Hybrid Pollen Sterility Research Articles

Genetic characterization and fine mapping of qHMS4 responsible for pollen sterility in hybrids between Oryza sativa L. and Oryza glaberrima Steud.

The online version contains supplementary material available at 10.1007/s11032-022-01306-8.

Domain Unknown Function DUF1668-Containing Genes in Multiple Lineages Are Responsible for F1 Pollen Sterility in Rice.

Postzygotic reproductive isolation maintains species integrity and uniformity and contributes to speciation by restricting the free gene flow between divergent species. In this study we identify causal genes of two Mendelian factors S22A and S22B on rice chromosome 2 inducing F1 pollen sterility in hybrids between Oryza sativa japonica-type cultivar Taichung 65 (T65) and a wild relative of rice species Oryza glumaepatula. The causal gene of S22B in T65 encodes a protein containing DUF1668 and gametophytically expressed in the anthers, designated S22B_j. The O. glumaepatula allele S22B-g, allelic to S22B_j, possesses three non-synonymous substitutions and a 2-bp deletion, leading to a frameshifted translation at the S22B C-terminal region. Transcription level of S22B-j and/or S22B_g did not solely determine the fertility of pollen grains by genotypes at S22B. Western blotting of S22B found that one major band with approximately 46 kDa appeared only at the mature stage and was reduced on semi-sterile heterozygotes at S22B, implying that the 46 kDa band may associated in hybrid sterility. In addition, causal genes of S22A in T65 were found to be S22A_j1 and S22A_j3 encoding DUF1668-containing protein. The allele of a wild rice species Oryza meridionalis Ng at S22B, designated S22B_m, is a loss-of-function allele probably due to large deletion of the gene lacking DUF1668 domain and evolved from the different lineage of O. glumaepatula. Phylogenetic analysis of DUF1668 suggested that many gene duplications occurred before the divergence of current crops in Poaceae, and loss-of-function mutations of DUF1668-containing genes represent the candidate causal genetic events contributing to hybrid incompatibilities. The duplicated DUF1668-domain gene may provide genetic potential to induce hybrid incompatibility by consequent mutations after divergence.

Production of embryo rescued hybrids between the landrace “Friariello” (Capsicum annuum var. annuum) and C. baccatum var. pendulum: phenotypic and cytological characterization

Capsicum baccatum var. pendulum is a valuable source of disease resistance and fruit quality traits that could play an important role in the genetic improvement and safeguard of local varieties of common peppers. However, its use for C. annuum var. annuum breeding has been scarce due to postzygotic incompatibilities. In this work, we obtained seven interspecific hybrids between the Italian landrace ‘Friariello’ of C. annuum var. annuum and C. baccatum var. pendulum through embryo rescue. The F1 hybrids were verified by SSR markers and by in situ hybridization, which confirmed their hybrid status and provided evidence for differences in the repetitive content between the parental genomes. These hybrids were vigorous and, for several morphological traits, were intermediate between their parents. However, they were male sterile. The cytological analysis revealed several abnormalities occurring at various stages of the male meiosis. A number of these anomalies were likely the result of structural differences (e.g. a reciprocal translocation, a paracentric inversion) between the two genomes. Due to hybrid pollen sterility, backcross progenies (BC1) were obtained when hybrids were used as female parents. A partial restoration of the fertility in BC1 makes promising the exploitation of this material in breeding programs.

Genetic Linkage Map Construction and QTL Analysis of Two Interspecific Reproductive Isolation Traits in Sponge Gourd.

The hybrids between Luffa acutangula (L.) Roxb. and L.cylindrica (L.) Roem. have strong heterosis effects. However, some reproductive isolation traits hindered their normal hybridization and fructification, which was mainly caused by the flowering time and hybrid pollen sterility. In order to study the genetic basis of two interspecific reproductive isolation traits, we constructed a genetic linkage map using an F2 population derived from a cross between S1174 [L. acutangula (L.) Roxb.] and 93075 [L. cylindrica (L.) Roem.]. The map spans 1436.12 CentiMorgans (cM), with an average of 8.11 cM among markers, and consists of 177 EST-SSR markers distributed in 14 linkage groups (LG) with an average of 102.58 cM per LG. Meanwhile, we conducted colinearity analysis between the sequences of EST-SSR markers and the genomic sequences of cucumber, melon and watermelon. On the basis of genetic linkage map, we conducted QTL mapping of two reproductive isolation traits in sponge gourd, which were the flowering time and hybrid male sterility. Two putative QTLs associated with flowering time (FT) were both detected on LG 1. The accumulated contribution of these two QTLs explained 38.07% of the total phenotypic variance (PV), and each QTL explained 15.36 and 22.71% of the PV respectively. Four QTLs for pollen fertility (PF) were identified on LG 1 (qPF1.1 and qPF1.2), LG 3 (qPF3) and LG 7 (qPF7), respectively. The percentage of PF explained by these QTLs varied from 2.91 to 16.79%, and all together the four QTLs accounted for 39.98% of the total PV. Our newly developed EST-SSR markers and linkage map are very useful for gene mapping, comparative genomics and molecular marker-assisted breeding. These QTLs for interspecific reproductive isolation will also contribute to the cloning of genes relating to interspecific reproductive isolation and the utilization of interspecific heterosis in sponge gourd in further studies.

Double‐Neutral Genes, Sa‐n and Sb‐n, for Pollen Fertility in Rice to Overcome Indica × Japonica Hybrid Sterility

A major obstacle in the development of superior intersubspecific hybrids between subspecies (indica and japonica) of Asian cultivated rice (Oryza sativa L.) is partial sterility of the F1 generation. Neutral genes, Sa‐n, Sb‐n, and Sc‐n, for pollen fertility have the potential to overcome F1 pollen sterility. The present study was planned to find double‐neutral genes for pollen fertility and to evaluate their effect on pollen fertility of intersubspecific hybrids. Five genotypes were crossed with eight isogenic lines to mine neutral genes for pollen fertility. Pollen fertility of hybrids was observed in F1 and F2, and segregation ratios were recorded in F2 to confirm the neutral genes. The results showed that DN18 and DN75 had double‐neutral genes (Sa‐n and Sb‐n) whereas DN22 had three neutral genes Sa‐n, Sb‐n, and Sc‐n at Sa, Sb, and Sc pollen sterility loci, respectively. Furthermore, we found a single neutral gene (Sc‐n) for pollen fertility at the Sc locus and neutral gene for embryo sac fertility at the S5 locus in DN13. DN18 and DN75 were crossed with numerous typical japonica and indica cultivars. Results showed that double‐neutral genes could overcome most of the intersubspecific hybrid pollen sterility. These lines could be used for the development of high yielding indica × japonica hybrids and neutral genes could be introgressed into elite cultivars of Asian cultivated rice for fertility restoration.

Identification of neutral alleles at pollen sterility gene loci of cultivated rice (Oryza sativa L.) from wild rice (O. rufipogon Griff.)

Oryza rufipogon Griff. is the ancestor of Asian cultivated rice (O. sativa L.) and possesses valuable genes for rice breeding. Pollen abortion is one of the major causes of indica–japonica hybrid sterility in rice and it happens due to allelic interaction at the pollen sterility gene loci. A total of six loci (Sa, Sb, Sc, Sd, Se, and Sf) have been found to be associated with F1 pollen sterility between indica and japonica rice, and five of them (all except Sf) have been mapped. Neutral alleles (Sn) at each locus have the potential to overcome the pollen sterility associated with the respective locus. Therefore, exploitation and utilization of neutral alleles have significant importance in overcoming indica–japonica hybrid sterility. In this study, an accession (IRW28) of O. rufipogon, native to China, was selected as paternal to cross with typical japonica (Taichung 65) and indica (Guanglu’ai 4) tester lines, and two F2 populations were developed. The simple sequence repeat markers tightly linked to five pollen sterility loci were applied for genotyping the F2 populations. Chi-squared tests were applied to examine the normal segregation/distortion at each locus. The expected and observed pollen sterility for each locus were estimated. As a result, the genotypes at five pollen sterility gene loci for IRW28 were identified as: Sai−1/Sai−1, Sbn/Sbn, Sci−2/Sci−2, Sdn/Sdn and Sen/Sen. Our results suggest that IRW28 (O. rufipogon) has the neutral alleles for pollen fertility at the Sb, Sd and Se loci, and these alleles have a good affinity with indica and japonica rice. These neutral alleles provide valuable gene resources to overcome the inter-subspecific hybrid pollen sterility in rice.

Mitochondrial gene in the nuclear genome induces reproductive barrier in rice

Hybrid incompatibility in F(1) hybrids or later generations is often observed as sterility or inviability. This incompatibility acts as postzygotic reproductive isolation, which results in the irreversible divergence of species. Here, we show that the reciprocal loss of duplicated genes encoding mitochondrial ribosomal protein L27 causes hybrid pollen sterility in F(1) hybrids of the cultivated rice Oryza sativa and its wild relative O. glumaepatula. Functional analysis revealed that this gene is essential for the later stage of pollen development, and distribution analysis suggests that the gene duplication occurred before the divergence of the AA genome species. On the basis of these results, we discuss the possible contribution of the "founder effect" in establishing this reproductive barrier.

Fine mapping of a gene causing hybrid pollen sterility between Yunnan weedy rice and cultivated rice (Oryza sativa L.) and phylogenetic analysis of Yunnan weedy rice

Weedy rice represents an important resource for rice improvement. The F(1) hybrid between the japonica wide compatibility rice cultivar 02428 and a weedy rice accession from Yunnan province (SW China) suffered from pollen sterility. Pollen abortion in the hybrid occurred at the early bicellular pollen stage, as a result of mitotic failure in the microspore, although the tapetum developed normally. Genetic mapping in a BC(1)F(1) population (02428//Yunnan weedy rice (YWR)/02428) showed that a major QTL for hybrid pollen sterility (qPS-1) was present on chromosome 1. qPS-1 was fine-mapped to a 110 kb region known to contain the hybrid pollen sterility gene Sa, making it likely that qPS-1 is either identical to, or allelic with Sa. Interestingly, F(1) hybrid indicated that Dular and IR36 were assumed to carry the sterility-neutral allele, Sa ( n ). Re-sequencing SaM and SaF, the two component genes present at Sa, suggested that variation for IR36 and Dular may be responsible for the loss of male sterility, and the qPS-1 sequence might be derived from wild rice or indica cultivars. A phylogenetic analysis based on microsatellite genotyping suggested that the YWR accession is more closely related to wild rice and indica type cultivars than to japonica types. Thus it is probable that the YWR accession evolved from a spontaneous hybrid between wild rice and an ancient cultivated strain of domesticated rice.

Identification and fine mapping of S-d, a new locus conferring the partial pollen sterility of intersubspecific F1 hybrids in rice (Oryza sativa L.)

The partial pollen abortion of hybrids between the indica and japonica subspecies of Asian cultivated rice is one of the major barriers in utilizing intersubspecific heterosis in hybrid rice breeding. Although a single hybrid pollen sterility locus may have little impact on spikelet fertility, the cumulative effect of several loci usually leads to a serious decrease in spikelet fertility. Isolating of the genes conferring hybrid pollen sterility is necessary to understand this phenomenon and to overcome the resulting genetic barrier. In this study, a new locus for F1 pollen sterility, S-d, was identified on the short arm of chromosome 1 by analyzing the genetic effect of substituted segments of the near-isogenic line E11-5 derived from the japonica variety Taichung 65 (recurrent parent) and the indica variety Dee-geo-woo-gen (donor parent). The S-d locus was first mapped to a 0.8 cM interval between SSR markers PSM46 and PSM80 using a F2 population of 125 individuals. The flanking markers were then used to identify recombinants from a population of 2,160 plants derived from heterozygotes of the primary F2 population. Simultaneously, additional markers were developed from genomic sequence divergence in this region. Analysis of the recombinants in the region resulted in the successful mapping of the S-d locus to a 67-kb fragment, containing 17 predicted genes. Positional cloning of this gene will contribute to our understanding of the molecular basis for partial pollen sterility of intersubspecific F1 hybrids in rice.

Two novel loci for pollen sterility in hybrids between the weedy strain Ludao and the Japonica variety Akihikari of rice (Oryza sativa L.)

Partial pollen sterility has been observed in hybrid progeny derived from a japonica cultivar, Akihikari and a weedy strain, Ludao, which naturally grows in Jiangsu province of east China. Cytological and histological analyses revealed that pollen abortion occurred largely at the bicellular pollen stage, primarily due to the gradual disaggregation of generative and vegetative cells. A genome-wide analysis was further carried out in a backcross population of Akihikari //Ludao/Akihikari using a total of 118 simple sequence repeat (SSR) markers and an expressed sequence tag (EST) marker distributed on the entire rice linkage map. Two loci controlling hybrid pollen sterility, designated as S33(t) and S34(t), were located on chromosomes 3 and 11, respectively. Both loci were putatively different from all the previously reported gametophyte genes and hybrid pollen sterility loci. Interaction between the Ludao and Akihikari alleles at each of the two loci resulted in reduction of fertility in the pollens carring the Ludao alleles. To map the precise location of the major locus, S33(t), we selected 165 plants of the backcross population with pollen fertility higher than 80.0%, and assayed the recombinant events surrounding the S33(t) locus using newly developed SSR markers. The S33(t) was delimited to an 86 kb region between SSR markers RM15621 and RM15627. Sequence analysis of this region indicated that there were ten open reading frames. These results will be valuable for cloning this gene and marker-assisted transferring of the corresponding neutral allele in rice breeding programs. Furthermore, the origin of the weedy strain Ludao is discussed.

Molecular mapping of S-c, an F1 pollen sterility gene in cultivated rice

Hybrids between indica and japonica rice varieties usually show partial sterility, and are a major limiting factor in the utilization of heterosis at subspecific level. When studying male-gamete (pollen) abortion, a possibly important cause for sterility, six loci (S-a, S-b, S-c, S-d, S-e and S-f) for F1 pollen sterility were identified. Here we report genetic and linkage analysis of S-c locus using molecular markers in a cross between Taichung 65, a japonica variety carrying allele S-cj, and its isogenic line TISL5, carrying alleleS-cj. Our results show that pollen sterility occurring in the hybrids is controlled by one locus. We used 208 RFLP markers, as well as 500 RAPD primers, to survey the polymorphism between Taichung 65 and TISL5. Six RFLP markers located on a small region of chromosome 3, detected different RFLP patterns. Co-segregation analysis of fertility and RFLP patterns with 123 F2 plants confirmed that the markers RG227, RG391, R1420 were completely linked with the S-c locus. The genetic distances between the markers C730, RG166 and RG369 and the S-c locus were 0.5 cM, 3.4 cM, and 3.4 cM respectively. Distorted F2 ratios were also observed for these 4 RFLP markers in the cross. This result suggests that the `one locus sporo-gametophytic' model could explain F1 hybrid pollen sterility in cultivated rice. RG227, the completely linked marker, has been converted to STS marker for marker-assisted selection.

The contribution of epistasis to species differences in annual sunflowers.

The contribution of epistasis to 15 morphological traits differentiating two annual sunflowers (Helianthus annuus and H. debilis ssp. cucumerifolius) and to hybrid pollen sterility was estimated in a first generation backcross (BC1) mapping population. Analysis of digenic interactions among quantitative trait loci (QTLs) with significant main effects revealed significant interaction effects for six of the 15 morphological traits and for pollen sterility. Likewise, a genome-wide scan of all possible two-locus combinations detected additional significant interactions for three of the traits with significant epistasis in the original analysis: stem pigmentation, phyllary pubescence, and pollen viability. However, these were the only traits of the 16 examined in which detected interactions explained more than 5% of phenotypic variance. The implications of these findings for adaptive evolution and for the introgression of advantageous morphological QTLs across a natural hybrid zone between these taxa are discussed.

Characterization of Foxtail Millet, Setaria italica (L.) P. Beauv., in Pakistan Based on Intraspecific Hybrid Pollen Semi-sterility.

Sixteen strains of foxtail millet, Setaria italica (L.) P. Beauv., originally collected from Pakistan were crossed with three strains which had previously been used as testers for classifying six landrace groups based on hybrid pollen sterility. Wide variation was observed in pollen fertility of the F1 hybrids, ranging from 14.2% to 94.7%, while pollen fertility of all the selfed Pakistani strains and testers was normal. Pakistani strains were classified into type C (8 strains), type BC (2) and type X (6) on the basis pollen fertility of more than 75% is normal fertility. No A, B or AC type strains were detected. Type C was found in land-races collected from Punjab Province, the southern part of the North-West Frontier Province (N. W. F. P.) and Baltistan. Type X was distributed in the northern part of N. W. F. P. and an adjacent area of the western part of Gilgit Agency, in the northeastern part of Gilgit Agency and in Baltistan. Different landrace groups were suggested among the strains of type X by the variation in hybrid sterility observed with each tester. This observation was strongly supported by an almost sterile hybrid between type X strains from N. W. F. P. and the northeastern part of Gilgit Agency. Type BC was found in Gilgit Agency and Baluchistan. Landrace groups classified by hybrid pollen fertility did not show any clear relationship with morphological groups which had a different geographical distribution. The present results imply that S. italica, with various phylogenetic backgrounds, might have been introduced into this region several times, and subsequently evolved into three distinct morphological groups.