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. 2025 May 27;18:43. doi: 10.1186/s12284-025-00803-6

Identification and Genetic Analysis of Collinearity Loci for Interspecific Hybrid Sterility in Genus Oryza

Ying Yang 1,#, Qiuhong Pu 1,#, Yonggang Lv 1,#, Jing Li 1, Jiawu Zhou 1, Xianneng Deng 1, Xuanchen Song 1, Yu Zhang 1,✉, Dayun Tao 1,✉
PMCID: PMC12116409  PMID: 40423911

Abstract

Background

Hybrid sterility is a common phenomenon in hybrids between the Asian cultivated rice (Oryza sativa L.) and its relatives with AA genome, which limits the utilization of interspecific heterosis and favorable gene introgression. Numerous loci for hybrid sterility have been identified between O. sativa and its relatives. However, it remains elusive whether hybrid sterility between different species is controlled by a set of conserved loci, and whether there are variations in the genetic mode of these loci.

Results

In this study, six novel hybrid sterility loci for pollen sterility were identified from different cross combinations between O. sativa and its three wild relatives. S59 caused hybrid pollen sterility in hybrids between O. sativa and O. rufipogon. S60 and S61 controlled the hybrid pollen sterility between O. sativa and O. glumaepatula. S62, S63 and S64 governed the hybrid pollen sterility between O. sativa and O. barthii. Genetic and linkage analysis showed that S59, S60, and S62 were located in near the same region on the short arm of chromosome 5. S61 and S63 were mapped near RM27460 on the short arm of chromosome 12. S64 was restricted into the 60.27 kb region between RM4853 and RM3372 on the short arm of chromosome 3. The genetic behavior of six novel hybrid sterility loci follows one-locus allelic interaction model, the male gametes carrying the alleles of O. sativa in the heterozygotes were selectively aborted except for S62.

Conclusions

The findings from this research would provide a better understanding for the genetic nature of interspecific hybrid sterility in rice.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12284-025-00803-6.

Keywords: Rice, Interspecific hybrid sterility, Segregation distortion, Collinearity, Orthologous

Background

Asian cultivated rice (Oryza sativa L.) is the primary food crop for half of the global population. Its genetic diversity has become increasingly narrow during domestication from wild ancestors to cultivated rice and modern improvements. The seven relatives of Asian cultivated rice with the same AA genome and similar sequence arrangement as O. sativa, are considered to be a valuable source for genetic improvement (Chang 1976; Ohmido et al. 1995; Xiao et al. 1998; Vaughan et al. 2003). However, the severe reproductive isolation of interspecific hybrids between O. sativa and its AA genome relatives hinders the utilization of favorable genes in wild rice.

Reproductive isolation is a natural biological phenomenon restricting gene flow between populations, indicating speciation and maintaining species identity (Sano 1986; Orr et al. 2000). Hybrid sterility is a major form of postzygotic reproductive isolation in rice. Since the first report on the phenomenon of hybrid sterility between O. sativa subsp. indica and O. sativa subsp. japonica nearly 100 years ago, approximately 60 hybrid sterility loci have been reported in interspecific and intraspecific of rice so far (Kato et al. 1928; Ouyang et al. 2018; Zhang et al. 2022). Till now, 15 hybrid sterility loci have been cloned and characterized. Among those loci, S1 (Xie et al. 2017; Koide et al. 2018; Xie et al. 2019), S5 (Chen et al. 2008; Yang et al. 2012), S7 (Yu et al. 2016), S13 (Myint et al. 2024), S22 A/S22B (Sakata et al. 2021), Sa (Long et al. 2008), Sc (Shen et al. 2017), ESA1 (Hou et al. 2019), hsa1 (Kubo et al. 2016), qHMS1 (You et al. 2023), qHMS7 (Yu et al. 2018), and pf12/RHS12/Se (Zhou et al. 2023; Wang et al. 2023a, b) fit the one-locus allelic interaction model. Three hybrid sterility pairs, including S27/S28 (Yamagata et al. 2010), DPL1/DPL2 (Mizuta et al. 2010), DSG1/DSG2 (Nguyen et al. 2017), followed the two-locus epistatic interaction model.

During the genetic mapping study of hybrid sterility loci between O. sativa and its AA genome relatives, researchers frequently identified six “hot spots” from different species. A comparison of the mapping results for all these hybrid sterility loci suggested that some loci arising from multiple species should be allelic to each other, since they were located to the same chromosome region and had similar genetic pattern (Li et al. 2020). Further research has identified some of these loci as homologous loci. One such locus is the qHMS7, which is responsible for the pollen semi-sterility in the hybrid between O. sativa and O. meridionalis. This locus contains two tightly linked genes, ORF2 and ORF3. ORF2 encodes a toxic genetic element that leads to the abortion of pollen in a sporophytic manner. On the other hand, ORF3 encodes an antidote that protects pollen in a gametophytic way. Researchers analyzed the haplotype of ORF2 and ORF3 in different AA genome species. They found that ORF2 was present in all sequenced accessions, while ORF3 only had one haplotype identified in parts of O. rufipogon and most of O. sativa. Still, ORF3 was not detected in O. meridionalis, O. glaberrima, O. barthii, and O. longistaminata (Yu et al. 2018). The mapping interval for qHMS7 is close to several hybrid sterility loci reported previously, such as S21 (detected from crosses between O. sativa and O. glaberrima, O. sativa and O. rufipogon, respectively) and S23 (identified from the cross between O. sativa and O. glumaepatula) (Doi et al. 1999; Miyazaki et al. 2007; Sobrizal et al. 2000a, b; Li et al. 2018). Compared to qHMS7, ORF2 and ORF3 of S23 also have the “Toxin-Antidote” function (Fang et al. 2019). However, there are significant differences between S23 and qHMS7 in their genetic effects. S23 and qHMS7 exhibit varying degrees of causing hybrid F1 pollen abortion, and the expression of S23 is influenced by the environment. Meanwhile, qHMS7 is not affected by the environment (Yu et al. 2018; Fang et al. 2019). This result suggests that qHMS7 and S23 were hybrid sterility orthologous loci between O. sativa and its wild relatives O. meridionalis, and O. glumaepatula. However, the allele from O. glumaepatula is expected to be a distinct allele from O. meridionalis.

Another example is the S1 locus, which plays a crucial role in determining the occurrence of hybrid sterility between two cultivated rice species, O. sativa and O. glaberrima (Koide et al. 2008b; Garavito et al. 2010; Zhou et al. 2010). The male and female gametes carrying the O. sativa allele, S1-s, are selectively aborted in the heterozygote, while those carrying the O. glaberrima allele (S1-g) survive. S1-g is composed of three adjacent genes S1 TPR, S1 A4 and S1 A6 (SSP), these three genes produce a complex gamete-killer system, but the O. sativa allele S1-s lacks all these genes (Xie et al. 2017, 2019; Koide et al. 2018). Several studies identified S10 as another locus that induces both male and female gamete abortion in a cross between japonica and indica. Interestingly, S10 was mapped in a similar position as S1 on chromosome 6 (Sano et al. 1994), and some further studies suggest that S1 and S10 were allelic to each other (Heuer and Miézan 2003; Zhu et al. 2005). A major locus qpsf6 for hybrid sterility was also identified near the SSR marker RM587 on the short arm of chromosome 6. This QTL is responsible for pollen and spikelet fertility in the cross between O. sativa and O. longistaminata. Comparing their mapping region and genetic effect indicated that this QTL coincides with S1 (Chen et al. 2009). In addition, three hybrid sterility loci were identified, and showed good collinearity with S1 in the hybrid combinations obtained from O. sativa crossed with O. rufipogon, O. nivara, and O. barthii, respectively (Yang et al. 2016). These results indicated that the hybrid sterility loci S1 between O. sativa and O. glaberrima, O. barthii, O. longistaminata, O. rufipogon, O. nivara should be an orthologous locus. Although some hybrid sterility loci located in the same interval were identified to be homologous loci through further research, while the genetic effects of these orthologous loci are still unclear as they were identified from distinct hybrid crosses in different genetic backgrounds.

In this study, six novel loci for interspecific hybrid male sterility in the hybrids between O. sativa and its three wild relatives were confirmed on chromosome 3, 5 and 12, respectively. Among them, S59, S60, and S62 were responsible for interspecific hybrid pollen sterility in the crosses between the Asian cultivated rice cultivar DJY1 and O. rufipogon, O. glumaepatula, and O. barthii, respectively. They were located near the RM1024 on chromosome 5 and showed good collinearity. S61 identified from the cross between DJY1 and O. glumaepatula, and S63 found from the cross between DJY1 and O. barthii were both mapped to the same region on chromosome 12. Another locus, S64, was located on the short arm of chromosome 3 in the cross between DJY1 and O. barthii. This study showed that the hybrid sterility loci identified from different hybrid combinations were mapped in the same or similar chromosome regions, and they may be allelic to each other. In addition, the different degree of segregation distortion and the mode of gamete transmission were observed among the collinearity loci in the same genetic background. These results would be beneficial for further understanding the nature of interspecific hybrid sterility in rice and help us understand the function of collinearity loci for hybrid sterility in Oryza genus divergence.

Results

Genetic Analysis of Hybrid Pollen Semi-Sterility

The recurrent parent DJY1 and NILs, harbored the different hybrid sterility loci, had normal pollen and spikelet fertility. In contrast, all six BC7F1 hybrids developed from the crosses between NILs and DJY1 exhibited pollen semi-sterility, and the sterile pollen type was stained abortion, while the spikelet fertility of all six BC7F1 hybrids was fertile (Fig. 1; Table 1). Pollen fertility in the six BC7F2 populations were segregated into fertile and semi-sterile classes, fitting a 1:1 segregation ratio. However, the spikelet fertility of all six BC7F2 populations was normal, similar to recurrent parent and NILs (Fig. 2). These results suggest that pollen sterility in the BC7F1 hybrids was controlled by a single locus, attributed to single locus male sterility.

Fig. 1.

Fig. 1

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Pollen grain diagrams of the recurrent parent DJY1, near-isogenic lines (NILs) for each sterility locus, and the hybrid F1 derived from crosses between the recurrent parent and the NILs for each sterility locus, as visualized by 1% (w/v) I2-KI staining (a–m). Scale bar, 100 μm

Table 1.

Pollen and spikelet fertility (%) of F1s and their parents

Parent and F1 Donor Generation Pollen ferility (%) Spikelet fertility (%)
Species Accession
DJY1 O. sativa 96.34 ± 1.73 97.08 ± 2.69
NIL-S59 O. rufipogon Acc.106,138 BC6F2 97.43 ± 1.98 93.91 ± 2.92
NIL-S60 O. glumaepatula Acc.105,662 BC6F4 95.03 ± 3.6 92.67 ± 3.54
NIL-S61 O. glumaepatula Acc.105,662 BC6F3 98.25 ± 0.63 91.62 ± 3.28
NIL-S62 O. barthii Acc.104,061 BC6F4 96.01 ± 2.12 92.04 ± 2.89
NIL-S63 O. barthii Acc.100,117 BC6F3 97.45 ± 0.90 92.58 ± 2.91
NIL-S64 O. barthii Acc.100,117 BC6F3 94.25 ± 2.77 94.86 ± 3.27
NIL-S59/DJY1 BC7F1 52.83 ± 2.37 93.53 ± 2.8
NIL-S60/DJY1 BC7F1 47.07 ± 3.52 95.06 ± 3.1
NIL-S61/DJY1 BC7F1 51.27 ± 1.23 94.12 ± 1.52
NIL-S62/DJY1 BC7F1 49.91 ± 0.31 94.57 ± 2.03
NIL-S63/DJY1 BC7F1 46.46 ± 2.49 91.77 ± 3.36
NIL-S64/DJY1 BC7F1 49.56 ± 0.74 92.51 ± 1.91
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Fig. 2.

Fig. 2

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Frequency distribution of pollen and spikelet fertility in BC7F2 populations. BC7F2 mapping populations derived from NILs × DJY1 for S59 (a), S60 (b), S61 (c), S62 (d), S63 (e) and S64 (f), respectively. The χ²(1:1) value reflects the Chi-square test performed on the segregation ratio of pollen fertility in the BC7F2 population, hypothesizing that the ratio of pollen fertile to sterile plants follows the theoretical 1:1 ratio

Molecular Mapping of Hybrid Sterility Loci

To map the hybrid sterility loci, polymorphic SSR markers from the introgression regions based on the Rice 6k Chips, were utilized to genotype the corresponding BC7F2 mapping populations (Table S2). The ANOVA analysis of genotypic and phenotypic data in the mapping populations indicated that there were six loci controlling pollen sterility on chromosome 3, 5, and 12, respectively (Table 2).

Table 2.

The segregation of marker genotypes in BC7F2 populations

Loci Species Accession Generation Chr Markera No. of individuals Marker segregation Inline graphic Aborted gamete
DDb DW WW
S59 O. rufipogon Acc.106,138 BC7F2 5 RM1024 463

7

(95.16 ± 4.17)c

225

(57.24 ± 7.13)

231

(98.35 ± 3.80)

 217.11 S-DJY1
S60 O. glumaepatula Acc.105,662 BC7F2 5 RM1024 488

36

(95.21 ± 11.90)

228

(51.99 ± 9.69)

224

(97.49 ± 5.86)

 146.95 S-DJY1
S61 O. glumaepatula Acc.105,662 BC7F2 12 RM27460 505

5

(81.41 ± 24.43)

249

(52.48 ± 8.89)

251

(96.24 ± 6.67)

 239.76 S-DJY1
S62 O. barthii Acc.104,061 BC7F2 5 RM1024 498

177

(98.54 ± 0. 92)

248

(56.15 ± 12.22)

73

(98.41 ± 1.44)

 43.45 S-barthii
S63 O. barthii Acc.100,117 BC7F2 12 RM27460 415

21

(94.33 ± 2.56)

218

(58.54 ± 7.42)

176

(97.48 ± 2.59)

 116.85 S-DJY1
S64 O. barthii Acc.100,117 BC7F2 3 RM3372 522

11

(96.67 ± 0.81)

235

(51.27 ± 6.69)

276

(96.87 ± 1.17)

 274.24 S-DJY1
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aMarker information is from the Gramene website (http://archive.gramene.org/markers)

, bDD, DW and WW indicated DJY1- homozygous, heterozygous and wild rice- homozygous genotypes, respectively

cPollen fertility (%)

Ten polymorphic SSR markers on the short arm of chromosome 5 were used to genotype the 463 individuals in the BC7F2 population derived from the cross between NIL-S59 and DJY1. As a result, the S59 locus was restricted to a 2.6 cM region flanked by RM17804 and RM1024, at genetic distance of 0.4 and 2.2 cM, respectively (Fig. 3b). The physical distance between the two SSR markers was about 87.8 kb based on the Gramene public database (http://www.gramene.org).

Fig. 3.

Fig. 3

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Genetic location and collinearity analysis for S59, S60, and S62 loci on chromosome 5. a The location interval of intraspecific hybrid sterility loci S24, Sb, and f5-Du which were identified from indica. b–d The genetic location of S59, S60 and S62, respectively

In order to map the hybrid sterility locus S60 which was identified from the cross between O. sativa and O. glumaepatula, twelve polymorphic SSR markers from the introgressed region on chromosome 5 were used to genotype 488 plants in the BC7F2 population from the cross between NIL-S60 and DJY1. Then, S60 was located within a 1.4 cM region flanked by RM1024 and RM17812, with genetic distances of 0.5 and 0.9 cM, respectively (Fig. 3c). Another hybrid sterility locus S61 identified from the cross between O. sativa and O. glumaepatula was mapped into a 0.5 cM region flanked by RM8109 and RM27478 on the short arm of chromosome 12, at genetic distances of 0.2 and 0.3 cM, respectively (Fig. 4d).

Fig. 4.

Fig. 4

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Genetic location and collinearity analysis for S61, S63 loci on chromosome 12. a–c S25, S36, S39(t), qs12, pf12, RHS12 and Se were mapped into similar region on chromosome 12. d–e the genetic location of S61 and S63, respectively

In this study, three hybrid sterility loci identified from the cross between O. sativa and O. barthii, were resigned as S62, S63, and S64, respectively. A total of 498 individuals from the BC7F2 generation crossed by NIL-S62 and DJY1 were used to detect the pollen fertility and genotype. Then, the S62 locus was located to a 2.5 cM region flanked by RM1024 and RM17834 on chromosome 5, at genetic distances of 0.9 and 1.6 cM, respectively (Fig. 3d). And S63 was anchored to a 4.4 cM region flanked by RM27460 and RM27478 on chromosome 12 (Fig. 4e). Nine polymorphic SSR markers were used to genotype 522 individuals in the BC7F2 population that derived from NIL-S64 and DJY1. S64 was preliminarily confined to a 1.15 cM region between RM3894 and RM22 on the short arm of chromosome 3, at a genetic distance of 1.08 and 0.07 cM, respectively (Fig. 5d). Next, S64 was mapped into a 60.29 kb region between RM4853 and RM3372 by map-based cloning (Figure S2).

Fig. 5.

Fig. 5

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Genetic location and collinearity analysis for S64 locus on chromosome 3. a–c The genetic location of S19 identified from O. glaberrima. d S64 from O. barthii was restricted into the 60.27 kb region between RM4853 and RM3372 in this study

To sum up, we identified six novel interspecific hybrid male sterility loci in the hybrids between O. sativa and its three wild relatives on chromosome 3, 5 and 12, respectively. Among them, S59, S60, and S62 were responsible for pollen sterility in the crosses between the Asian cultivated rice cultivar DJY1 and O. rufipogon, O. glumaepatula, and O. barthii, respectively. Compared with the previous report, S59, S60, and S62 had good collinearity with S24, Sb, and f5-Du for intersubspecific hybrid sterility in Asian cultivated rice (Kubo et al. 2008; Li et al. 2006; Wang et al. 2006). S61 identified from the cross between DJY1 and O. glumaepatula, and S63 found from the cross between DJY1 and O. barthii were both mapped to the same region on chromosome 12. They were located near several previously identified QTL for hybrid pollen sterility from multiple crosses between indica and japonica varieties (S25, qs12, pf12, RHS12, and Se), and between O. sativa and O. nivara (S36), as well as between O. sativa and O. glaberrima (S39(t) (Win et al. 2009; Xu et al. 2014; Kubo et al. 2017; Zhou et al. 2023; Wang et al. 2023a, b). Another locus, S64, was located on the short arm of chromosome 3 in the cross between DJY1 and O. barthii. The mapping region of S64 was similar to the region of S19 identified in cross between O. sativa and O. glaberrima (Taguchi et al. 1999; McCouch et al. 2002; Zhang et al. 2011).

Segregation Distortion of the Six Interspecific Hybrid Sterility Loci

Hybrid sterility often results in segregation distortion due to the abortion of gametes from one of the parents. In this study, the degree of segregation distortion and the mode of gamete transmission for the six interspecific hybrid sterility loci were analyzed by detecting the segregation of three genotypes in BC7F2 populations and the transmission ratio of hybrid sterility loci for male and female gamete in the reciprocal cross populations using the tightly linked SSR markers.

Linkage analysis showed that the S59, S60 and S62 for interspecific hybrid sterility were tightly linked with RM1024 on the short arm of chromosome 5. The interaction between S59-sativa and S59-rufipogon led to the partial abortion of male gametes carrying the allele of S59-sativa in the heterozygotes, and only 1.51% homozygotes of S59-sativa existed in the BC7F2 population. A similar genetic behavior of gametes abortion was observed in S60, with 7.38% homozygotes of O. sativa in the BC7F2 population. Conversely, for S62, the male gametes from O. barthii were partially aborted in the heterozygotes, and the male gametes from O. sativa were preferentially transmitted to the next generation. Hence, as for S59 and S60, wild species alleles were preferentially transmitted to progeny, whereas the wild species allele at the S62 locus was selectively aborted in the heterozygotes. Reciprocal cross populations were raised to further analyze the genetic effect of the S59, S60 and S62, respectively (Table 3). The results showed that for S59 and S60, when F1 was used as the male parent to cross with DJY1 and NIL, respectively, the ratio of the surviving male gamete S59-sativa and S60-sativa deviated significantly from the expected ratio of 1:1 in the progeny. However, when F1 was used as the female parent, the ratio of heterozygous and homozygous plants in the progeny was consistent with the segregation ratio of 1:1 as expected. These findings suggest that male gamete of S59-sativa and S60-sativa cannot be transmitted normally, while the female gamete of S59-sativa and S60-sativa can be transmitted normally. Regarding S62, when F1 was used as a female parent crossed with recurrent parent DJY1 and NIL, respectively, the ratio of heterozygous and homozygous plants fitted into 1:1, suggesting that female gametes of both S62-sativa and S62-barthii can be transmitted equally. Interestingly, when F1 was used as male parent, it was found that the transmission ratio of the male gamete S62-sativa was significantly higher than that of S62-barthii. It was suggested the male gamete of S62-barthii cannot be transmitted normally (Table 3). Taken together, although S59, S60 and S62 were located within a similar region, the degree and direction of gamete segregation distortion varied among the different loci.

Table 3.

The transmission ratio of hybrid sterility loci S59, S60 and S62 on chromosome 5 for male and female gamete in the reciprocal cross populations

Loci Marker (RM1024, Chr. 5) Progeny genotype Inline graphic Transmission ratio
Female genotype Male genotype DD WW DW Total D W
S59 DD DW 21 93 114 45.474 0.18 0.82
DW DD 68 75 143 0.343 0.48 0.52
WW DW 108 17 125 65.73 0.14 0.86
DW WW 126 127 253 0.004 0.50 0.50
S60 DD DW 26 159 185 95.616 0.14 0.86
DW DD 78 107 185 4.546 0.42 0.58
WW DW 76 7 83 57.361 0.08 0.92
DW WW 119 106 225 0.751 0.47 0.53
S62 DD DW 87 4 91 75.703 0.95 0.05
DW DD 48 40 88 0.727 0.55 0.45
WW DW 10 75 85 49.706 0.88 0.12
DW WW 36 48 84 1.714 0.57 0.43
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DD, DW and WW indicated DJY1- homozygous, heterozygous and wild rice- homozygous genotypes, respectively

S61 and S63 were located on the same interval on the short arm of chromosome 12. Extreme segregation distortion was found in S61, only 0.99% homozygotes of S61-sativa were found in the BC7F2 population. The interaction between S61-sativa and S61-glumaepatula led to the nearly complete abortion of male gametes carrying the allele of S61-sativa in the heterozygotes (Table 2). Compared to S61, the gamete elimination effect of S63 was lower, 5.06% homozygotes of S63-sativa were found in the BC7F2 population. In addition, the gamete transmission ratio of S61 and S63 were investigated by reciprocal crosses. For S61, the ratio of heterozygous and homozygous plants deviated significantly from the expected ratio of 1:1 in four reciprocal cross populations, regardless of whether the heterozygote was used as either the male or the female parent. And the transmission rate of O. sativa-type gamete was significantly higher when heterozygous plants were used as female parents. The results showed that both male and female gamete of S61-sativa cannot be transmitted normally. For S63 locus, when F1 was used as the male parent to cross with DJY1 and NIL, respectively, the ratio of the surviving male gamete S63-sativa deviated significantly from the expected ratio of 1:1 in backcross populations. However, when F1 was used as the female parent, the ratio of heterozygous and homozygous plants in the progeny was consistent with the expected ratio of 1:1. These findings suggest that male gamete of S63-sativa cannot be transmitted normally, while the female gamete of S63-sativa can be transmitted equally (Table 4).

Table 4.

The transmission ratio of hybrid sterility loci S61 and S63 on chromosome 12 for male and female gamete in the reciprocal cross populations

Loci Marker (RM27460, Chr. 12) Progeny genotype Inline graphic Transmission ratio
Female genotype Male genotype DD WW DW Total D W
S61 DD DW 7 93 100 73.96 0.07 0.93
DW DD 29 71 100 17.64 0.29 0.71
WW DW 50 8 58 30.414 0.14 0.86
DW WW 38 12 50 13.52 0.24 0.76
S63 DD DW 21 179 200 124.82 0.11 0.89
DW DD 34 45 79 1.532 0.43 0.57
WW DW 80 6 86 63.674 0.07 0.93
DW WW 130 89 219 7.676 0.41 0.59
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DD, DW and WW indicated DJY1- homozygous, heterozygous and wild rice- homozygous genotypes, respectively

S64 is a pollen sterility locus located on the short arm of chromosome 3. The interaction between S64-sativa and S64-barthii resulted in the partial abortion of male gametes carrying the allele of S64-sativa in the heterozygotes, the homozygous O. sativa plants were less than homozygous O. barthii plants in the BC7F2 population (Table 2). The reciprocal cross test also indicated that the male gamete transmission rate of O. barthii was significantly higher when heterozygous plant was used as male parent (Table 5). This result indicated that pollen semi-sterility in hybrid and segregation distortion at S64 locus were due to the selective abortion of pollen grains carrying the O. sativa allele.

Table 5.

The transmission ratio of hybrid sterility locus S64 on chromosome 3 for male and female gamete in the reciprocal cross populations

Locus Marker (RM3372, Chr. 3) Progeny genotype Inline graphic Transmission ratio
Female genotype Male genotype DD WW DW Total D W
S64 DD DW 21 146 187 93.563 0.11 0.89
DW DD 94 102 196 0.327 0.48 0.52
WW DW 113 16 129 72.938 0.12 0.88
DW WW 104 87 191 1.513 0.46 0.54
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DD, DW and WW indicated DJY1- homozygous, heterozygous and wild rice- homozygous genotypes, respectively

Discussion

Six Novel Hybrid Sterility Loci Identified in O. sativa and its Three Wild Relatives

In this study, six loci responsible for interspecific hybrid sterility were identified from the crosses between O. sativa and its three wild relatives, including S59 identified from the cross between O. sativa and O. rufipogon, S60 and S61 identified from the cross between O. sativa and O. glumaepatula, S62, S63, and S64 identified from the crosses between O. sativa and O. barthii (Figs. 3, 4 and 5). By now, three loci responsible for hybrid sterility between O. sativa and O. rufipogon have been reported, including S6, S21(t) and qss6-b (Sano 1990; Miyazaki et al. 2007; Koide et al. 2008a, 2012; Yang et al. 2016). S6 and qss6-b were mapped on chromosome 6, and S21(t) was located on the long arm of chromosome 7. Therefore, S59 should be a new locus controlling pollen sterility in hybrids between O. sativa and O. rufipogon. Six loci responsible for hybrid pollen sterility between O. sativa and O. glumaepatula were reported, including S12, S22 A, S22B, S23(t), S27, S28, and S56(t) (Sano 1994; Sobrizal. et al. 2000a, b, 2001, 2002; Zhang et al. 2018). The chromosomal location of S12 was unknown (Sano 1994). S22 A and S22B were mapped on chromosome 2 (Sakata et al. 2014, 2021), S23(t) was mapped on chromosome 7 (Sobrizal et al. 2000b). S27 and S28 were located on chromosome 4 and chromosome 8, respectively (Sobrizal et al. 2001; Sobrizal et al. 2002; Yamagata et al. 2010), and S56(t) was mapped into the region between RM20797 and RM1093 on chromosome 7 (Zhang et al. 2018). Thus, S60 located on chromosome 5 and S61 located on chromosome 12 were novel loci for interspecific hybrid sterility between O. sativa and O. glumaepatula. The qss6-c locus was located on chromosome 6, which was responsible for both pollen and spikelet sterility between O. sativa and O. barthii. It had a good co-linear relationship with S1 (Sano 1990; Yang et al. 2016). Therefore, S62, S63, and S64 are novel loci that control male gamete development in hybrids between O. sativa and O. barthii.

Different Alleles of the Orthologous Loci Showed Different Genetic Effect on Hybrid Sterility

In this study, S59, S60, and S62 were located on chromosome 5 and shared the similar chromosome region with S24, Sb, and f5-Du, which were mapped as pollen killer for intersubspecific hybrid sterility in the Asian cultivated rice (Kubo et al. 2008; Li et al. 2006; Wang et al. 2006). Another two loci, S61 and S63, the comparison of map positions showed that S61 and S63 on chromosome 12 had good co-linear with S25, qs12, pf12, RHS12, and Se identified from multiple crosses between indica and japonica varieties (Kubo et al. 2017; Zhou et al. 2023; Wang et al. 2023a, b), and S36 identified from the cross between O. sativa and O. nivara (Win et al. 2009), as well as S39(t) identified from the cross between O. sativa and O. glaberrima (Xu et al. 2014). The mapping region of S64 coincided with S19, which was identified from the cross between O. sativa and O. glaberrima (Taguchi et al. 1999; McCouch et al. 2002; Zhang et al. 2011). Furthermore, the sterile pollen of these loci heterozygotes showed stained abortion phenotype by I2-KI staining. Comparing the mapping results and type of sterile pollen in heterozygotes indicated that these loci from different species should be allelic to each other. In previous studies, researchers speculated that hybrid sterility loci consistently detected at the same or similar positions on chromosomes across different species should be orthologous loci, and the hypothesis was confirmed by the molecular characterization of some hybrid sterility loci, such as S1, S5, S27, Sa, and qHMS7 (Long et al. 2008; Yamagata et al. 2010; Win et al. 2009; Yang et al. 2012; Xie et al. 2017; Koide et al. 2018; Yu et al. 2018; Xie et al. 2019; Fang et al. 2019).

Although some orthologous loci for hybrid sterility were identified, it was difficult to investigate the genetic effects for different alleles because those loci were derived from different crosses in distinct genetic backgrounds. In this study, the near-isogenic lines harboring the hybrid sterility loci in the same background were used to compare the genetic effect of orthologous loci. The male gametes carrying O. sativa alleles at S59 and S60 loci were selectively aborted in the heterozygotes, while the wild species allele at the S62 locus was selectively aborted in the heterozygotes. In addition, the male gametes carrying the japonica allele at S24/Sb/f5-Du were selectively aborted in heterozygous plants (Kubo et al. 2008; Li et al. 2006; Wang et al. 2006). Thus, this hybrid sterility “hot spot” locus was observed in the different hybrid combinations of interspecific and intersubspecific, suggesting that this conserved locus originated before the divergence of AA genome species and had a significant impact on the reproductive isolation with the distinct genetic pattern.

At another hybrid sterility “hot spot” on chromosome 12, the male gametes from O. glumaepatula and O. barthii in the heterozygotes were preferentially transmitted to progeny. However, even when heterozygous plants of S61 as maternal parent to cross with DJY1 and NIL-S61, the transmission rate of O. sativa-type gamete and O. glumaepatula-type does not fit the expected ratio 1:1 in the offspring of the hybrid (Table 4). It is suggested that S61 conferred a different segregation distortion pattern from S63. S25, qs12, pf12, RHS12, and Se were identified from indica and japonica crosses and showed significant preferential transmission of the indica alleles, while some loci identified from the cross between japonica and its wild relatives showed significant preferential transmission of the wild relatives alleles (Win et al. 2009; Xu et al. 2014; Kubo et al. 2017; Zhou et al. 2023; Wang et al. 2023a, b). In addition, S64 was identified for interspecific hybrid sterility between O. sativa and O. barthii, which was located in the same region as S19 identified from the cross between O. sativa and O. glaberrima (Taguchi et al. 1999; McCouch et al. 2002; Zhang et al. 2011). Similar to S19, the O. sativa allele of S64 were not completely transmitted to the progeny via male gamete. The presence of co-linearity among various sterility loci suggests that similar loci in different species of the Oryza genus may be responsible for controlling reproductive barriers among AA genome species. Identifying and characterizing more hybrid sterility loci from other Oryza species will help shed light on the difference in genetic effect of those allelic loci on hybrid sterility and understand the function of allelic hybrid sterile loci in Oryza speciation.

Strategies for Overcoming the Interspecific Hybrid Sterility

The abundant genetic diversity of AA genome species in genus Oryza played an important role in the Asian cultivated rice improvement. Interspecific hybrid offspring exhibit stronger heterosis compared to intersubspecific hybrids. However, the severe reproductive isolation of interspecific hybrid makes it difficult to utilize interspecific heterosis. Understanding the genetic nature was the basis for utilizing interspecific heterosis or transferring favorable genes to the Asian cultivated rice. By now, great progress has been made in the cloning and molecular characterization of the hybrid sterility loci, for example, S5 (Chen et al. 2008; Yang et al. 2012), S7 (Yu et al. 2016), and ESA1 (Hou et al. 2019) for female sterility, Sa (Long et al. 2008), Sc (Shen et al. 2017), qHMS1 (You et al. 2023), qHMS7 (Yu et al. 2018), pf12/RHS12/Se (Zhou et al. 2023; Wang et al. 2023a, b) for male sterility, S1 for both male and female sterility (Xie et al. 2017; Koide et al. 2018; Xie et al. 2019). Based on the above information, artificial wide-compatible lines can be made to overcome the hybrid sterility by CRISPR/Cas9 gene editing and RNA interference technologies (Yu et al. 2016; Shen et al. 2017; Xie et al. 2017; Koide et al. 2018; Hou et al. 2019; Xie et al. 2019).

Furthermore, transferring neutral allele of the major hybrid sterility loci to the target parent to raise wide-compatibility lines as bridge parent is an effective strategy to overcome hybrid sterility. For example, scientists have proved that the utilization of wide-compatible genes of S5 and f5, as well as the japonica alleles at Sc and pf12 locus could overcome intersubspecific hybrid sterility (Zhou et al. 2023). Pyramiding the indica alleles at Sb, Sc, Sd and Se loci and the neutral allele at S5 locus in japonica genetic background through MAS are compatible with indica rice varieties, the F1 hybrids showed fertile pollen and spikelet (Guo et al. 2016).

In interspecific of AA genome species in the Oryza genus, there have been successful cases of improving the fertility of both pollen and spikelet in interspecific hybrids by using either single locus or multiple hybrid sterility loci pyramiding lines as bridge parents (Deng et al. 2010; Li et al. 2023). Additionally, our study suggests that employing a single allele to raise a bridge parent to overcome hybrid sterility has its limitations due to allelic differentiation. Therefore, bridge parents developed by pyramiding hybrid sterility loci identified from more species in O. sativa genetic background would effectively improve the interspecific hybrid sterility between O. sativa and more than two species.

Conclusions

In this study, six novel hybrid pollen sterility loci for interspecific hybrids were identified and designed as S59, S60, S61, S62, S63, and S64, respectively. These six loci could be classified into three groups based on collinearity analysis. The first group contained three loci, S59, S60, and S62 responsible for interspecific hybrid pollen sterility in the crosses between DJY1 and O. rufipogon, O. glumaepatula, and O. barthii, respectively. All of them were located in the same region on chromosome 5. The second group included two loci, S61 and S63, they were mapped to the same position on chromosome 12 and identified from the crosses between O. sativa and O. glumaepatula, O. barthii, respectively. S64 from O. barthii was located on chromosome 3 and belonged to the third group. Meanwhile, the alleles from different species at the same locus differed in the degree of segregation distortion and gamete transmission mode because of allelic differentiation. This study provides insight into the genetic basis of conserved loci for interspecific hybrid sterility, which played an important role in understanding the hybrid sterility and divergence of genus Oryza.

Materials and Methods

Plant Materials

An O. rufipogon accession, Acc.106,138; an O. glumaepatula accession, Acc.105,662; two accessions of O. barthii, Acc. 104,061 and Acc. 100,117, introduced from the International Rice Research Institute (IRRI) as male and donor parents, were crossed with a japonica variety of O. sativa, Dianjingyou 1 (DJY1). The F1 as female parent was backcrossed with DJY1 as a male parent to obtain BC1F1 progenies. The plants with pollen fertility below 90% were selected to make successively backcross from BC1F1 until BC5F1/BC6F1. Subsequently, the semi-sterile plants identified in the BC5F1/BC6F1 generations were subjected to multiple rounds of self-fertilization to establish 24 distinct populations for further study and analysis. Mixed pools of pollen fertile and semi-sterile plants for each population were constructed and screened using Rice 6k Chips (Illumina, USA) for introgressed segments associated with pollen sterility (Figure S1, Table S1). After comparing the introgressed segments associated with pollen sterility of these populations with the localization intervals of reported hybrid sterility loci, six pollen semi-sterile families were selected for further research, including one from the cross between O. sativa and O. rufipogon, two from the cross between O. sativa and O. glumaepatula, one from the cross between O. sativa and Acc. 104,061 of O. barthii, and two from the cross between O. sativa and Acc. 100,117 of O. barthii. The introgressed segments associated with pollen sterility were identified on chromosome 3, 5, and 12, respectively. Since no hybrid sterility loci have been reported in these chromosome regions between O. sativa and its three wild relatives (Table S1), the hybrid sterility locus identified from the cross between DJY1 and O. rufipogon was designed as S59, and another two loci identified from the cross between DJY1 and O. glumaepatula were named as S60 and S61, respectively. Three loci controlled the hybrid sterility between DJY1 and O. barthii were designed as S62, S63, and S64, respectively.

Polymorphic screening was conducted using SSR markers in the introgression regions based on the results of genome-wide screening by Rice 6k Chips in the six populations mentioned above (Table S2). The individuals harboring the homozygous genome fragments from wild relatives were selected as NILs in BC6F3/BC6F4 populations by SSR polymorphic markers. NILs were crossed with the recurrent parent DJY1, and then were self-fertilized to produce the BC7F2 populations. The individuals of BC7F2 populations were used to map the interspecific hybrid sterility loci. To study the gamete elimination and transmission pattern, reciprocal crosses were produced between heterozygous plants in BC7F1 and DJY1, and between heterozygous plants in BC7F1 and NILs.

All plant materials were grown in paddy field in Breeding Field, Yunnan Academy of Agricultural Sciences (YAAS), Xishuangbanna (100° E, 20°N), Yunnan Province, P. R. China.

Phenotype Evaluation

Five to ten spikelets were collected from the upper and middle branches of the main panicle of each individual 1–2 days before anthesis and fixed in 70% ethanol (Doi et al. 1998). To evaluate the pollen fertility of each plant, 3–6 anthers from each spikelet were mixed and stained with a 1% iodine-potassium iodide (I2-KI) solution. The observed pollen grains were classified into four categories: typical abortion, spherical abortion, stained abortion, and fertile. At least 300 pollen grains were observed under three independent microscopic fields. The pollen fertility score was determined as the average percentage of fertile pollen grains across the three views. The spikelet fertility was investigated by scoring the fertilized spikelet rate of three panicles on each plant.

DNA Extraction and PCR Protocol

Genomic DNA for simple sequence repeat (SSR) analysis was extracted from the fresh leaves of each plant using a simple DNA extraction method (Edwards et al. 1991). Rice SSR markers were chosen from previously published SSR markers in rice (McCouch et al. 2002). The PCR reaction was performed in a total volume of 10 µl, containing about 40 ng of template DNA, 0.2µM of each primer, and 5 µl 2×Taq PCR StarMix Loading Dye-free (Vazyme, Nanjing, China). The reaction mixture was incubated at 94℃ for an initial 5 min, followed by 32 cycles of 94℃ for 30 s, 55 ℃ for 30 s, and 72 ℃ for 40 s, with a final extension step of 7 min at 72℃. The PCR products were separated on an 8% non-denaturing polyacrylamide gel and detected using silver staining.

Genomic DNA for Rice 6k Chips was extracted from the fresh/frozen leaf samples using the CTAB method (Murray and Thompson 1980). The protocol of Rice 6k Chips as Infinium HD Assay Ultra Protocol Guide (https://support.illumina.com.cn/downloads/infinium_hd_ultra_assay_protocol_guide_(11328087_b).html) was used.

Electronic Supplementary Material

Supplementary Material 1. (13.5KB, xlsx)
Supplementary Material 2. (12.4KB, xlsx)
Supplementary Material 3. (10.5KB, xlsx)
Supplementary Material 4. (10.8KB, xlsx)
Supplementary Material 5. (10.1KB, xlsx)
Supplementary Material 6 (1.9MB, jpg)
Supplementary Material 7 (888.2KB, jpg)

Author contributions

TDY and ZY conceived and designed the experiments. YY carried out most of the experiments and wrote the manuscript. PQH, LYG, SXC participated in phenotyping and genotyping. LJ, ZJW and DXN participated in developing plant materials. All authors have read and agreed to the published version of the manuscript.

Funding

This study was funded by National Natural Science Foundation of China (Grant No. 31991221, U2002202, 32460503), Yunnan Provincial Science and Technology Department, China (Grant Nos. 202101 AT070193, 202201 AS070072, 202205 AR070001-04), Yunnan Provincial Government (530000210000000013809).

Data availability

No datasets were generated or analysed during the current study.

Declarations

Institutional review board statement

Not applicable.

Informed consent

Not applicable.

Competing interests

The authors declare no competing interests.

Footnotes

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Ying Yang, Qiuhong Pu and Yonggang Lv have contributed equally to this work.

Contributor Information

Yu Zhang, Email: zhangyu_rice@163.com.

Dayun Tao, Email: taody12@aliyun.com.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary Material 1. (13.5KB, xlsx)
Supplementary Material 2. (12.4KB, xlsx)
Supplementary Material 3. (10.5KB, xlsx)
Supplementary Material 4. (10.8KB, xlsx)
Supplementary Material 5. (10.1KB, xlsx)
Supplementary Material 6 (1.9MB, jpg)
Supplementary Material 7 (888.2KB, jpg)

Data Availability Statement

No datasets were generated or analysed during the current study.

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