica HSA1a-is-HSA1b-is alleles into japonica showed female gamete abortion at an early mitotic stage. The fact that the recombinant haplotype HSA1a-j-HSA1b-is caused semi-sterility in the heterozygous state with the HSA1a-is-HSA1b-is haplotype suggests that variation in the hsa1 locus is a possible cause of the wide-spectrum sterility barriers seen in F1 hybrids and successive generations in rice. We propose a simple genetic model to explain how a single causal mechanism can drive both F1 and F2 hybrid sterility.

Cytological Behavior of Hybridization Barriers Between Oryza sativa and Oryza officinalis

Oryza officinalis is one of the important wild species in the tertiary gene pool of Oryza sativa. It has a number of elite genes for rice breeding in resistance or tolerance. However, breeding barriers are so serious that the gene transfer is much difficult by sexual cross method between O. sativa and O. officinalis. Characteristics of the breeding barriers were systemically studied in this paper. When both the diploid （AA, 2n=2x=24） and autotetraploid （AAAA, 2n=4x=48） cultivated rice were crossed as maternal parents with O. officinalis （CC, 2n=2x=24）, none F1 hybrid seeds were obtained. The young hybrid ovaries aborted at 13-16 d after pollinations （DAP）. By rescuing hybrid embryos, in vitro F1 plantlets were obtained in 2x×2x combinations with the crossabilities lower than 0.5%. Lower rates of double-fertilization and abnormal development of hybrid embryo and endosperm were mainly observed in both combinations of 2x×2x and 4x×2x. Free endosperm nuclei in hybrid degenerated early at 1 DAP in a large scale. Almost no normal endosperm cells formed at 3 DAP. Development of a lot of embryos ceased at globularor pear-shaped stage as well as some degenerated gradually. The hybrid plantlets were both male and female sterility. Due to the abnormal development, a diversity of abnormal embryo sacs formed in hybrids, and hybrid pollen grains were typically abortive. It showed that conflicts of genome A and C in hybrid induced abnormal meioses of meiocytes.

Molecular Mapping of Sterility QTLs qSS-3, qSS-6a and qSS-7 as Single Mendelian Factors via NIL strategy

Hybrid sterility between Oryza glaberrima and O. sativa seriously hampers the introgression of favorable genes from each other. In order to further understand this issue, identification and isolation of hybrid sterility QTLs as single Mendelian factors are an effective strategy. A genetic map was constructed using a BC1 F1 population derived from a cross between an O. safiva japonica cultivar and an O. glaberrima accession. Four main-effect QTLs for pollen sterility were detected in the BC1F1. Five BC8F1 advanced backcross populations were developed via successive backcrosses based on phenotype and molecular selections. The BC8F1 populations showed bimodal distribution for pollen fertility and could be classified into semi-sterile and fertile types, fitting single Mendilian factor inheritance ratios. Three QTLs detected in the BC1F1 corresponding to qSS-3, qSS-6a and qSS-7 were mapped on chromosomes 6, 3 and 7, respectively, as single Mendilian factors.

A hybrid sterile locus leads to the linkage drag of interspecific hybrid progenies

Interspecific hybridization plays an important role in rice breeding by broadening access to desirable traits such as disease resistance and improving yields.However,interspecific hybridization is often hindered by hybrid sterility,linkage drag,and distorted segregation.To mine for favorable genes from Oryza glaberrima,we cultivated a series of BC4 introgression lines(ILs)of O.glaberrima in the japonica rice variety background(Dianjingyou 1)in which the IL-2769(BC4F10)showed longer sterile lemmas,wider grains and spreading panicles compared with its receptor parent,suggesting that linkage drag may have occurred.Based on the BC5F2 population,a hybrid sterility locus,S20,a long sterile lemma locus,G1-g,and a new grain width quantitative trait locus(QTL),qGW7,were mapped in the linkage region about 15 centimorgan(cM)from the end of the short arm of chromosome 7.The hybrid sterility locus S20 from O.glaberrima eliminated male gametes of Oryza sativa,and male gametes carrying the alleles of O.sativa in the heterozygotes were aborted completely.In addition,the homozygotes presented a genotype of O.glaberrima,and homozygous O.sativa were not produced.Surprisingly,the linked traits G1-g and qGW7 showed similar segregation distortion.These results indicate that S20 was responsible for the linkage drag.As a large number of detected hybrid sterility loci are widely distributed on rice chromosomes,we suggest that hybrid sterility loci are the critical factors for the linkage drag in interspecific and subspecific hybridization of rice.

Prospects of utilization of inter-subspecific heterosis between indica and japonica rice

The Asian cultivated rice(Oryza sativa L.) grown worldwide is divided into two subspecies, indica and japonica. It is well known that the heterosis of inter-subspecies is usually stronger than that of intra-subspecies. Since the 1970 s, indica hybrid rice, an intra-subspecific hybrid rice, has being widely used in China and even in the world. However, the inter-subspecific hybrid rice between indica and japonica is still unavailable. The major obstacle is the hybrid sterility of the inter-subspecies. In recent decades, the genetic and molecular basis of indica-japonica hybrid sterility was understood more and more clearly. Some breeding approaches for overcoming inter-subspecific hybrid sterility were proposed and used to develop the indicajaponica hybrid rice. The updated understanding will offer new approaches for development of breeding lines for overcoming indica-japonica hybrid sterility, which facilitates developing of inter-subspecific hybrid rice.

Why are There Indica Type and Japonica Type in Rice?——History of the Studies and a View for Origin of Two Types

On the bases of archaeological discoveries, the earliest domestication of rice has been confirmed in the middle and lower Changjiang River basin, while in the region wild rice populations are found in shallow swamps under a climate with freezing winter cold. These findings lead us to-examine the past ideas about domestication and differentiation of rice. Historically, in 1930s two sub-species, indica and japonica, were proposed on the basis of sterility in F1 hybrids between them. Soon after that, the two types were classified by the associations of a number of genetically independent traits. The characteristic associations of traits have been explained by the hybrid sterility or reproductive barriers which were assumed to comprise a set of duplicate recessive lethal genes and to be an inner genetic mechanism to lead to the varietal differentiations In 1980s, the hybrid sterility between Indica and Japonica types was analyzed, and Indica, Japonica, and wide-compatibility type which gives fertile hybrids when cross to Indica and Japonica types, are proved to contain an allele, S5＇, S5＇ and S5^n, respectively at a locus on chromosome 6. And those gametes having Sj allele are found to be partially aborted in the hybrid genotypes of S5＇/S5＇ while no gamete abortion occurs in S5＇/S5^n and S5＇/S5^n genotypes. Since then, the gene S5^n has been used in hybrid rice breeding to obtain fertile and vigorous hybrids between subspecies, and the long-disputed problem of hybrid sterility has been solved. Also in such studies the characteristic association of traits found in each of vadetal groups is better explained by founder effects. On the other hand, a large number of native cultivars of rice were surveyed with enzyme polymorphism in 1980s and later with molecular markers. As a result, profound genetic diversity is found in cultivated rice as well as in wild rice. These findings seem to lead us to the idea of multiple independent domestications of rice. However, before reaching such a conclusion, at least two factors, i.e., long-distance-dissemination of some genotypes and the possibilities of introgression by local wild rice to primitive cultivars need to be examined. Taking the two factors as well as the historical events into consideration, it is considered here that the perennial japonica cultivars which are close to wild rice in the Changjiang River basin were disseminated to East India through Assam or along the Bengal Bay, where they were transformed under the introgressions of local wild rice and formed a secondary center, from which some genotypes seem to be disseminated to colonies in Southeast Asia under the influence of Hinduism. Later some of the genotypes were introduced into China and constituted so called Indica type. This may be a reasonable picture for the varietal differentiation.

Suppression or knockout of SaF/SaM overcomes the Sa-mediated hybrid male sterility in rice

Hybrids between the indica and japonica subspecies of rice （Oryza sativa） are usually sterile, which hinders utilization of heterosis in the inter-subspecific hybrid breeding. The complex locus Sa comprises two adjacently located genes, SaF and SaM, which interact to cause abortion of pollen grains carrying the japonica allele in japonica-indica hybrids. Here we showed that silencing of SaF or SaM by RNA interference restored male fertility in indica-japonica hybrids with heterozygous Sa. We further used clustered regularly interspaced short palindromic repeats （CRISPR）/Cas9-based genome editing to knockout the SaF and SaM alleles, respectively, of an indica rice line to create hybrid-compatible lines. The resultant artificial neutral alleles did not affect pollen viability and other agricultural traits, but did break down the reproductive barrier in the hybrids. We found that some rice lines have natural neutral allele Sa-n, which was compatible with the typical japonica or indica Sa alleles in hybrids. Our results demonstrate that SaF and SaM are required for hybrid male sterility, but are not essential for pollen development. This study provides effective approaches for the generation of hybrid-compatible lines by knocking out the Sa locus or using the natural Sa-n allele to overcome hybrid male sterility in rice breeding.

Molecular Control of Male Reproductive Development and Pollen Fertility in Rice

Anther development and male fertility are essential biological pro- cesses for flowering plants and are important for crop seed produc- tion. Genetic manipulation of male fertility/sterility is critical for crop hybrid breeding. Rice （Oryza sativa L.） male sterility phenotypes, including genic male sterility, hybrid male sterility, and cytoplasmic male sterility, are generally caused by mutations of fertility-related genes, by incompatible interactions between divergent allelic or non-allelic genes, or by genetic incompatibilities between cytoplas-mic and nuclear genomes. Here, we review the recent advances in the molecular basis of anther development and male fertility-sterility conversion in specific genetic backgrounds, and the interactions with certain environmental factors. The highlighted findings in this review have significant implications in both basic studies and rice genetic improvement.

The molecular and evolutionary basis of reproductive isolation in plants

Reproductive isolation is defined as processes that prevent individuals of different populations from mating, survival or producing fertile offspring. Reproductive isolation is critical for driving speciation and maintaining species identity, which has been a fundamental concern in evolutionary biology. In plants,reproductive isolation can be divided into prezygotic and postzygotic reproductive barriers, according to its occurrence at different developmental stages. Postzygotic reproductive isolation caused by reduced fitness in hybrids is frequently observed in plants, which hinders gene flow between divergent populations and has substantial effects on genetic differentiation and speciation, and thus is a major obstacle for utilization of heterosis in hybrid crops. During the past decade, China has made tremendous progress in molecular and evolutionary basis of prezygotic and postzygotic reproductive barriers in plants. Present understandings in reproductive isolation especially with new data in the last several years well support three evolutionary genetic models, which represent a general mechanism underlying genomic differentiation and speciation. The updated understanding will offer new approaches for the development of wide-compatibility or neutral varieties, which facilitate breeding of hybrid rice as well as other hybrid crops.

Artificial Selection in Domestication and Breeding Prevents Speciation in Rice

Speciation has long been regarded as an irreversible process once the reproductive barriers had been established.However,unlike in natural populations,artificial selection might either accelerate or prevent speciation processes in domesticated species.Asian cultivated rice is a target crop for both domestication and artificial breeding;it contains two subspecies of indica and japonica,which usually produce sterile inter-subspecific hybrids due to reproductive barriers.In this study,we constructed the evolutionary trajectory of a reproductive isolation system S5,which regulates fertility in indica-japonica hybrids via three adjacent genes,based on the data of 606 accessions including two cultivated and 11 wild rice species.Although hybrid sterility haplotypes at S5 lead to establishment of a killer-protector reproductive barrier,origin of wide-compatibility haplotypes by complex hybridization and recombination provides an opposing force to reproductive isolation and thus prevents speciation during domestication.Analysis in a diallel set of 209 crosses involving 21 parents showed that the wide-compatibility genotypes largely rescued fertility of indica-japonica hybrids,indicating that the wide-compatibility gene would enable gene flow to maintain species coherence.This counteracting system indicates that combined effects of natural evolution and artificial selection may result in reversible processes of speciation in rice,which may also have implications for genetic improvement of rice.