Improving bridge effect to overcome interspecific hybrid sterility by pyramiding hybrid sterile loci from Oryza glaberrima

doi:10.21203/rs.3.rs-3232995/v1

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Improving bridge effect to overcome interspecific hybrid sterility by pyramiding hybrid sterile loci from Oryza glaberrima

https://doi.org/10.21203/rs.3.rs-3232995/v1

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published 27 Dec, 2023

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In order to evaluate the hybrid sterile effect and the bridging effect caused by the hybrid sterile locus, total nine NILs carrying one, two and three alleles of O. glaberrima were developed and used as test lines to cross with O. sativa and O. glaberrima accessions. The results showed that the degree of pollen sterility of F₁ hybrids between the NILs and Dianjingyou 1 has deepened with the increase of the number of alleles from O. glaberrima at the hybrid sterile loci. Under the action of three hybrid sterile loci, F₁ hybrids between the two cultivated rice species are almost completely sterile. On the other hand, the NILs carrying a single allele of O. glaberrima had limited effect on improving pollen fertility of interspecific hybrid. The pyramided lines containing multiple sterile alleles of O. glaberrima showed increased pollen fertility when crossing with O. glaberrima accessions. Further the backcrossing can be used to improve pollen and spikelet fertility of interspecific hybrids after the first cross. The report has potential for understanding the nature of interspecific hybrid sterility, and overcoming the interspecific hybrid F₁ pollen grain sterility between O. sativa and O. glaberrima.

Biological sciences/Genetics/Agricultural genetics

Biological sciences/Genetics/Plant breeding

bridge effect

hybrid sterility

interspecific hybrid

near isogenic lines

Oryza sativa

Oryza. glaberrima

Asian cultivated rice (Oryza sativa L.) is a prime food crop world-wide. However, advance in genetic improvement of rice has encountered problems owing to the narrow genetic diversity and the bottleneck of further yield increase[1]. African cultivated rice (O. glaberrima Steud.) is deemed to be a potential source of useful genes for improving Asian cultivated rice by hybridization as both cultivated species have the same AA genome and similar sequence arrangement[2, 3]. However, there are strong reproductive barriers in the interspecific hybrids between the two cultivated species[4]. The F₁ hybrids barely produce any fertile pollen grains and as a result the valuable genes disappear along with hybrid sterility. Therefore, hybrid sterility is one of the main hindrances against the utilization of useful genes from the African cultivated rice into Asian cultivated rice.

To date, at least 10 hybrid sterile loci were reported as gamete eliminators or pollen killers between O. sativa and O. glaberrima[5], and two of them were cloned[6, 7, 8]. The cumulative effects of these sterile loci lead to complete male sterility in F₁ hybrids. O. glaberrima and O. sativa varieties possessed the genotype S-glab and S-sati, respectively, at the sterile S locus. Homozygotes of S-sati/S-sati and S-glab/S-glab show normal fertility, while S-sati gametes are aborted when the sporophytic plants have the heterozygous genotype S-sati/S-glab in an O. sativa background. It was reported that the O. sativa lines carrying the S1-glab allele from O. glaberrima can be used as bridge parents to improve the fertility of hybrids between O. glaberrima and O. sativa[9]. However, the bridge effect of others sterile loci and their pyramided lines remain unknown.

In our previous study, six hybrid sterile loci, S1, S19, S20, S37(t), S38(t), S39(t) were identified from the crosses between O. glaberrima and O. sativa[10, 11, 12]. The S1 and S37(t) loci function as the “gamete eliminator”: both male and female gametes carrying the allele of O. sativa are aborted in the heterozygotes. The S19, S20, S38(t), S39(t) locus functions as the “pollen killer”: only male gametes carrying the allele of O. sativa are aborted in the heterozygotes.

In order to improve the bridge effect to overcome interspecific reproductive barrier, the near isogenic lines (NILs) carrying the single and multiple alleles from O. glaberrima on the hybrid sterility loci S1, S19, S20, S37(t), S38(t) and S39(t) were developed. The hybrid sterile effect and bridge effect of the hybrid sterility loci were investigated in this study.

NIls with single and multiple hybrid sterile alleles from O. glaberrima were developed.

A set of NILs, designated as NILS1, NILS19, NILS20, NILS37(t), NILS38(t), NILS39(t), with the O. glaberrima allele at the locus for hybrid sterility S1, S19, S20, S37(t), S38(t) and S39(t) in the genetic background of DJY1 were developed by successive backcross and molecular marker selection using DJY1 as the recurrent parent after hybridization with five O. glaberrima assessions[10, 11, 12]. These NILs were detected by a survey of the genome with the whole-genome SNP array. All of them carried the target chromosomal segment from the O. glaberrima accession and the genetic background was similar to the recurrent parent DJY1 (Fig. 1).

Three NILs, NILS1, NILS20 and NILS37(t), carrying the allele of O. glaberrima on the hybrid sterile loci S1, S20, S37(t) were used to develop the pyramided sterile loci lines with molecular marker-assistant method and phenotype selection. The pollen and spikelet fertility for all the F₁ plants were evaluated and the SSR molecular markers linked with S1, S20, and S37(t) were used to confirm the true hybrid. Those multiple S allele lines were self-pollinated and the marker-assistant method was used to obtain homozygous S alleles lines. Experiments to create the targeted locus pyramid started with two crosses NILS1×NILS20 and NILS1×NILS37(t). Subsequently, the F₁ plants from the cross NILS1×NILS37(t) were used as the female to cross with the NILS20 for the S1, S20, S37(t) pyramiding.

The F₂ self-pollinated plants from the cross of NILS1×NILS37(t) showing the normal pollen and spikelet fertility, and homozygous genotype of O. glaberrima on SSR markers linked with S1 and S37(t) were selected as the pyramid line for S1 and S37(t) loci. We designated the pyramided line as the NILS1S37(t). In fact, all the F₂ self-pollinated plants showing the homozygous genotype of O. glaberrima on S1 and S37(t) loci because the S1 and S37(t) loci function as the “gamete eliminator” and as the result only the gametes carrying the allele of O. glaberrima survived (Table 1).

With the assistance of SSR markers linked with the S1 and S20 loci, the F₂ self-pollinated plants from the cross of NILS1×NILS20 showing the homozygous genotype of O. glaberrima on the S1 and S20 loci were selected and the pyramid lines for the S1 and S20 alleles from O. glaberrima in O. sativa genetic background were obtained. There were two genotypes in the F₂ self-pollinated offspring from the cross of NILS1×NILS20: the one is homozygotes of O. glaberrima at S1 and S20 loci; and another is homozygote of O.glaberrima at S1 locus while heterozygous at S20 locus. Only the male gamete of S1-glab and S20-glab survived while the female gamete of S1-glab, S20-glab and S20-sati survived in the F₁ plants because S1 locus functions as the “gamete eliminator” and S20 locus functions as the “pollen killer”. The selected plants with homozygous genotypes of O. glaberrima on the S1 and S20 loci also showed the normal pollen and spilelet fertility (Table 1). We designated the pyramided line as the NILS1S20.

For the pyramided line of alleles from O. glaberrima on the S1, S20 and S37(t) loci in O. sativa genetic background, the SSR markers linked with S1, S20 and S37(t) alleles were used to genotype the F₂ plants in the cross of of (NILS1×NILS37(t))×NILS20. The plants with homozygous genotype of O. glaberrima on the all three loci and showing the normal pollen and spikelet fertility were obtained as the pyramided line with the three alleles from O. glaberrima (Table 1). We designated the pyramided line as the NILS1S20S37(t).

Totally, nine NILs carrying one, two and three allele of O. glaberrima were used as test lines to cross with O. sativa and O. glaberrima accessions. The hybrid sterile effect and the bridging effect caused by the hybrid sterility locus were studied.

Table 1

Pollen grain, spikelet fertility and genotype of F1 plants in the crosses between DJY1 and the NILs
Material name	Generation	Pollen fertility (%)	Spikelet fertility (%)	S1 Chr.6^a		S19 Chr.3		S20 Chr.7		S37(t) Chr.1		S38(t) Chr.4		S39(t) Chr.12
Material name	Generation	Pollen fertility (%)	Spikelet fertility (%)	RM190	RM587	RM3372	RM22	RM20847	RM20852	RM449	RM513	RM16260	RM5414	RM1880	RM4
DJY1	P	99.05 ± 0.25	92.10 ± 3.07	s^b	s	s	s	s	s	s	s	s	s	s	s
NILS1	P	99.25 ± 0.31	91.19 ± 4.62	g	g	s	s	s	s	s	s	s	s	s	s
NILS19	P	98.43 ± 1.46	86.42 ± 8.95	s	s	g	g	s	s	s	s	s	s	s	s
NILS20	P	99.31 ± 0.29	93.12 ± 3.89	s	s	s	s	g	g	s	s	s	s	s	s
NILS37(t)	P	98.84 ± 0.57	92.13 ± 3.77	s	s	s	s	s	s	g	g	s	s	s	s
NILS38(t)	P	99.00 ± 0.42	90.11 ± 1.53	s	s	s	s	s	s	s	s	g	g	s	s
NILS39(t)	P	97.63 ± 1.08	93.30 ± 5.64	s	s	s	s	s	s	s	s	s	s	g	g
NILS1S20	P	99.06 ± 0.38	77.08 ± 5.00	g	g	s	s	g	g	s	s	s	s	s	s
NILS1S37(t)	P	98.78 ± 0.54	78.75 ± 3.87	g	g	s	s	s	s	g	g	s	s	s	s
NILS1S20S37(t)	P	98.93 ± 0.39	79.37 ± 4.43	g	g	s	s	g	g	g	g	s	s	s	s
DJY1×NILS1	F₁	47.83 ± 1.85	51.97 ± 2.12	H	H	s	s	s	s	s	s	s	s	s	s
DJY1×NILS19	F₁	48.60 ± 3.56	89.96 ± 4.02	s	s	H	H	s	s	s	s	s	s	s	s
DJY1×NILS20	F₁	49.10 ± 4.07	88.40 ± 2.34	s	s	s	s	H	H	s	s	s	s	s	s
DJY1×NILS37(t)	F₁	58.91 ± 8.27	44.12 ± 1.32	s	s	s	s	s	s	H	H	s	s	s	s
DJY1×NILS38(t)	F₁	46.79 ± 1.51	82.65 ± 5.11	s	s	s	s	s	s	s	s	H	H	s	s
DJY1×NILS39(t)	F₁	47.32 ± 2.82	75.72 ± 12.71	s	s	s	s	s	s	s	s	s	s	H	H
DJY1×NILS1S20	F₁	33.81 ± 1.71	42.69 ± 9.30	H	H	s	s	H	H	s	s	s	s	s	s
DJY1×NILS1S37(t)	F₁	31.46 ± 10.0	50.19 ± 4.74	H	H	s	s	s	s	H	H	s	s	s	s
DJY1×NILS1S20S37(t)	F₁	3.35(n = 1)	0.00	H	H	s	s	H	H	H	H	s	s	s	s
^a Locus name and chromosome
^b s, H and g indicated DJY1-homozygous, heterozygous and O. glaberrima-homozygous genotypes, respectively.

Hybrid sterile effect caused by S loci

DJY1, the recurrent parent and tested line of O. sativa, was crossed with its nine NILs, NILS1, NILS19, NILS20, NILS37(t), NILS38(t), NILS39(t), NILS1S20, NILS1S37(t) and NILS1S20S37(t). As each NIL had the same DJY1 background and the difference was only on the specific chromosome fragment harboring the specific sterile S allele, the effect of genetic background could be readily eliminated and the difference between DJY1 and the NILs originated from the S locus. For the NILs with the single hybrid sterile locus, the pollen and spikelets fertility of F₁ plants were semi-sterile in the crosses between DJY1 and the NILS1, NILS37(t); the pollen grains were semi-sterile and the spikelet fertility were normal in the crosses between DJY1 and the NILS19, NILS20, NILS38(t) and NILS39(t) (Table 1). These results indicated that the S1 and S37(t) acted as the “gamete eliminator” and S19, S20, S38(t) and S39(t) acted as the “pollen killer” which is correspond with previous report. The genetic pattern of those sterility loci followed one-locus sporophyte-gametophyte interaction model[13].

When the NILs with multiple S alleles were crossed with DJY1, the pollen fertility was significantly lower than that of the single S allele. The NILS1S20 and NILS1S37(t) containing two hybrid sterile genes caused about two-thirds of pollen abortion in hybrids and the F₁ pollen grain fertility were 33.81% and 31.46%, respectively. In particular, the pollen grain of F₁ plants was 3.35% in the cross between DJY1 and the NILS1S20S37(t) (Table 1), which was the lowest among the hybrid cross combinations. These results showed that the cumulative effect of these sterile loci in F₁ hybrids was remarkable. When NILS1 and NILS37(t) crossed with DJY1, the pollen and spikelet fertility of F₁ plant were semi-fertility (Table 1) which followed the gamete eliminate model. However, the spikelet fertility of F₁ plants was still semi-fertility when the NILS1S37(t) crossed with DJY1 (Table 1). It is unclear how these two loci are involved in interaction.

Bridge effect of the S loci

In addition, DJY1 and its nine NILs were used as female and male parents to make reciprocal crosses with two O. glaberrima accessions in order to evaluate the bridge effect of the S loci, and the crosses of O. glaberrima with DJY1 were used as the control. The F₁ plants were backcrossed as females to their corresponding NILs untill the BC₂F₁ generation was achieved.

Interspecific hybrids between the NILs and two O. glaberrima accessions were obtained and hybrids between DJY1 and O. glaberrima accessions were used as the control. The average pollen fertility of each cross was evaluated in the F₁ generation.

There was no significant difference in pollen fertility between reciprocal F₁s of all combinations (P = 0.94), indicating that no cytoplasmic effect was involved in hybrid sterility between the two Africa cultivated rice accessions and the Asian cultivated rice variety DJY1 (data not shown). The difference of F₁ pollen fertility between the two interspecific test crosses, DJY1 1×IRGC102263 and DJY1×IRGC103469 was not significant (P = 0.08), indicating that the compatibility of DJY1 with the two African cultivated rice varieties was consistent. The F₁ pollen fertility data of reciprocal crosses between each NIL (or recurent parent) and two Afrian rice accessions was merged to obtain larger sample size.

The F₁ progenies between the six NILs carrying the single O. glaberrima hybrid sterile alleles and the O. glaberrima accessions showed significantly different on pollen fertility (Table 2). Student's t test indicated that when NILS19, NILS20, and NILS37(t) were crossed with the O. glaberrima accessions, the average pollen fertility of F₁ was 0.49%, 1.72%, 0.85%, respectively, which was notably higher than that of the control (0.15%). Among them, S20 locus had the largest effect on improving pollen fertility of interspecific F₁ hybrids, followed by S37(t), and S19 is only significant at 0.05 level. While the progenies of other NILs, NILS1, NILS38(t) and NILS39(t), were similar to the control.

Table 2

Pollen fertility of F1 plants from the cross between two O. glaberrima accessions and NILs with single hybrid sterile genes.
Crosses	Mean ± SD (%)	Least value (%)	Maximum value (%)	Plant number
DJY1xO. glaberrima	0.15 ± 0.39	0.00	1.31	19
NILS1xO. glaberrima	0.12 ± 0.25	0.00	0.72	13
NILS19xO. glaberrima	0.49 ± 0.86*	0.00	2.05	7
NILS20xO. glaberrima	1.72 ± 2.39**	0.00	8.42	20
NILS37(t)xO. glaberrima	0.85 ± 0.94**	0.00	4.10	23
NILS38(t)xO. glaberrima	0.00 ± 0.00	0.00	0.00	5
NILS39(t)xO. glaberrima	0.10 ± 0.24	0.00	0.60	6
* means significant at 0.05 level; ** means significant at 0.01 level

The pollen fertility of F₁ hybrids between the NILs with multiple sterile loci and the African rice accessions were notably higher than that of control (the crosses between recurrent parent and the African accessions). The F₁ pollen fertility was 5.96% in the hybrids between NILS1S37(t) and O. glaberrima accessions; and 4.73% in the hybrids between NILS1S20 and O. glaberrima accessions. The tri-gene polymerization NILS1S20S37(t) produced notably higher pollen fertility (3.90%) of F₁ hybrids than that of NILs with single locus. However, the F₁ pollen fertility of NILS1S20S37(t) showed no significant difference from those of the F₁ progenies of two gene NILS1S20 and NILS1S37(t) (Table 3).

Table 3

Pollen fertility of F1 hybrids between two O. glaberrima accessions and NILs with multiple hybrid sterile genes
Crosses	Mean ± SD(%)	Least value(%)	Maximum value(%)	Plant number
DJY1xO. glaberrima	0.15 ± 0.39	0.00	1.31	19
NILS1S20xO. glaberrima	4.73 ± 3.11**	1.67	8.85	5
NILS1S37(t)xO. glaberrima	5.96 ± 0.70**	5.46	6.45	2
NILS1S20S37(t)xO. glaberrima	3.90 ± 2.10**	1.83	7.35	7
* means significant at 0.05 level; ** means significant at 0.01 level

The results indicated that the multiple hybrid sterile alleles from O. glaberrima gave better bridging effective than those of single allele.

Backcrossing can be used to improve pollen fertility of interspecific hybrids after the hybridization

For the NILs with single hybrid sterile locus from O. glaberrima, the highest pollen fertility reached 85.81% in the BC₁F₁ population from the cross of NILS1 and O. glaberrima, which was more than three times of the control (Table 4). Compared with the F₁ generation, the average pollen fertility of BC₁F₁ generation increased significantly. The average pollen fertility of the BC₁F₁ individuals involving six NILs carrying the single sterile allele ranged from 8.14–13.77% and the average pollen fertility of the control’s is 5.06%. However, the average pollen fertility of the six NILs carrying the single sterile allele did not notably differ from the control. It is because of the large standard deviation value in the BC₁F₁ segregation population. The average pollen fertility in BC₁F₁ generations showed that the plants harboring the multiple sterile alleles from O. glaberrima were notably more fertile than that of lacking them. When O. glaberrima accessions were crossed with NILS1S20 and NILS1S37(t) which harboring two sterile alleles from O. glaberrima, their average pollen fertility in the BC₁F₁ population was 44.04% and 49.50%, respectively; and the pollen fertility was range from 3.11–97.00% and 19.57–81.71%, respectively (Table 4). The result indicated that the pyramided lines with multiple hybrid sterility alleles still had the more prominent bridge effect in the BC₁F₁ generation.

Table 4

Pollen fertility of BC1F1 from the crosses between NILs and two O. glaberrima accessions
Crosses	Average(%)	Least value(%)	Maximum value(%)	Plant number
DJY1xO. glaberrima	5.06 ± 4.89	0.00	25.85	69
NILS1xO. glaberrima	11.87 ± 15.65	0.00	85.81	207
NILS19xO. glaberrima	9.87 ± 11.19	0.00	42.96	43
NILS20xO. glaberrima	10.27 ± 11.41	0.00	55.08	98
NILS37(t)xO. glaberrima	8.14 ± 10.21	0.00	46.34	47
NILS38(t)xO. glaberrima	13.77 ± 18.27	0.00	53.69	14
NILS39(t)xO. glaberrima	8.36 ± 10.99	0.00	36.09	24
NILS1S20xO. glaberrima	44.04 ± 26.60**	3.11	97.00	50
NILS1S37(t)xO. glaberrima	49.50 ± 25.90**	19.57	81.71	4
* means significant at 0.05 level; ** means significant at 0.01 level

The average pollen fertility was notably higher in the BC₂F₁ generation than those of in the BC₁F₁ generation. In the crosses between NILS1 and the O. glaberrima accessions, the average pollen fertility (51.01%) is notable higher than that of the control. The pyramiding lines with two hybrid sterile alleles still showed good performance in the BC₂F₁ generation. The hybrids between NILS1S20 and O. glaberrima accessions had the highest average pollen grain fertility (78.92%). It is worth mentioning that the maximum pollen fertility reached normal range (> 95%) in all the hybridization crosses in BC₂F₁ generation (Table 5).

Table 5

Pollen fertility of BC2F1 from the crosses between NILs and two O. glaberrima accessions
NILs	Average (%)	Standard deviation	Least value (%)	Maximum value (%)	Plant number
DJY1xO. glaberrima	30.93	21.89	0.00	99.29	516
NILS1xO. glaberrima	51.01**	34.38	0.00	99.40	1018
NILS19xO. glaberrima	37.47	23.45	0.00	99.18	65
NILS20xO. glaberrima	39.53	26.48	0.00	99.40	340
NILS37(t)xO. glaberrima	31.83	20.69	0.00	98.91	248
NILS38(t)xO. glaberrima	26.71	27.43	0.00	99.39	152
NILS39(t)xO. glaberrima	31.23	20.53	0.00	98.51	163
NILS1S20xO. glaberrima	78.92**	26.92	0.00	99.41	663
* means significant at 0.05 level; ** means significant at 0.01 level

Hybrid sterility is the biggest obstacle in the rice hybridization breeding project. Hybrid sterility is controlled by multiple hybrid sterile nuclear genes, most of these genes conform to the inheritance of a single Mendelian factor, and some hybrid sterile genes have epistatic interaction. So far, more than 50 hybrid sterile loci were reported, among which more than 10 hybrid sterile loci were reported between the two cultivated species, O. sativa and O. glaberrima[5]. Our study showed that the presence of a single hybrid sterile gene can cause about 50% of gametes sterility, while the presence of two hybrid sterile genes can cause about two-thirds of male gametes sterility. If three hybrid sterile genes are involved, the pollen fertility of that hybrid F₁ will be less than 5%. Up to now, it is known that interspecific hybrid sterility of cultivated rice involves at least 10 genes. When Asian cultivated rice is crossed with African cultivated rice, its F₁ generation has almost no fertile pollen. Therefore, interspecific hybrid sterility has always been the focus and difficulty in the rice genetic improvement.

The hybrid sterility also occurs frequently in the inter-subspecific hybridization crosses in rice and the hybrid sterility is mainly affected by five loci involving four for F₁ male sterility and one for F₁ female sterility[14]. The indica-compatible japonica lines (ICJLs) were developed by pyramiding four indica allele and one neutral allele in japonica genetic background through marker-assisted selection. When the indica-compatible japonica lines were test-crossed with a set of typical indica and japonica varieties, the results indicated that the ICJLs were compatible with indica while incompatible with japonica rice. The study showed a great promise of overcoming the intersubspecific hybrid sterility by developing pyramiding lines at hybrid sterility loci[14].

Based on known genetic information, if the sterile loci in a given O. sativa background could be substituted by the neutral alleles or the corresponding alleles from O. glaberrima, it would be possible to overcome the interspecific hybrid sterility between O. sativa and O. glaberrima[15, 16]. Deng et al. (2010) tested the feasibility of developing a “bridge parent” carrying the S1-glab allele from O. glaberrima in an O. sativa background[9]. The O. sativa lines carrying the S1-glab allele from O. glaberrima produced F₁ progeny with notably higher pollen grain fertility(3.52–5.37%) and egg fertility༈9.33–10.54%༉ than their controls (1.88% and 6.31%). This suggested that the O. sativa lines carrying the S1-glab allele from O. glaberrima could be used to improve the fertility of hybrids between O. glaberrima and O. sativa while the bridge effect of other loci remained unknown. In our case, it was confirmed that some of the “bridge parents” carrying a single sterile allele from O. glaberrima produced interspecific F₁ progenies with improved pollen fertility. Furthermore, present study showed that the pyramiding lines of multiple hybrid sterile alleles could significantly improve the F₁ pollen fertility than those with single hybrid sterility locus. In the presence of two alleles of O. glaberrima, the pollen fertility of hybrid F₁ increased 5–10 times compared with that of single gene. In fact, in the interspecific hybrid breeding project, increasing pollen fertility from 0 to 5% has no remarkable breeding value. Because the interspecific hybridization involves more hybrid sterile genes than that of the inter-subspecific[15], the “bridge effect” of a single hybrid sterile allele is very limited, even the pyramiding lines with two or three hybrid sterile alleles cannot obtain ideal "bridge effect".

In the test crosses of the indica-compatible japonica lines with indica, the result showed that the F₁ pollen and spikelet fertility reversed close to complete fertility when the indica-compatible japonica lines pyramided with four loci for “pollen killer” and one for “embryo sac killer”[14]. It can be deduced that the pyramiding lines with O. glaberrima alleles on five loci is still not enough on reversing the pollen and spikelet fertility to normal. It is worth further study from the perspective of practical application that how many hybrid sterile loci need to be pyramided to achieve the ideal bridging effect.

Backcrossing is a methods used to improve homogeneity of genetic background, and minimize the genetic variation inducing by genetic background. In our case, pollen fertility in BC₁F₁ generations showed that the plants harboring the sterile allele from O. glaberrima were notably more fertile than plants lacking the sterile allele from O. glaberrima when backcrossed to the DJY1. Furthermore, the average pollen fertility of the six NILs carrying a single fertile allele did not notably differ from each other, which indicated that all single alleles had near same effect on improving fertility of interspecific hybrids in BC₁F₁. Conversely, similar to most phenotypic variations, interspecific hybrid sterility was shown to be a quantitative trait controlled by multiple genes. Although the bridge lines with the single O. glaberrima allele were shown to produce interspecific hybrids with improved fertility, other sterile loci were still heterozygous, which reduced fertility in all the F₁ populations. It has been shown that the introgression at a single sterile locus is not enough to overcome F₁ sterility via elimination of single sterile locus action, which is similar to previous reports[9, 17]. Backcrossing can increase pollen and spikelet fertility of the progeny, while the valuable genes linkage with the sterile loci contained in O. glaberrima will naturally disappear along with hybrid sterility in the first cross. All three NILs with multiple O. glaberrima alleles produced interspecific F₁ progeny with notably higher pollen grain fertility than the control. This indicates that the strategy of developing O. sativa lines possessing as multiple sterile alleles from O. glaberrima as possible to overcome the hybrid sterility between these two species was effective. However, the NILS1S20S37(t) produced interspecific F₁ progeny with notable lower pollen grain fertility than the F₁ progeny of NILS1S20 and NILS1S37(t). The epistatic interaction between different loci may involved in the interspecific hybrid. Several epistatic interactions between different loci causing hybrid sterility and hybrid breakdown were reported[10, 18, 19, 20, 21]. More efforts are needed to elucidate the effect of various combinations of multiple pyramiding hybrid sterile bridge parents, and to dissect their interaction or epistatic effect among hybrid sterility loci.

Plant materials and growth conditions

An O. sativa japonica subsp. cultivar Dianjingyou 1 (DJY1) from Yunnan Academy of Agricultural Sciences (YAAS) was used as the recurrent parent. Two O. glaberrima accessions, IRGC102263 and IRGC103469, from International rice Research Institute (IRRI) were used as the tested lines of O. glaberrima in this study.

All materials were planted at the Winter Breeding Station, YAAS, Sanya, Hainan Province, P. R. China. The first cropping season was from November to April of the following year, and the second cropping season was from July to October.

Phenotypic evaluation

Pollen fertility for all parental lines, F₁, BC₁F₁ and BC₂F₁ plants was investigated following the instructions of Zhu[22]. Pollen grain fertility was measured using anthers collected from spikelets at 1 to 2 days before anthesis and stored in 70% ethanol[23]. Three to four anthers per floret per plant were mixed and stained with 1% I-KI solution, and more than 300 pollen grains were observed under a light microscope. Sterile types were further classified as typical, spherical or stained abortion types[24]. Three independent microscopic fields were scored for estimation of the percentage of the four types of pollen grains in each plant. Spikelet fertility was scored as the fertilized spikelet rate of three to five panicles on each plant.

Molecular marker and assay

The SSR markers used in this study for developing the NILs carrying single and mutiple alleles of O. glaberrima were selected on rice microsatellite maps[25]. A whole-genome SNP array(6k) of rice designed by Cornell University was used to survey the genome of the NILs.

Statistical analysis

Statistical analysis of the data was performed using one-way ANOVA, and the Student's test was used for further pairwise comparisons if ANOVA differences were significant. Pollen and spikelet fertility data as a percentage was transformed by function arcsine square root before the analysis but are listed as percentages.

Data availability statement: All relevant data are within the paper.

Acknowledgments: We would like to thank Dr. Huihui Yu for his help in the process of making chromosome maps based on R Language. This research was funded by National Natural Science Foundation of China, grant number C130501, 31860372, 32160489, and 31000704; Basic Research Foundation of Yunnan Provincial Science and Technology Department, China, grant number 202201AS070072, 202101AS070286; Technology Talent and Platform program of Yunnan Provincial Science and Technology Department, China, grant number 202205AC160057; Applied Basic Research Foundation of Yunnan Academy of Agricultural Sciences JZ201801; and the Yunnan Seed Industrialization Laboratory Program. We would like to thank Dr. Huihui Yu for his help in the process of making chromosome maps based on R Language.

Author Contributions: Conceptualization, D.T.and J. L.; experiment implementation, J.L. and J.Z.; investigation, J.L., P.X., X.D., W.D., Y.Y, Y.Z., Y.L., Q.P.; writing—original draft preparation, J.L.; writing—revise and editing, D.T. and J.Z.. All authors have read and agreed to the published version of the manuscript.

Conflicts of Interest: The author(s) declare no competing interest.

Ethics declarations: The plant collection and use was in accordance with all the relevant guidelines.

Permissions statement: The rice cultivars involved in this paper have permission.

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No competing interests reported.

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Improving bridge effect to overcome interspecific hybrid sterility by pyramiding hybrid sterile loci from Oryza glaberrima

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Abstract

Figures

Introduction

Results

Hybrid sterile effect caused by S loci

Bridge effect of the S loci

Backcrossing can be used to improve pollen fertility of interspecific hybrids after the hybridization

Discussion

Materials and Methods

Plant materials and growth conditions

Phenotypic evaluation

Molecular marker and assay

Statistical analysis

Statements and Declarations

Ethics declarations: The plant collection and use was in accordance with all the relevant guidelines.

References

Additional Declarations

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Journal Publication

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