QTLs for hybrid fertility and their association with female and male sterility in rice

Transcription

1 Genes & Genomics (2012) 34: DOI /s RESEARCH ARTICLE QTLs for hybrid fertility and their association with female and male sterility in rice Reflinur Joong Hyoun Chin Sun Mi Jang Backki Kim Joohyun Lee Hee-Jong Koh 1) Received: 25 October 2011 / Accepted: 28 December 2011 / Published online: 25 June 2012 The Genetics Society of Korea and Springer 2012 Abstract Hybrid sterility is one of the major barriers to the application of wide crosses in plant breeding and is commonly encountered in crosses between indica and japonica rice varieties. Ten mapping populations comprised of two reciprocal F 2 and eight BC 1F 1 populations generated from the cross between Ilpumbyeo (japonica) and Dasanbyeo (indica) were used to identify QTLs and to interpret the gametophytic factors involved in hybrid fertility or sterility between two subspecies. Frame maps were constructed using a total of 107 and 144 STS markers covering 12 rice chromosomes in two reciprocal F 2 and eight BC 1F 1 populations, respectively. A total of 15 main-effect QTLs and 17 significant digenic-epistatic interactions controlling spikelet fertility (SF) were resolved in the entire genome map of F 2 and BC 1F 1 populations. Among detected QTLs responsible for hybrid fertility, four QTLs, qsf5.1 and qsf5.2 on chromosome 5, qsf6.2 on chromosome 6, and qsf12.2 on chromosome 12, were identified as major QTLs since they were located at corresponding positions in at least three mapping populations. Loci qsf5.1, qsf6.1 and qsf6.2 were responsible for both female and male sterility, whereas qsf3.1, qsf7 and qsf12.2 affected the spikelet fertility only through embryosac factors, and qsf9.1 did through pollen factors. Five new QTLs identified in this study will Reflinur S. M. Jang B. Kim H.-J. Koh ( ) Department of Plant Science, Plant Genomics and Breeding Institute, Research Institute for Agriculture and Life Sciences, Seoul National University, Seoul, Korea heejkoh@snu.ac.kr J. H. Chin IRRI, Los Banos, Philippines J. Lee Department of Applied Bioscience, Konkuk University, Seoul , Korea Reflinur Indonesian Center for Agricultural Biotechnology and Genetic Resources Research and Development, Bogor, Indonesia be helpful for understanding the hybrid sterility and for breeding programs via inter-subspecific hybridization. Keywords Rice; Hybrid sterility; QTL; Digenic-epistatic interaction; Gametophytic factors Introduction Asian cultivated rice (Oryza sativa L.) is a predominantly self-pollinating species that has been differentiated into two subspecies, indica and japonica based on differences in morphological and physiological characteristics, such as grain length and width, apiculus hair length, KClO 3 resistance, phenol reaction (Oka, 1991), isozymes (Second, 1982; Glaszmann, 1987), various molecular markers (Wang and Tanksley, 1989; Second and Wang, 1992; Zhang et al., 1992; Ishii et al., 1995; Mackill, 1995; Liu et al., 1996; Zhang et al., 1997) and their tendency to exhibit hybrid sterility when crossed (Kato et al., 1930). The differences between the two subspecies both at a morphological and at a molecular level are associated with their adaptability to different environments (Oka, 1988; Wang and Tanksley, 1989; Li and Rutger, 1997). The use of potential gene sources from each type of rice can be very useful for improving rice quality and quantity in a plant breeding program. However, several reproductive barriers such as hybrid sterility, hybrid weakness, hybrid breakdown, and gametophytic competition in different indica and japonica crosses are frequently encountered. These barriers limit the utilization and exploitation of useful genes because they prevent gene exchange or gene flow between these two subspecies. Hybrid sterility has long been known as one of the most common barriers in crossing subspecies of rice that are quite different at the genomic level (Oka, 1953; Wan et al., 1993; Liu et al., 2004; Song et al., 2005). In order to overcome these barriers, it is very important to elucidate the underlying molecular mechanisms of hybrid sterility (Li et al., 2006). The advent of molecular markers, especially in rice where the whole

2 356 Genes & Genomics (2012) 34: genome is covered, has given the geneticists involved in identifying, characterizing, and cloning the genes related to hybrid sterility hope that the molecular mechanisms underlying hybrid sterility can be elucidated. When the molecular mechanism of hybrid sterility is finally fully understood, the use of beneficial traits from each type of rice subspecies can be easily utilized to improve breeding variety. Harushima et al. (2001) reported that one application of DNA markers is to analyze quantitative trait loci (QTL) that seem to be responsible for reproductive isolation. Several genetic analyses have been conducted to identify a large number of loci affecting hybrid sterility (Yanagihara et al., 1992; Wan et al., 1993; Wan and Ikehashi, 1995; Taneichi et al., 2005; Zhu et al., 2005; Zhao et al., 2006; Zhao et al., 2007; Chen et al., 2008; Long et al., 2008), and only two genes out of the many loci involved in the sterility of the indica-japonica hybrid have been cloned (Chen et al., 2008; Long et al., 2008). Although several research groups have reported many loci involved in hybrid sterility, most of the results were not informative enough to elucidate the cause of sterility, male or female gametes, because data were obtained from a single cross population (or multiple crosses with different genetic backgrounds) to identify the QTLs and genetic factors responsible for hybrid sterility. Moreover, cytological studies are also required to identify whether hybrid sterility is caused by female or male gamete abortions. Oryza sativa cv. Ilpumbyeo (Korean japonica variety) and Dasanbyeo (Korean indica variety) possess many advantageous agronomic traits. Ilpumbyeo cultivar is a high-quality, high-yielding semi-dwarf japonica rice cultivar, showing a desirable canopy architecture and lodging resistance, but susceptible to rice disease. Meanwhile, Dasanbyeo is known as a high yield potential indica cultivar, short plant height with desirable canopy architecture, strong lodging tolerance, and multiple-resistance to several rice disease, such as blast, bacterial blight and stripe virus but susceptible to insect pest (Choi et al., 1997). Therefore, in this study we used reciprocal F 2 and BC 1F 1 populations derived from crosses of Ilpumbyeo and Dasanbyeo to thoroughly explore hybrid sterility loci inherited from each parent. Since BC 1F 1 types conferring embryo sac-segregating and pollen segregating populations were used to detect QTLs for hybrid sterility, genetic factors affecting hybrid sterility, such as female and/or male could be determined. Information about the loci and genetic factors responsible for hybrid sterility explored using these parents will be valuable for improving these two elite rice cultivars in the future by facilitating the transfer of desirable and superior alleles between them. The objectives of this study were to identify main-effect QTLs (M-QTLs) and digenic-epistatic interactions responsible for hybrid sterility in an inter-subspecific cross between indica and japonica varieties, and to determine the gametophytic factors involved in hybrid sterility. Materials and Methods Plant materials A total of ten reciprocal crosses consisting of two F 2 and eight BC 1F 1 populations generated from Korean elite indica variety, Dasanbyeo and Korean japonica variety, Ilpumbyeo were used to identify the QTL-related hybrid sterility gene(s) in inter-subspecific crosses in rice. A total of 210 F 2 progeny derived from the cross of Ilpumbyeo (I; female parent) and Dasanbyeo (D; male parent), hereafter ID (I/D), and 199 F 2 progeny of its reciprocal cross, hereafter DI (D/I), were produced. Meanwhile, eight reciprocal backcross populations that consisted of 84 BC 1F 1 plants of I/D//D, 92 plants of I/D//I, 71 plants of D//D/I, 77 plants of D/I//I, 25 plants of I//I/D, 27 plants of I//D/I, 57 plants of D//I/D, and 75 plants of D/I//D populations were generated. In 2009, these populations, including parental lines, were grown at the experimental farm of Seoul National University in Suwon. Fresh leaf tissues of the parents and each mapping population at the maximum tillering stage were collected for DNA extraction. DNA was extracted following the CTAB method described by Causse et al. (1994), with minor modifications. Fertility observation Spikelet fertility was measured as the number of fertile (filled) grains per total number of spikelets in a panicle, expressed as a percentage. Three fully mature panicles from each plant for all tested populations were observed, and a mean value for the spikelet fertility was calculated. Molecular marker analysis A total of 107 STS markers dispersed throughout the rice genome and designed in Crop Molecular Breeding Lab, Seoul National University (Chin et al., 2007) were used to construct a linkage map of two F 2 populations. While constructing frame maps of the eight BC 1F 1 populations, a total of 144 STS primers were employed. PCR analysis in the F 2 populations was performed in a 20 μl total reaction volume containing 20 ng of template DNA, 0.2 μm of each primer, 10 mm Tris-HCl (ph 8.3), 50 mm KCl, 0.01% gelatin, 1.5 mm MgCl 2, mm dntps, and 1 unit of Taq DNA polymerase. Template DNA was initially denatured at 94 C for 5 minutes, followed by 35 cycles of PCR amplification under the following parameters: 30 sec of denaturation at 94 C, 30 sec of primer annealing at 55 C (depending upon the annealing temperature of the primers), and 30 sec of primer extension at 72 C. A final 7 min incubation at 72 C was programmed to allow for the completion of primer extension on a PTC220 dual 96-well thermo-cycler (MJ Research, USA). The amplified products were electrophoretically resolved on a 6% polyacrylamide non-denaturing gel using 1 TBE buffer. Meanwhile, the PCR for the BC 1F 1 populations was subjected to high-resolution melting (HRM) analysis as an alter-

3 Genes & Genomics (2012) 34: native rapid genotyping technique. PCR was carried out under in a 20 μl reaction mixture consisting of 20 ng of template DNA, 0.2 μm of each primer, 10 mm Tris-HCl (ph 8.3), 50 mm KCl, 0.01% gelatin, 1.5 mm MgCl 2, mm dntps, 50 μm Syto-9 (Invitrogen, USA), and 1 unit of Taq DNA polymerase. PCR amplification was performed on a PTC220 dual 96-well thermo-cycler (MJ Research, USA) as follows: 5 min at 94 C, followed by 35 cycles of 30 sec at 94 C, 30 sec at 55 C (depending upon the annealing temperature of the primers), 30 sec at 72 C, and 7 min at 72 C for the final extension. PCR products were analyzed by performing a Hi-Res melting curve analysis on a LightScanner (Idaho Technology Inc.). To avoid evaporation of the samples during Hi-Res melting, a 10 μl mineral oil overlay was applied to each sample (BioRad, USA). Data analysis The linkage map construction in the F 2 and BC 1F 1 populations was performed with the Mapmaker/EXP 3.0 program, where a LOD score of 3.0 was used as the threshold for declaring linkage (Lander et al., 1987; Lincoln et al., 1992), and the Kosambi function was used to convert recombinant values to genetic distances between the markers (Kosambi, 1944). The chromosomal locations of putative QTLs through linkage relationship analysis between the loci of the traits and molecular markers were resolved according to the method of composite interval mapping (CIM) by using the computer package Windows QTL Cartographer version 2.5 (Wang et al., 2006). CIM was run with the default setting for model 6 in the program (five background markers and a window size of 10 cm). Experiment-wise significance (a=0.05) likelihood ratio test (LR) thresholds for QTL identification were determined with 1,000 permutations, and expressed as a LOD. The percent variation explained (PVE) by the underlying QTL was derived from the R-squared values. Digenic-epistatic interactions between all pairs of loci in the F 2 populations were evaluated by a two-way analysis of variance using the SAS EPISTACY command (Holland, 1998). A liberal P-value (p < 0.001) was used to declare epistatic interactions significant, and these interactions were further incorporated into multiple locus models with the SAS GLM procedure. While the analysis of the epistatic effects in the BC 1 population was carried out using QTL Mapper 1.6 software (Wang et al., 1999), the nomenclature of QTLs followed that described by McCouch et al. (1997). Results Phenotypic evaluation The phenotypic data for spikelet fertility in the parents, reciprocal F 1, F 2, and BC 1F 1 populations are shown in Table 1. The spikelet fertility of the parents, Ilpumbyeo and Dasanbyeo, was normally high, and did not differ significantly between Table 1. Descriptive statistics of spikelet fertility in parents and reciprocal F 1, F 2, and BC 1F 1 populations generated from crosses between Ilpumbyeo and Dasanbyeo. Parent/ Fertility (%) Population a (mean ± SD) b Range (%) Skewness c I 91.9 ± 2.21c D 93.4 ± 1.72c F1-ID 1.72 ± 1.65d F1-DI 4.05 ± 4.8d ID ± DI ± ID//D ± ID//I ± DI//D ± DI//I ± I//ID ± I//DI 7.80 ± D//ID ± D//DI ± a I refers to Ilpumbyeo; D refers to Dasanbyeo b SD is the abbreviation for standard deviation. Spikelet fertility between the parents was not significantly different at the 0.05 level of significance (c), or between the two reciprocal F 1 populations (d). c The skewness value was obtained by applying a normality test in SAS. parents at the 0.1% level. However, significantly reduced spikelet fertility was observed in the F 1 plants, and there was no effect of crossing directions on spikelet fertility, indicated by the non-significant difference between reciprocal F 1 plants at the 0.1% level. This suggests that there might be no cytoplasmic effect on the expression of spikelet fertility. All traits varied widely in the two F 2 and eight BC 1F 1 populations and the skewness value for almost all of the traits, except in I//DI, was less than 1.0, indicating that the populations fit a normal distribution (Fig. 1) and were appropriate for use in QTL analysis related to hybrid fertility. Among ten populations used in this study, the highest mean value of spikelet fertility was observed in the progeny of DI//D, and the lowest was observed in I//DI (Fig. 2). Identification of main-effect QTLs for spikelet fertility Ten linkage maps were constructed using two reciprocal F 2 and eight BC 1F 1 populations generated from cross between Ilpumbyeo (japonica) and Dasanbyeo (indica) to identify the QTLs underlying hybrid sterility between the two subspecies. Total length of linkage map and average distance between adjacent markers varied with mapping populations (data not shown). However, our linkage map in general covered 97% of the total length of the Nipponbare/Kasalath map as the reference (Harushima et al., 1998). QTLs associated with spikelet fertility were identified and are summarized in Table 2 and Table 3 for the two reciprocal F 2 and eight BC 1F 1 populations,

4 358 Genes & Genomics (2012) 34: Figure 1. Frequency distribution of spikelet fertility in the two reciprocal F 2 and eight BC 1F 1 populations generated from the parents, Ilpumbyeo and Dasanbyeo. Population types are indicated by A (ID), B (DI), C (ID//D), D (ID//I), E (DI//D), F (DI//I), G (I//ID), H (I//DI), I (D//ID), and J (D//DI). The spikelet fertility of F 1, the parents, IP (Ilpumbyeo) and DS (Dasanbyeo) is indicated by arrows in the black bar (F 1), red bar (Ilpumbyeo), and blue bar (Dasanbyeo). Table 2. Main-effect QTLs affecting spikelet fertility (SF) in the reciprocal F 2 generated from Ilpumbyeo and Dasanbyeo hybrids. Chr Loci Marker interval a LOD a b d c Gene R 2e Favorable action d LOD a b d c Gene R 2e Favorable (%) allele action d (%) allele Ilpumbyeo Dasanbyeo Dasanbyeo Ilpumbyeo 3 qsf3.1 S03027-S OD 6.2 D 5 qsf5.1 S05004B-S OD 10.2 I OD 9.3 I 5 qsf5.2 S05009-S OD 7.8 I OD 8.7 I 6 qsf6.2 S06040-S D 9.7 D A 34.3 D 7 qsf7 S07050A-S OD 8.4 I 9 qsf9.1 S09004-S09040B OD 8.0 D 12 qsf12.1 S12005-S12009A D 27.2 I OD 6.6 I 12 qsf12.2 S12009A-S12011B D 7.1 I D 5.4 I total a Underlined markers refer to the nearest loci of a putative QTL. b Additive (a) effect. c Dominance (d) effect. A positive sign means dominance for a higher value of the trait, and a negative value means dominance for a lower value of the trait. d A=additive or partial dominance (0< d/a <0.55); D=partial dominance or dominance (0.55< d/a <1.20), OD=overdominance ( d/a >1.20). The scale of ratio d/a is adopted from (Edwards et al., 1987). e The percentage of individual phenotypic variance explained, value determined by Windows QTL Cartographer, Version 2.5.

5 Genes & Genomics (2012) 34: Figure 2. Mean value of spikelet fertility observed in the two reciprocal F 2 and eight BC 1F 1 populations generated from the parents, Ilpumbyeo and Dasanbyeo. SF is an abbreviation for spikelet fertility. The highest mean value of spikelet fertility was observed in DI//D (67.89%), whereas the lowest was observed in I//DI (7.80%). respectively. A total of 15 QTLs controlling spikelet fertility (SF) were resolved in the entire genome map of the F 2 and BC 1F 1 populations. These QTLs were mapped on chromosomes 1, 3, 4, 5, 6, 7, 9, 11, and 12 (Table 2 and Table 3). Eight out of 15 QTLs were detected in reciprocal F 2 populations, while 14 out of 15 QTLs were identified in reciprocal BC 1F 1 populations. In the ID population, six QTLs were detected (Table 2), whereas seven QTLs were detected in the DI population. Five QTLs, qsf5.1, qsf5.2, qsf6.2, qsf12.1 and qsf12.2 were identified in both F 2 populations, in which only Ilpumbyeo allele had positive effects on spikelet fertility. The other three QTLs were detected either in ID or DI population. The individual phenotypic variance explained (PVE) by each QTL ranged from 7.1% to 27.2% in ID population, and from 5.4% to 34.3% in DI population. The gene action of the QTLs for hybrid fertility identified in F 2 populations was under-dominance at all loci (Table 2). This indicates that alleles responsible for decreased spikelet fertility were dominant over the alleles increasing spikelet fertility. The dominance of these alleles was confirmed by the frequency distribution in the reciprocal F 2 population, showing that the progenies were mostly in the lower range of the spikelet fertility distribution (Fig. 1). In the backcross populations (Table 3), one QTL in I//DI and D//ID, two QTLs in each of ID//D and I//ID, three QTLs in each of ID//I, DI//D and DI//I, and four QTLs in D//DI were identified. PVE of the QTLs ranges from 6.3% to 76.0%. Table 3. Main-effect QTLs affecting spikelet fertility (SF) in the hybrid reciprocal backcrosses. a b Population Chr Loci Marker Interval a LOD b A c R 2d (%) Positive effect ID//D 3 qsf3.1 S03027-S D 6 qsf6.2 S06040B-S D Total 64.1 ID//I 1 qsf1 S01011-S I 5 qsf5.1 S05004B-S I 5 qsf5.3 S05032-S I Total 45.7 DI//D 6 qsf6.1 S06031-S06040B D 6 qsf6.2 S06040B-S D 11 qsf11 S11018-S I Total 93.7 DI//I 5 qsf5.1 S05004B-S I 7 qsf7 S07050A-S I 12 qsf12.2 S12011B-S I Total 63.4 I//ID 4 qsf4 S04058-S04077A I 5 qsf5.2 S05009-S I Total 44.4 I//DI 3 qsf3.2 S03048-S D D//ID 6 qsf6.2 S06040B-S D D//DI 1 qsf1 S01011-S D 5 qsf5.1 S05004B-S D 9 qsf9.1 S09006-S09026B D 9 qsf9.2 S09026B-S09040B D Total 78.4 Underlined markers refer to the nearest loci of a putative QTL. LOD thresholds were determined using Windows QTL Cartographer based on 1000 permutations. c Refers to the additive (a) effect. I = Ilpumbyeo, D = Dasanbyeo. d The percentage of individual phenotypic variance explained, value determined by Windows QTL Cartographer, Version 2.5.

6 360 Genes & Genomics (2012) 34: Table 4. Digenic-epistatic QTL pairs affecting spikelet fertility (SF) in two reciprocal F2 populations generated from crosses between Ilpumbyeo and Dasanbyeo. Pop a chr Locus 1 chr Locus 2 aa da ad dd PVE (%) ID 1 S S *** S S **** 0.92 ** S S * 9.4 *** 9.6 Total 27.6 DI 5 S05004B (qsf5.1) 12 S12009A (qsf12.1) **** **** -27 **** * 3.9 a Pop is an abbreviation for population, I refers to Ilpumbyeo, and D refers to Dasanbyeo. Component epistasis is additive-by-additive (aa ), dominance-by-additive (da ), additive-by-dominance (ad ), and dominance-by-dominance (dd ). Significance levels of P<0.05, P<0.01, P<0.005, and P<0.001 are denoted by *, **, ***, and ****, respectively. In most QTLs except qsf11 in DI//D and qsf3.2 in I//DI, alleles of recurrent parents contributed positively to the increase of spikelet fertility despite the crossing direction. Of the fourteen QTLs identified for hybrid sterility in the reciprocal BC 1F 1 populations, qsf5.1 was located at the corresponding position in the three populations (ID//I, DI//I, and D//DI). The qsf6.2 was also located at a similar position in three other populations (ID//D, DI//D, and D//ID). The qsf1 was detected at the corresponding position in both the ID//I and D//DI populations. Overall, in comparison to other QTL regions detected in our mapping populations, qsf5.1 and qsf5.2 on chromosome 5, qsf6.2 on chromosome 6, and qsf12.2 on chromosome 12 were highly conserved regions responsible for hybrid ferility since they were located at the same positions in both the F 2 and BC 1F 1 populations (Table 2 and Table 3). In addition, one major QTL on chromosome 12, qsf12.2, was also thought to be a highly conserved QTL responsible for spikelet fertility Figure 3. Distribution of QTLs responsible for hybrid sterility identified in the two reciprocal F 2 and eight BC 1F 1 populations generated from the parents, Ilpumbyeo and Dasanbyeo. The QTLs related to hybrid sterility were identified throughout the rice chromosomes, except chromosomes 2, 8, and 10. In comparison to other regions, the QTLs for hybrid sterility located on chromosomes 3, 5, 6, and 12 were commonly identified in at least three populations. The marker order in constructing the graph was derived from the physical position of DNA markers on each chromosome, based on the Rice Genome Research Program data ( The flanking markers nearest to the QTL regions are also shown (below the graph).

7 Genes & Genomics (2012) 34: Table 5. Digenic epistasis QTLs affecting spikelet fertility (SF) identified in eight reciprocal backcross populations between Ilpumbyeo and Dasanbyeo. a b c d Pop chr Marker interval i b chr Marker interval j LOD Ai a Aj a AAij c PVE (%) d ID//D 3 S03140-S S11071B-S ID//I 1 5 S05009-S05004B 12 S12011B-S S05004B-S S09040B-S S05064-S05077A 7 S07114-S Total 21.1 S01022-S01038 (qsf1) 5 S05009-S05029 (qsf5.1) S02052-S02057B 9 S09004-S S03027-S S12039B-S S03136-S S08112-S08121A Total 14.9 DI//D 8 S08090-S S10051-S10058A S10001-S10003A 12 S12071-S Total 7.8 D//DI 1 S01011-S01022 (qsf1) 3 S03020-S S01160-S01181B 4 S04058-S S10072-S S12039-S Total 27.9 Ai and Aj represent the additive effect on intervals i and j, respectively. Markers indicated in bold are those nearest or flanking putative main-effect QTLs. AAij represents the epistatic effect between intervals i and j as defined by (Mei et al., 2003). PVE (%) is the proportion of phenotypic variation explained by AAij, significant at P< inherited from the parents since it was located in the same position in the reciprocal F 2 and DI//I populations. We found that some of detected QTLs felt into neighbor intervals (Table 2 and Table 3). Despite several algorithms including inclusive composite interval mapping (ICIM) and mix-model composite interval mapping (MCIM) were employed to verify whether or not they represented two QTL, however, the certain QTLs still fell into the same position. This suggests that two adjacent peaks at neighbor intervals represent two QTLs. Identification of digenic-epistatic interactions for spikelet fertility A total of 17 significant digenic-epistatic interactions (hereafter Ep-QTLs) were identified in the reciprocal F 2 and BC 1F 1 populations (Table 4 and Table 5). In reciprocal F 2 populations, four Ep-QTLs for spikelet fertility were detected, and there was no main-effect QTL involved (Table 4). In the BC 1F 1 populations, a total of 13 Ep-QTLs were detected (Table 5). Of them, only two Ep-QTLs, S01022-S01038 (qsf1) with S05009-S05029 (qsf5.1) in ID//I and S01011-S01022 (qsf1) with S03020-S03027, contained main-effect QTLs. PVE of each Ep-QTL ranged from 2.8% to 12.5%, which was relatively lower comparing to main-effect QTLs. Discussion Main-effect QTLs for spikelet fertility in comparison with previous studies A large number of loci or QTLs affecting hybrid sterility in rice have been identified. Several QTLs causing female gamete abortion (Yanagihara et al., 1992; Wan et al., 1993; Wan and Ikehashi, 1995; Wan et al., 1996; Zhu et al., 2005; Singh et al., 2006; Zhao et al., 2006; Li et al., 2007; Zhao et al., 2007; Chen et al., 2008), male gamete abortion (Sano, 1983; Sano, 1990; Wang et al., 1998; He and Xu, 2000; kubo et al., 2000; Liu et al., 2001; Sobrizal et al., 2001; Sobrizal et al., 2002; Song et al., 2005; Wang et al., 2006 ; Jing et al., 2007; Koide et al., 2008; Li et al., 2008; Long et al., 2008; Chin et al., 2011), and spikelet fertility/sterility (Li et al., 1997a; Wang et al., 1998; He et al., 1999; He and Xu, 2000; Kubo and Yoshimura, 2001; Mei et al., 2003; Marri et al., 2005; Mei et al., 2005; Song et al., 2005; Wang et al., 2005; Qiao et al., 2008; Chin et al., 2011) have been reported. In our study, we identified 15 QTLs associated with hybrid fertility/sterility. Of these, five loci, qsf5.1, qsf7, qsf9.2, qsf11, and qsf12.1, were novel. The QTLs found in this study compared with already reported QTLs are shown in Table 6. The qsf1 locus detected on chromosome 1, located at

8 362 Genes & Genomics (2012) 34: Table 6. Comparison of QTLs for hybrid sterility with previous studies in rice. QTL Chromosome Previous studies that identified common regions Parents a Population type b qsf1 1 f1 (Wang et al., 1998) Balilla/Dular//Nanjing 11 BC 1F 1 qsf3.1 3 L3b (He and Xu, 2000); S3b (He et al., 1999) Zhenshan 97/Minghui 63; Peiai 645/Peiai 8902S qsf3.2 3 sf3.1(marri et al., 2005) IR58025A/Oryza rufipogon BC 2F 1 qsf4 4 qfrp4-1(hittalmani et al., 2003) IR64/Azucena DH qsf qsf5.2 5 f5 (Li et al., 1997c; Song et al., 2005; Wang et al., 2006 ; Qiao et al., 2008; Chin et al., 2011) Lemont/Teqing 02428/Nanjing 11//Balilla Zhenshan 97/Dular//Balilla Dasanbyeo/Ilpumbyeo//Dasanbyeo Dasanbyeo/TR22183 qsf5.3 5 pfi5.2 and sfj5.2 (Chin et al., 2011) Dasanbyeo/TR22183 RIL qsf6.1 6 S5 (Yanagihara et al., 1995; Liu et al., 1997; Chen et al., 2008) IR36/Nekken/Akihikari 02428/Nanjing 11//Balilla CO4T1/Balilla; CO4T1/Nanjing 11; CO2T1/Balilla; CO2T1/Nanjing 11 qsf6.2 6 esa-1(liu et al., 2001) ZYQ8/JX17 DH qsf6.3 6 L6 (He and Xu, 2000); qsf6 (Qiao et al., 2008) Zhenshan 97/Minghui 63 Dasanbyeo/Ilpumbyeo//Dasanbyeo F 2 F 2 F 2, F 3, F 4 F 1 three-way cross Testcross BC 1F 1 RIL F 1 three-way cross F 1 three-way cross Test-crosses of T 1 (transgenic plants) F 2 BC 1F 1 qsf7 7 - qsf9.1 9 qpss9.1(lanceras et al., 2004) CT9993/IR62666 DH qsf qsf qsf qsf qsf12.1(qiao et al., 2008) Dasanbyeo/Ilpumbyeo//Dasanbyeo BC 1F 1 a Balilla, Zhenshan 97, Azucena, Lemont, 02428, Ilpumbyeo, TR22183, Akihikari, JX17, and CT9993 belong to japonica type; Dular and Nekken are wide compatibility varieties (WCV); Nanjing 11, Minghui 63, Peiai 645, Peiai 8902S, IR58025A, IR64, Teqing, Dasanbyeo, IR36, ZYQ8, and IR62266 belong to indica type. b DH is an abbreviation for doubled-haploid; RIL is an abbreviation for recombinant inbred lines Mbp of the Nipponbare Pseudomolecule annotated by IRGSP Build5, is likely to be similar to that of the f1 locus (Wang et al., 1998). Although this locus is not novel, we found that it is associated with both female and male sterility, and this fact has not been reported in previous studies. We came to this conclusion because this locus was detected in both ID//I (embryo sac-segregating population) and D//DI (pollen-segregating population). The qsf3.1 locus was physically located at Mbp of the Nipponbare Pseudomolecule, which is similar to the location of L3b and S3b (He et al., 1999; He and Xu, 2000). Locus qsf3.2, located at Mbp of the Nipponbare Pseudomolecule, shared a similar location with the sf3.1 locus on chromosome 3 (Marri et al., 2005). The physical location of QTL qsf4 responsible for spikelet fertility on chromosome 4 is Mbp of the Nipponbare Pseudomolecule, corresponding to qfrp4-1 for spikelet fertility reported by Hittalmani et al. (2003). The qsf5.2, which is located at Mbp of the Nipponbare Pseudomolecule on chromosome 5, shared a similar location with the f5 locus (Li et al., 1997c; Song et al., 2005; Wang et al., 2006 ; Qiao et al., 2008; Chin et al., 2011). We found a novel QTL qsf5.1, located very close to qsf5.2 at Mbp; a position that was different from that reported in previous studies. In addition, the qsf5.3 locus located at Mbp shared a similar location with the locus specifying pollen (pfi5.2) and spikelet fertility (sfj5.2) on chromosome 5, reported by Chin et al. (2011). Furthermore, the loci responsible for spikelet fertility on chromosome 6, qsf6.1 and qsf6.2, are located at Mbp, and Mbp, respectively. Locus qsf6.1 corresponded to S5 for wide compatibility (Yanagihara et al., 1995; Liu et al., 1997; Chen et al., 2008), and qsf6.2 corresponded to esa1 for embryo sac abortion (Liu et al., 2001). The locus qsf7 is located at Mbp of the Nipponbare Pseudomolecule on chromosome 7, distant from the two spikelet sterility QTLs reported by previous studies (Li et al., 1997b; He et al., 1999), suggesting that this locus is novel. The qsf9.1 locus on chromosome 9 corresponded to qpss9.1 specifying spikelet sterility reported by Lanceras et al. (2004), whereas qsf9.2, located at Mbp of the Nipponbare Pseudomolecule, was a new QTL for hybrid sterility on chromosome 9. The qsf11 locus is located at Mbp of the Nipponbare

9 Genes & Genomics (2012) 34: Pseudomolecule, and to our knowledge this locus is also a new QTL responsible for spikelet fertility as previous studies have not reported a similar QTL for spikelet fertility at this location. In addition, the qsf12.2 locus corresponded to qsf12.1 reported by (Qiao et al., 2008), whereas qsf12.1 was a novel locus responsible for spikelet fertility found in the present study. The QTLs for hybrid fertility identified on chromosomes 5 and 6 exhibited high LOD and PVE values, implying that they had a major effect on spikelet fertility and highly conserved across genetic backgrounds and mapping populations (Table 2 and Table 3). Several potential Ep-QTLs were identified in six out of the ten mapping populations (Table 4 and Table 5). However, their contributions to PVE were relatively low comparing to the main effect QTLs, suggesting that specific loci are more effective in determining spikelet fertility in hybrid progenies. Gametophytic factors affecting QTLs for spikelet fertility Since only either of the female (embryosac) or male (pollen) gamete is segregating in BC 1F 1 populations depending on the crossing direction, it is possible to estimate which gamete might affect the spikelet fertility. For example, Although both of embryosac and pollen will be segregating in DI or ID F 2 populations, only embryosacs will be segregating in ID//I or DI//I BC 1F 1 population, while in I//DI or I//ID BC 1F 1 population only pollens will be segregating. In this context, it is natural that most of the QTLs detected in F 2 populations were also present in BC 1F 1 populations, because in F 2 all of the factors affecting hybrid fertility are mixed up including the sterility source of embryosac and pollen origin. The two close peaks of QTLs (qsf5.1 and qsf5.2) observed on chromosome 5 in the F 2 population might suggest that this is only one locus, however, in some BC 1F 1 populations, only one peak was observed (Fig. 3). This means that there are two loci responsible for hybrid ferility at this position on chromosome 5. The locus qsf5.2 affects only male sterility, as it was identified only in the pollen-segregating population (I//ID), and qsf5.1 affects both male and female factors (Fig. 3), confirming that these are two completely different QTLs. Similarly, qsf6.1 and qsf6.2 affected spikelet fertility through both male and female gametes. However, qsf3.1, qsf7 and qsf12.2 are likely to affect only the spikelet fertility through embryosac factors. On the other hand, qsf9.1 affected the spikelet fertility through pollen factors. 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