Enhanced grain yield in rice hybrids through complementation of fertility restoration by Rf3 and Rf4 genes as revealed by multilocation evaluation of tropical japonica derived rice (Oryza sativa) hybrids

Thirty-one tropical japonica derived Rf gene carrying rice hybrids were classified into three classes as Rf3, Rf4 and Rf3 + Rf4 hybrids. These hybrids were tested under three different mega-environments. Between Rf3 class and both the classes of hybrids possessing Rf4 genes, significant variation for spikelet and pollen fertility and grain yield was found. The pollen fertility was five times higher among Rf4 hybrids than that of hybrids carrying Rf3 alone. Likewise, spikelet fertility among Rf4 hybrids was two times higher than that of Rf3 hybrids. Parallel grain yield increase in Rf4 hybrids was 2.4 times than in Rf3 hybrids. However, Rf3 gene was found complementing Rf4 to truncate the range of pollen and spikelet fertility. Hybrids possessing Rf3 alone exhibited partial pollen and spikelet sterility, and significant negative standard heterosis for grain yield. The high yielding hybrid, “HYB36” carrying both the Rf genes was found to be widely adapted. The present study established that Rf4 gene is essential either alone or in combination with Rf3 for fertility restoration to achieve enhanced grain yield in WA-CMS based hybrids.     

Identification of stable restorers and genetics of fertility restoration in late‐maturing pigeonpea [Cajanus cajan (L.) Millspaugh]

A total of sixty‐six germplasm lines were crossed with five CMS lines, where two belong to A₄ cytoplasm, while other three belong to A₂ cytoplasm. On the basis of pollen fertility test as well as good pod setting, of 330 hybrids, 34 restorer lines were observed in ICPA 2043 and 19 in ICPA 2092. Thirteen germplasm lines restored fertility in both the A₄ CMS lines, viz. ICPA 2043 and ICPA 2092; however, none of the lines restored fertility in A₂ CMS lines. For confirmation of result, restoration competence of identified lines tested subsequently 2 years at two different temperatures. The segregation patterns for fertility restoration studied in F₂ and BC₁F₁ generations of selected ten crosses. Six crosses indicated the involvement of two major genes with recessive epistasis, three crosses confirmed dominant epistasis, and one cross indicated the involvement of duplicate recessive epistasis. The obtained results from this study will hasten the future three‐line breeding programme and lead the hybrid technology to the farmers' field with the better exploitation of CMS lines.     

Inheritance and molecular mapping of restorer‐of‐fertility (Rf) gene in A2 hybrid system in pigeonpea (Cajanus cajan)

Inheritance of fertility restorer gene in pigeonpea was studied using F₂ and BC₁F₁ populations derived from cross AL103A × IC245273. It was found to be controlled by single dominant gene. Out of 228 SSR primer pairs, 33 primer pairs showed parental polymorphism, while four primers were found polymorphic in bulk segregant analysis (BSA). These four primers viz., CcM 1615, CcM 0710, CcM 0765 and CcM 1522 were used for genotyping of F₂ population and were found to be placed at 3.1, 5.1, 28.1 and 45.8 cM, respectively. Two of them, CcM 1615 and CcM 0710, evinced clear and unambiguous bands for fertility restoration in F₂ population. The Rf gene was mapped on linkage group 6 between the SSR markers CcM 1615 and CcM 0710 with the distances of 3.1 and 5.1 cM, respectively. The accuracy of the CcM 1615 was validated in 18 restorers and six maintainer lines. The marker CcM 1615 amplified in majority of male restorer lines with a selection accuracy of 91.66%.     

Identification of the Rf gene conferring fertility restoration of the CMS Dian-type 1 in rice by using simple sequence repeat markers and advanced inbred lines of restorer and maintainer

Cytoplasmic male sterility of Dian‐type 1 (CMS‐D1) was developed 30 years ago in Yunnan. A major gene conferring fertility restoration for the CMS‐D1 system was detected by microsatellite markers in advanced inbred lines consisting of 196 maintainers and 62 restorers developed in breeding programmes of hybrid rice involving the CMS‐D1 system. The gene was mapped between two simple sequence repeat markers, OSR33 and RM228, on chromosome 10, and was temporarily designated as Rf‐D1(t). The genetic distances of the gene to the two microsatellite markers were 3.4 and 5.0 cM, respectively. This linkage was confirmed by using an F2 population derived from a cross between a CMS‐D1 line and a restorer. This study also demonstrated that using OSR33 was reliable and efficient for identification of restoring lines in hybrid rice breeding with the CMS‐D1 system.     

Combining ability of non‐adapted sources for male‐fertility restoration in Pampa CMS of hybrid rye*

Hybrid rye breeding and seed production relies on the cytoplasmic male sterility‐inducing Pampa (P)‐Cytoplasm. High levels of restoration were recently found in non‐adapted rye accessions from Argentina (Pico Gentario, Pastoreo Massaux) and Iran (IRAN IX). To analyse their relative superiority, five seed‐parent lines in P cytoplasm were crossed with five inbred lines of these sources and four adapted restorer lines. The 45 F1 hybrids were tested for male‐fertility restoration in three locations. Significant (P = 0.01) general and specific combining ability (GCA and SCA respectively) for male‐fertility restoration were found. The non‐adapted restorer lines were able to restore all seed‐parent lines similarly, resulting in hybrids with 55‐90% pollen shedding (mean 78%) compared with 2‐74% pollen shedding (mean 44%) when adapted restorer lines were used. Significant (P = 0.05) SCA effects were detected in 11 of 45 combinations, nine of them were crosses with adapted pollinator lines. Non‐adapted restorer lines showed a high phenotypic stability of male‐fertility restoration across locations. Introgression of these exotic sources into the adapted restorer gene pool by repeated backcrossing should result in environmentally stable male fertility in Pampa‐based rye hybrids.     

Frequency and fertility restoration efficiency of Rf3 and Rf4 genes in Indian rice

Identification of new parental lines is crucial for developing ecology‐specific hybrids with ideal agronomic performance. We screened a total of 570 different ecology‐specific Indian rice varieties for the presence of fertility restorer genes, Rf3 and Rf4 using tightly linked markers DRRM Rf3‐10 and RM6100, respectively. Among these varieties, 13% carried Rf3Rf3/Rf4Rf4, 31% carried rf3rf3/rf4rf4, 6% carried Rf3Rf3/rf4rf4 and remaining 50% carried Rf4Rf4/rf3rf3 allelic combinations. A mini set of 40 varieties with variable allelic combinations of fertility restorer genes were testcrossed with WA and Kalinga‐based CMS lines. All the 80 F₁s were evaluated for spikelet fertility and fertility restoration ability. Rf3Rf3/rf4rf4 genotypes mostly behaved as partial maintainers or partial restorers. In contrast, rf3rf3/Rf4Rf4 genotypes were partial or effective restorers. However, double dominant genotypes showed better fertility restoration than the genotypes containing Rf3 or Rf4 individually. Some of the genotypes showed unexpected restoration pattern implying occurrence of other fertility restorer(s) apart from Rf3 and Rf4. The perfect restorers and maintainers identified in this study can be directly used in hybrid rice breeding.     

Fine mapping of Rf2, a major locus controlling pollen fertility restoration in sorghum A1 cytoplasm,encodes a PPR gene and its validation through expression analysis

Sorghum is one of the pioneering cereal crops where cytoplasmic male sterility (CMS) was successfully exploited for mass production of F₁ hybrid seed. Mapping genes for fertility restoration (Rf) is an important aspect of understanding the molecular basis of fertility restoration in crop plants. In this study, we fine‐mapped a fertility restoration locus, Rf2 of sorghum reported earlier (Jordan, Mace, Henzell, Klein, & Klein, 2010 ), involving two F₂ populations (296A × RS29 and 296A × DSV1) and newly developed SSR markers delimited Rf2 locus to 10.32‐kb region on chromosome 2. The Rf2 locus was tightly linked with two new SSRs, MS‐SB02‐3460 (0.14 cM) and MS‐SB02‐3466 (0.75 cM) on both sides, and hosted only one gene (Sobic.002G057050) of PPR gene family. Another new SSR marker developed in the study, MS‐SB02‐37912, forms the part of PPR gene and could act as a perfect marker in marker‐assisted breeding for fertility restoration involving Rf2 in sorghum breeding. The strong involvement of Sobic.002G057050 gene in fertility restoration was supported through RNA expression analysis.     

Influence of water availability on fertility restoration of CMS lines with the 'M35', A4 and '9E' CMS-inducing cytoplasms of sorghum

Male fertility restoration in new types of sorghum cytoplasmic male sterility‐inducing cytoplasms (A4, ‘9E’, ‘M35’), characterized by the formation of non‐dehiscent anthers, is difficult. Lines with fertility‐restorer genes for these unique cytoplasms do occur, but rarely, and when found tend to be unstable in their inheritance and expression. The aim of this research was to explore reasons for this instability. Seven lines in three unique cytoplasms, ‘9E’, A4 and ‘M35’, and six lines that restore with these cytoplasms were grown at the Agricultural Research Institute for South‐East Region in Saratov, Russia from 1993 to 2004. Levels of male fertility restoration and various environmental factors were recorded. It is reported that for sorghum hybrids in the A4, ‘9E’ and ‘M35’ male‐sterile cytoplasms, the level of plant male fertility is determined by the level of water available to plants during anther and pollen formation that which ‘switches on’ the expression of fertility‐restoring genes, and is possibly involved in an unusual type of male fertility inheritance in these cytoplasms. The creation of reliable line‐fertility restorers capable of the restoration of male fertility of F₁ hybrids in ‘M35’ cytoplasm under conditions of water stress is also reported. Current research explore mechanisms involved possible in responses to water levels at various growth stages and their influence on fertility within these cytoplasms.     

Identification of restorers and maintainers for developing basmati and non-basmati hybrids in rice, Oryza sativa

Twenty‐seven improved aromatic lines of germplasm and 18 non‐aromatic disease‐resistant genotypes of rice were test‐crossed with four cytoplasmic male‐sterile lines (IR 58025A, IR 62829A, PMS 3A and PMS 10A). Thirteen aromatic and 10 non‐aromatic genotypes were selected based on pollen fertility, and crosses were repeated to confirm sterility‐maintaining and fertility‐restoring ability. Genotypes were categorized as effective restorers (> 80% spikelet fertility), partial restorers (21‐79% spikelet fertility) and maintainers (< 1% spikelet fertility). The effective basmati restorers identified were Basmati 385, Chandan, P1031‐8‐5‐1, HKR 241‐IET‐12020, SAF Khalsa 7 and Karnal Local. The basmati maintainers identified were Basmati 370, Pusa basmati 1, P615‐K‐167‐13 and P1173‐4‐1. The frequency of restorers obtained was higher for the non‐aromatic than the aromatic basmati type. The performance of restorers varied with cytoplasmic male‐sterile (CMS) line, location and season of testing. The differential ability to restore fertility in the CMS lines that have the wild abortive (WA) cytosterile system could result from different nuclear backgrounds of the CMS lines. These restorers and maintainers possess acceptable grain dimensions, a desirable degree of aroma, volume expansion through linear kernel elongation and cooking quality characteristics of basmati rice. These genotypes will contribute to developing basmati hybrids and provide restorers and maintainers with acceptable key basmati quality characteristics.     

Inheritance of root traits and phosphorus uptake in common bean (Phaseolus vulgaris L.) under limited soil phosphorus supply

Heterosis is an important way to improve yield and quality for many crops. Hybrid rice and hybrid maize contributed to enhanced productivity which is essential to supply enough food for the increasing world population. The success of hybrid rice in China has led to a continuous interest in hybrid wheat, even when most research on hybrid wheat has been discontinued in other countries for various reasons including low heterosis and high seed production costs. The Timopheevii cytoplasmic male sterile system is ideal for producing hybrid wheat seeds when fertility restoration lines with strong fertility restoration ability are available. To develop PCR-based molecular markers for use in marker-assisted selection of fertility restorer lines, two F₂ populations derived from crosses R18/ND36 and R9034/ND36 were used to map fertility restoration genes in the two elite fertility restorer lines (R-lines) R18 and R9034. Over 678 SSR markers were analyzed, and markers closely linked to fertility restoration genes were identified. Using SSR markers, a major fertility restoration gene, Rf3, was located on the 1B chromosome in both populations. This gene was partially dominant in conferring fertility restoration in the two restorer lines. SSR markers Xbarc207, Xgwm131, and Xbarc61 are close to this gene. These markers may be useful in marker-assisted selection of new restorer lines with T. timopheevii cytoplasm. Two minor QTL conferring fertility restoration were also identified on chromosomes 5A (in R18) and 7D (in R9034) in two R-lines.     

Analysis of QTL responsible for grain iron and zinc content in doubled haploid lines of rice (Oryza sativa) derived from an intra-japonica cross

Quantitative trait loci (QTL) related to the grain iron and zinc contents of brown rice were mapped by using a doubled haploid population derived from an intra-japonica cross between 'Hwaseonchal' and 'Goami 2'. QTL–QTL, background–QTL, and background–background interactions and candidate genes that affect grain iron and zinc contents were preliminarily identified. Twenty-one iron- and zinc-related QTL were found. The major-effect QTL qFe7 and qZn7 provided the highest contribution to phenotypic variance for grain iron and zinc contents. The colocation of zinc- and iron-related QTL on chromosomes 1, 4, 7 and 11 may account for the strong correlation between iron and zinc contents. A region on chromosome 7 and epistatic interaction between loci on chromosomes 2 and 10 affected iron content. qZn7 and qZn11.3 exerted additive effects on zinc content. Eleven iron- and zinc-related candidate genes colocated with qFe7, qZn7 and the region on chromosome 7 with an additive effect on iron content. The major-effect QTL identified here may be useful for breeding biofortified rice.     

Molecular mapping of the fertility restoration locus Rf1 in sunflower and development of diagnostic markers for the restorer gene

Fertility restoration by dominant nuclear genes is essential for hybrid breeding based on cytoplasmic male sterility (CMS) to obtain heterotic effects and high seed yields. In sunflower, only the PET1 sterility inducing cytoplasm has been used in commercial hybrid breeding until now. This particular male sterility was derived from an interspecific hybrid Helianthus petiolaris × H. annuus. For the recent work we used the segregating population RHA325(CMS) × HA342, based on the PET1 cytoplasm. Molecular markers were mapped within 1.1 cM around the restoration locus Rf1. At the distal side, the marker OP-K13_454 mapped at a distance of 0.9 cM and E32M36-155R at 0.7 cM from Rf1. At the proximal side the markers E44M70-275A, E42M76-125A and E33M61-136R were mapped at 0.1, 0.2, and 0.3 cM from the restorer locus, respectively. These markers provide an excellent basis for a map based cloning approach and for marker-assisted sunflower breeding.     

Comparison of fertility between the F1, F2 and anther derived lines in the crosses of indica/japonica and japonica/indica in rice (Oryza sativa L.)

Summary Response of anthers in in vitro culture was examined in the indica-japonica hybrids of rice (Oryza sativa L.). Significant genotypic differences were observed for callus induction and regeneration among the different interracial hybrids of indica-japonica races. Induction frequency of haploids ranged from 57.7 to 72.9 per cent and doubled haploid androgenic lines ranged from 27.1 to 42.3 per cent in the anther culture of the different hybrids. The indica-japonica hybrids recorded partial pollen grain and spikelet fertility in F1 (29.9 to 41.5% and 19.4 to 48.7% respectively) as well as in F2 (42.7 to 50.6% and 37.1 to 54.4% respectively). In contrast, the androgenic doubled haploid lines recorded significant increase and the pollen grain and spikelet fertility was 76.3 and 78.6 per cent respecitively. The results suggested that the sterility barriers for realising genetic recombinants and fixation of fertile homozygous lines in indica-japonica hybridization programme could be overcome through F1 anther culture technique.Abbreviations BAP Benzyl Amino Purine - 2,4-D 2,4-Dichlorophenoxy acetic acid - MS Murashige & Skoog medium - IAA Indole Acetic Acid     

Characterization of kafirin composition and in vitro digestibility in CMS‐lines,fertility restorers and F1 hybrids with the new types of CMS‐inducing cytoplasms of sorghum

One of the reasons of poor nutritive value of sorghum grain is resistance of its seed storage proteins (kafirins) to protease digestion. To reveal sorghum entries with increased kafirin digestibility, the sodium dodecyl sulfate – polyacrylamide gel electrophoresis (SDS‐PAGE) of endosperm proteins of 10 lines [cytoplasmic male sterility (CMS)‐lines and fertility restorers] and five F₁ hybrids before and after pepsin digestion was carried out. For quantitative estimation of kafirin digestibility the SDS‐PAGE banding patterns were scanned by laser densitometer. Significant variability for both individual fractions and total kafirin digestibility was found. The line KVV‐45, fertility restorer for the Indian ‘M35‐1A’ type of CMS, had the highest level of kafirin digestibility (30% and 25% of undigested γ‐ and α₁‐kafirins, respectively), while in some entries 80–90% of kafirins remained undigested. Increased α₁‐kafirin digestibility coincided with relatively high γ‐kafirin digestibility. High‐molecular weight kafirins (HMWK) (45 kDa and 66 kDa) resistant to pepsin digestion were found in some lines, the F₁ hybrids had the same HMWK as parental lines. These data demonstrate possibility for isolation of sorghum genotypes with increased nutritive value by screening their flour for in vitro protein digestibility.     

Comparative genetic analysis and molecular mapping of fertility restoration genes for WA,Dissi, and Gambiaca cytoplasmic male sterility systems in rice

The genetic relationship among three cytoplasmic male sterility (CMS) systems, consisting of WA, Dissi, and Gambiaca, was studied. The results showed that the maintainers of one CMS system can also maintain sterility in other cytoplasmic backgrounds. The F₁ plants derived from crosses involving A and R lines of the respective cytoplasm and their cross-combination with other CMS systems showed similar pollen and spikelet fertility values, indicating that similar biological processes govern fertility restoration in these three CMS systems. The results from an inheritance study showed that the pollen fertility restoration in all three CMS systems was governed by two independent and dominant genes with classical duplicate gene action. Three F₂ populations, generated from the crosses between the parents of good-performing rice hybrids, that possess WA, Dissi, and Gambiaca CMS cytoplasm, were used to map the Rf genes. For the WA-CMS system, Rf3 was located at a distance of 2.8 cM from RM490 on chromosome 1 and Rf4 was located at 1.6 cM from RM1108 on chromosome 10. For the Dissi-CMS system, Rf3 was located on chromosome 1 at 1.9 cM from RM7466 and Rf4 on chromosome 10 was located at 2.3 cM from RM6100. The effect of Rf3 on pollen fertility appeared to be stronger than the effect of Rf4. In the Gambiaca-CMS system, only one major locus was mapped on chromosome 1 at 2.1 cM from RM576. These studies have led to the development of marker-assisted selection (MAS) for selecting putative restorer lines, new approaches to alloplasmic line breeding, and the transfer of Rf genes into adapted cultivars through a backcrossing program in an active hybrid rice breeding program.     

Molecular mapping of quantitative trait loci for plant growth, yield and yield related traits across three diverse locations in a doubled haploid rice population

A doubled haploid (DH) population of 125lines derived from IR64 × Azucena, an indica – japonica cross were grown in three different locations in India during the wet season of 1995. The parents of mapping population had diverse phenotypic values for the eleven traits observed. The DH lines exhibited considerable amount of variation for all the traits. Transgressive segregants were observed. Interval analysis with threshold LOD > 3.00 detected a total of thirty four quantitative trait loci (QTL) for eleven traits across three locations. The maximum number of twenty QTL were detected at Punjab location of North India. A total of seven QTL were identified for panicle length followed by six QTL for plant height. Eight QTL were identified on three chromosomes which were common across locations. A maximum of seven QTL were identified for panicle length with the peak LOD score of 6.01 and variance of 26.80%. The major QTL for plant height was located on Chromosome 1 with peak LOD score of 16.06 flanked by RZ730-RZ801 markers. Plant height had the maximum number of common QTL across environment at the same marker interval. One QTL was identified for grain yield per plant and four QTL for thousand grain weight. Clustering of QTL for different traits at the same marker intervals was observed for plant height, panicle exsertion, panicle number, panicle length and biomass production. This suggests that pleiotropism and or tight linkage of different traits could be the plausible reason for the congruence of several QTL. Common QTL identified across locations and environment provide an excellent opportunity for selecting stable chromosomal regions contributing to yield and yield components to develop QTL introgressed lines that can be deployed in rice breeding program. This revised version was published online in July 2006 with corrections to the Cover Date.     

Fine mapping of a QTL for the number of spikelets per panicle by using near‐isogenic lines derived from an interspecific cross between Oryza sativa and Oryza minuta

We constructed a high‐resolution physical map for the qSPP7 QTL for spikelets per panicle (SPP) on rice chromosome 7 across a 28.6‐kb region containing four predicted genes. Using a series of BC₇F₄ near‐isogenic lines (NILs) derived from a cross between the Korean japonica cultivar ‘Hwaseongbyeo’ and Oryza minuta (IRGC Acc. No. 101144), three QTLs for the number of SPP, grains per panicle and primary branches were identified in the cluster (P ≤ 0.01). All three QTLs were additive, and alleles from the O. minuta parent were beneficial in the ‘Hwaseongbyeo’ background. qSPP7 was mapped to a 28.6‐kb region between the two simple sequence repeat (SSR) markers RM4952 and RM21605. The additive effect of the O. minuta allele at qSPP7 was 23 SPP, and 43.6% of the phenotypic variance was explained by the segregation of the SSR marker RM4952. Colocalization of the three QTLs suggested that this locus was associated with panicle structure and had pleiotropic effects. The NIL populations and molecular markers are useful for cloning qspp7.     

Molecular cytogenetic characterization and phenotypic evaluation of new wheat–rye lines derived from hexaploid triticale ‘Certa’ × common wheat hybrids

Hexaploid triticale contains valuable genes from both tetraploid wheat and rye and plays an important role in wheat breeding programmes. In order to explore the potential of hexaploid triticale ‘Certa’ in wheat improvement, two crosses were made using ‘Certa’ as female parent, and common wheat cultivars ‘Jinmai47’ (JM47) and ‘Xinong389’ (XN389) as male parents. The karyotyping of BCF_4:5 lines from Certa/JM47//JM47 and F_5:6 lines from Certa/XN389 was investigated using sequential fluorescence in situ hybridization (FISH). One 1B(1R) substitution line and five 1BL.1RS whole‐arm translocation lines were identified, one of which was found lacking ω‐secalin locus. Many structural alterations on wheat chromosomes were detected in the progeny. Great morphologic differences resulting from genetic variations were observed, among which the photosynthetic capability was increased while grain quality was slightly improved. Compared with both parents, the stripe rust resistance at adult stage was increased in lines derived from Certa/JM47//JM47, while it was decreased in lines derived from Certa/XN389. These newly developed lines might have the potential to be utilized in wheat improvement programmes.