Complete Fertility Restoration Research Articles

Identification of Good Restorer Lines through Molecular Confirmation of Rf3 and Rf4 Genes in Rice Hybrids

Pollen fertility can be restored by nuclear-encoded genes known as fertility restorer (Rf) genes. The initial stage in creating high-yielding heterotic hybrids involves the identification of restorers capable of effectively restoring the fertility of CMS lines. Hybrid rice breeding programs have proven effective in achieving these goals by utilizing cytoplasmic male sterility (CMS) systems in combination with restorer genes. Among these, Rf3 and Rf4 genes play critical roles in restoring male fertility and are essential components of successful hybrid rice breeding. In this study, we aimed to investigate the molecular conformation of Rf3 and Rf4 genes in a set of restorer lines and hybrids in rice. DNA was isolated from 10 restorer lines and 30 rice hybrids, two checks and KMR3R were used for comparison for positive alleles. Two functional markers, RM SF21-5 for Rf3 and RMS-PRR-9-1 for Rf4, were employed to identify the allelic status of the fertility restorer genes. Results revealed that among the 42 rice entries screened, five rice hybrids lacked both Rf3 and Rf4 genes and Four rice restorer lines and four hybrids were found to possess both Rf3 and Rf4 genes, making them valuable for hybrid breeding programs. Conversely, five hybrids lacked restorer genes and were unsuitable for such breeding programs. These findings establish Rf3and Rf4 as major fertility restoring genes in rice, consistently contributing to complete fertility restoration. The genotyping results provide valuable insights for selecting appropriate parental lines and restorers in hybrid rice breeding programs. The presence or absence of these fertility-restoring genes plays a crucial role in developing high-yielding and productive rice varieties through effective breeding strategies.

Fertility and Conception Following Kidney Transplantation: A Narrative Review

Individuals with chronic kidney disease often experience sexual disorders and infertility, which can significantly impact their quality of life. The cause of infertility in CKD patients is multifactorial, resulting from sexual dysfunction, hormonal disturbance, and uraemia-induced gonadal toxicity. Kidney transplantation can significantly improve this pathological state, although complete restoration of fertility may not occur. Conceiving after kidney transplantation carries a slightly higher maternal and fetal risk compared to the normal population, with neonates of kidneytransplanted mothers showing an increased risk of prematurity and low birth weight. The use of steroids in infants was found to be safe, while mycophenolate mofetil was associated with fetal and infant toxic events and is, therefore, not recommended for breastfeeding mothers following kidney transplantation. Nevertheless, studies have indicated the safety of using azathioprine in breastfeeding kidney-transplanted mothers. Notably, limited data is available on the use of immunosuppressive drugs during pregnancy and poses an area for further research.

An Induced Mutation in HvRECQL4 Increases the Overall Recombination and Restores Fertility in a Barley HvMLH3 Mutant Background.

Plant breeding relies on the meiotic recombination or crossing over to generate the new combinations of the alleles along and among the chromosomes. However, crossing over is constrained in the crops such as barley by a combination of the low frequency and biased distribution. In this study, we attempted to identify the genes that limit the recombination by performing a suppressor screen for the restoration of fertility to the semi-fertile barley mutant desynaptic10 (des10), carrying a mutation in the barley ortholog of MutL-Homolog 3 (HvMLH3), a member of the MutL-homolog (MLH) family of DNA mismatch repair genes. des10 mutants exhibit reduced recombination and fewer chiasmata, resulting in the loss of obligate crossovers (COs) leading to chromosome mis-segregation. We identified several candidate suppressor lines and confirmed their restored fertility in an Hvmlh3 background in the subsequent generations. We focus on one of the candidate suppressor lines, SuppLine2099, which showed the most complete restoration of fertility. We characterized this line by using a target-sequence enrichment and sequencing (TENSEQ) capture array representing barley orthologs of 46 meiotic genes. We found that SuppLine2099 contained a C/T change in the anti-CO gene RecQ-like helicase 4 (RECQL4) resulting in the substitution of a non-polar glycine to a polar aspartic acid (G700D) amino acid in the conserved helicase domain. Single nucleotide polymorphism (SNP) genotyping of F3 populations revealed a significant increase in the recombination frequency in lines with Hvrecql4 in the Hvmlh3 background that was associated with the restoration of fertility. The genotyping also indicated that there was nearly double the recombination levels in homozygous Hvrecql4 lines compared to the wild type (WT). However, we did not observe any significant change in the distribution of CO events. Our results confirm the anti-CO role of RECQL4 in a large genome cereal and establish the possibility of testing the utility of increasing recombination in the context of traditional crop improvement.

Поиск восстановителей фертильности для системы ЦМС типа Polima рапса ярового (Brassica napus L.) и оценка их качества

One of the prior directions in modern rapeseed breeding is making heterotic hybrids. Seed production of most hybrids F1 used in production are often carried out by two types of cytoplasmic male sterility, there are Polima and Ogura. At the All-Russian Rapeseed Research Institute, fertility restorers on sterile cytoplasm were made and estimated for the main valuable characteristics and biochemical properties. The purpose of the study was to create reducing agents necessary for obtaining highly productive hybrids on the Polima type cytoplasmic male sterility (CMS). The object of research was 56 varieties of Brassica napus L., which were chosen in 2013. The setting of experiments, observations, records and analyzes were carried out using the field plot technique, the methodology of field experiments, the method of statistical data processing. It is noted that hybrids F1 had more productivity than the parental forms and the standard (Ratnik and Rif). Promising reducing agents of cytoplasmic male sterility of the Polima type were identified, which ensured complete restoration of fertility in sterile lines with CMS of the Polima type. The reducing agent LHR-1 is of practical interest for the creation of hybrids on the Polima CMS.

Inheritance of fertility restoration of A4 cytoplasm in pearl millet [Pennisetum glaucum (L.) R. Br.

Present investigation was carried out to study the mode of inheritance of fertility restoration for A4 cytoplasm using pollen fertility and seed set per cent as criterion in determining the fertile and sterile plants. Two CMS lines of A4 cytoplasm were crossed with two fertility restorers generating four F1 crosses, namely, ICMA 99111 x PPMI 1003, ICMA 99111 x PPMI 1087, ICMA 03999 x PPMI 1003 and ICMA 03999 x PPMI 1087, their F2s and backcross generations. All the F1s were completely fertile indicating complete fertility restoration. F2s and backcross generations were evaluated at IARI, New Delhi and IARI Regional Centre, Dharwad during summer 2017 and χ 2 test was applied to test the significance. At both the locations, all the F2 segregating populations fit well into a Mendelian ratio of 15:1 indicating digenic duplicate dominance of fertility restoring genes with χ 2 value of 0.82, 2.90, 0.04, 3.97, 4.86, 4.98, 0.02, 1.26, 3.15, 4.98, 3.15 and 0.02. The F2 hypothesis was verified with the observed frequency of segregating plants fitting well into 3:1 ration with χ 2 value of 5.45, 1.93, 4.93, 0.60, 2.83, 0.44, 4.94, 2.77, 3.33, 0.13, 4.08 and 1.51. It is further confirmation of the findings that fertility restoration is indeed governed by two duplicate genes. Association between pollen fertility and seed set per cent was significant and positive.

Genotype × Environment Interaction for Fertility Restoration and Yield Traits in Pigeonpea (Cajanus cajan) Hybrids

In cytoplasmic genetics male-sterility (CGMS)-based hybrid seed production technology, instability of fertility restoration is one of the prime causes behind instable yield performance of hybrids over the wide range of environments which makes better hybrids adaptive to only a specific environment. Therefore, the present study was conducted to investigate the stability of fertility restoration and yield traits of 10 promising CGMS-based pigeonpea hybrids during 2012–2013 at three different environments of India. The results revealed a significant variability among hybrids for fertility restoration and yield traits over the environments. All the hybrids except ICPH 3494 and ICPH 3491 exhibited high (> 80%) pollen fertility and fully male-fertile plants across the environments indicating complete fertility restoration in these hybrids. Hybrids ICPH 2671, ICPH 2740, ICPH 3933, and ICPH 3461 were stable for pollen fertility, fertility restoration, and for days to maturity except ICPH 3461. Hybrids ICPH 2671, ICPH 2751, and ICPH 3461 were stable for number of pods plant−1, whereas ICPH 2671, ICPH 2740, ICPH 3933, ICPH 3461, and ICPH 3762 were stable for grain yield plant−1 with high mean, unit regression coefficient, and non-significant deviation from the regression line. The varying fertility status of hybrids across the environments indicates that fertility restoration is sensitive to environmental conditions.

Conversion of partial restorer Swarna into restorer by transferring fertility restorer Rf gene(s) through marker assisted back cross breeding (MABB) in rice

The major constraints in hybrid rice breeding are availability of limited number of parental lines with specific desirable traits and lower frequency of restorers among elite breeding lines. The popular, high-yielding mega-rice variety Swarna, has been identified to be a partial restorer (as it has only one of major fertility restorer genes, Rf4) and hence cannot be utilized directly in the hybrid rice breeding. To convert the partial restorer to complete restorer, a cross was made between Swarna and a stable restorer KMR3R possessing Rf3 and Rf4 genes and developed BC1F5 and BC2F4 populations by marker-assisted back cross breeding (MABB). The SSR marker DRRM-RF3-10 linked to Rf3 gene located on chromosome 1, clearly distinguished restorers from partial restorers. All the improved lines of Swarna possessing Rf3 and Rf4 genes showed complete fertility restoration in test crosses with higher grain yield heterosis. Few rice hybrids developed by using converted restorers were evaluated in multi location testing under the All India Co-ordinated Rice Improvement Project (AICRIP). The results indicated that new rice hybrids expressed higher heterosis with matching grain quality attributes like Swarna. This study provides significantly novel and relevant restorers to enhance and economize future hybrid rice breeding programs.

Identification of new stable and high iron rich fertility restorers in pearl millet

Biofortification of pearl millet (Pennisetum glaucum (L.) R. Br.) with improved iron (Fe) and zinc (Zn) will have great impact as it is an indispensable component of nutritional security of inhabitants of arid and semi-arid regions. Ten genotypes along with checks were evaluated in RBD in six locations during kharif, 2016 under rainfed conditions. Significant differences were observed in genotype, environment and genotype × environment interaction mean squares for grain Fe and Zn contents, indicating differential nutrient accumulation by the genotypes. The first two principal components obtained in AMMI analysis were significant and cumulatively explained the total variation were 81.47 % for Fe and 73.97 % for Zn. A positive and moderately high correlation (r=0.6) between Fe and Zn contents suggests good prospects of simultaneous improvement for both micronutrients. Among the ten genotypes, PPMI 953 was found to be more stable with high mean Fe (90 ppm) and Zn (59 ppm) contents. On crossing with designated A lines of pearl millet, the line PPMI 953 found to be restorer for A1 system with complete fertility restoration of F1 panicle of the cross, ICMA(1) 863 x PPMI 953 under bagged condition and resulting F1 with 78-84% fertility measured by seed setting % under bag. The F2 individuals showed 9:7 fertility-sterility ratio (χ 2 value=0.002, P value=0.964). The promising line, PPMI 953 may be used as source for further genetic improvement with respect to grain micronutrient content or can be directly used as male parent in development of high iron pearl millet hybrids.

Marker-assisted pyramiding of bacterial blight and gall midge resistance genes into RPHR-1005, the restorer line of the popular rice hybrid DRRH-3

Bacterial blight (BB) of rice caused by the pathogen Xanthomonas oryzae pv. oryzae and the insect gall midge (GM) (Orseolia oryzae) are two major constraints of rice production. The present study was carried out to improve RPHR-1005, a stable restorer line of the fine-grain-type rice hybrid DRRH-3, for BB and GM resistance through marker-assisted backcross breeding (MABB). Two major GM resistance genes, Gm4 and Gm8, and a major BB resistance gene, Xa21, were selected as target genes for transfer to RPHR-1005. Two sets of backcrosses were carried out to combine either Xa21 + Gm4 or Xa21+ Gm8 into RPHR-1005 using breeding lines in the genetic background of ISM possessing either Gm4 or Gm8 along with Xa21. Foreground selection was performed for Xa21, Gm4, Gm8, and the major fertility restorer genes Rf3 and Rf4 using gene-specific markers, while 61 polymorphic simple sequence repeat (SSR) markers were used for background selection and marker-assisted backcrossing was continued until BC2 generation. A promising homozygous backcross-derived plant at the BC2F2 generation possessing Xa21 + Gm4, and another possessing Xa21 + Gm8, were intercrossed to stack the target resistance genes. At ICF 4 (inter-crossed F4) , three promising lines possessing the three target resistance genes in a homozygous condition along with fine-grain type, complete fertility restoration, and better panicle exsertion than RPHR-1005 have been identified. Among these, a single line, # RPIC-16-65-125, showed better yield, was highly resistant to BB and GM, was of medium–slender grain type, and had complete fertility restoration along with better panicle exsertion and taller plant type than RPHR-1005. This is the first report of combining resistance against BB and GM in the genetic background of a hybrid rice parental line.

Marker-assisted improvement of the elite restorer line of rice, RPHR-1005 for resistance against bacterial blight and blast diseases.

This study was carried out to improve the RPHR-1005, a stable restorer line of the popular medium slender grain type rice hybrid, DRRH-3 for bacterial blight (BB) and blast resistance through marker-assisted backcross breeding (MABB). Two major BB resistance genes, Xa21 and Xa33 and a major blast resistance gene, Pi2 were transferred to RPHR-1005 as two individual crosses. Foreground selection for Xa21, Xa33, Pi2, Rf3 and Rf4 was done by using gene-specific functional markers, while 59 simple sequence repeat (SSR) markers polymorphic between the donors and recipient parents were used to select the best plant possessing target resistance genes at each backcross generation. Backcrossing was continued till BC2F2 and a promising homozygous backcross derived line possessing Xa21+ Pi2 and another possessing Xa33 were intercrossed to stack the target resistance genes into the genetic background of RPHR-1005. At ICF4, 10 promising lines possessing three resistance genes in homozygous condition along with fine-grain type, complete fertility restoration, better panicle exertion and taller plant type (compared to RPHR-1005) were identified.

Development of Gene-Pyramid Lines of the Elite Restorer Line, RPHR-1005 Possessing Durable Bacterial Blight and Blast Resistance.

RPHR-1005, the stable restorer line of the popular medium slender (MS) grain type rice hybrid, DRRH-3 was improved in this study for resistance against bacterial blight (BB) and blast diseases through marker-assisted backcross breeding (MABB). In this study, four major resistance genes (i.e., Xa21 and Xa33 for BB resistance and Pi2 and Pi54 for blast resistance) have been transferred to RPHR-1005 using RPBio Patho-1 (possessing Xa21 + Pi2), RPBio Patho-2 (possessing Xa21 + Pi54) and FBR1-15EM (possessing Xa33) as the donors. Foreground selection was carried out using PCR-based molecular markers specific for the target resistance genes and the major fertility restorer genes, Rf3 and Rf4, while background selection was carried out using a set of parental polymorphic rice SSR markers and backcrossing was continued uptoBC2 generation. At BC2F2, plants possessing the gene combination- Xa21 + Pi2, Xa21 + Pi54 and Xa33 in homozygous condition and with >92% recovery of the recurrent parent genome (RPG) were identified and intercrossed to combine all the four resistance genes. Twenty-two homozygous, pyramid lines of RPHR-1005 comprising of three single-gene containing lines, six 2-gene containing lines, eight 3-gene containing lines, and five 4-gene containing lines were identified among the double intercross lines at F3 generation (DICF3). They were then evaluated for their resistance against BB and blast, fertility restoration ability and for key agro-morphological traits. While single gene containing lines were resistant to either BB or blast, the 2-gene, 3-gene, and 4-gene pyramid lines showed good level of resistance against both and/or either of the two diseases. Most of the 2-gene, 3-gene, and 4-gene containing pyramid lines showed yield levels and other key agro-morphological and grain quality traits comparable to the original recurrent parent and showed complete fertility restoration ability, with a few showing higher yield as compared to RPHR-1005. Further, the experimental hybrids derived by crossing the gene-pyramid lines of RPHR-1005 with APMS6A (the female parent of DRRH-3), showed heterosis levels equivalent to or higher than DRRH-3. The results of present study exemplify the utility of MABB for targeted improvement of multiple traits in hybrid rice.

A Gene Encoding Pentatricopeptide Repeat Protein Partially Restores Fertility in RT98-Type Cytoplasmic Male-Sterile Rice.

Cytoplasmic male sterility (CMS) lines in rice, which have the cytoplasm of a wild species and the nuclear genome of cultivated rice, are of value for the study of genetic interactions between the mitochondrial and nuclear genomes. The RT98-type CMS line RT98A and the fertility restorer line RT98C carry the cytoplasm of the wild species Oryza rufipogon and the nuclear genome of the Taichung 65 cultivar (Oryza sativa L.). Based on a classical crossing experiment, fertility is reported to be restored gametophytically by the presence of a tentative single gene, designated Rf98, which is derived from the cytoplasm donor. Fine mapping of Rf98 revealed that at least two genes, which are closely positioned, are required for complete fertility restoration in RT98A. Here, we identified seven pentatricopeptide repeat (PPR) genes that are located within a 170 kb region as candidates for Rf98 Complementation tests revealed that the introduction of one of these PPR genes, PPR762, resulted in the partial recovery of fertility with a seed setting rate up to 9.3%. We conclude that PPR762 is an essential fertility restorer gene for RT98-type CMS. The low rate of seed setting suggested that some other genes near the Rf98 locus are also necessary for the full recovery of seed setting.

Marker-assisted introgression of the major bacterial blight resistance gene, Xa21 and blast resistance gene, Pi54 into RPHR-1005, the restorer line of the popular rice hybrid, DRRH3

The elite Indian rice hybrid, DRRH3 is highly susceptible to two major diseases, bacterial blight (BB) and blast, which limit its productivity significantly. In the present study, we have introgressed two major genes, viz., Xa21 and Pi54 conferring resistance against BB and blast, respectively into RPHR-1005, the male parent of DRRH3 through marker-assisted backcross breeding (MABB) and analyzed the backcross derived plants for their resistance against BB and blast. RPBio Patho-2 was used as a donor for both the resistance genes. Gene-specific markers were used for the foreground selection of Xa21 and Pi54 at each stage of backcrossing and markers specific for the major fertility restorer genes, Rf3 and Rf4 were used only at BC1F1 generation for foreground selection. Background selection was done using 62 polymorphic SSR markers and marker-assisted backcrossing was continued till BC3 generation. At BC3F4, through intensive phenotype-based selections 15 promising lines (ABLs) possessing high level of resistance against BB and blast, high yield, fine-grain type, complete fertility restoration along with better panicle exsertion and taller plant type as compared to RPHR-1005 were identified and test crossed with APMS 6 A, the female parent of DRRH3. The newly derived hybrids (i.e. improved versions of DRRH3) were observed to possess high level of resistance against BB and blast along with medium-slender grain type and yield level better than or equivalent to that of DRRH3. Our study exemplifies the utility of MABB for targeted improvement of multiple traits in hybrid rice.

Prolonged Oral Administration of a Pan-Retinoic Acid Receptor Antagonist Inhibits Spermatogenesis in Mice With a Rapid Recovery and Changes in the Expression of Influx and Efflux Transporters.

We have previously shown that oral administration of a pan-retinoic acid receptor antagonist in mice daily at 2.5 mg/kg for 4 weeks reversibly inhibited spermatogenesis, with no detectable side effects. To elucidate the lowest dose and the longest dosing regimen that inhibits spermatogenesis but results in complete restoration of fertility upon cessation of administration of the drug, we examined the effects of daily doses as low as 1.0 mg/kg with dosing periods of 4, 8, and 16 weeks. We observed 100% sterility in all regimens, with restoration of fertility upon cessation of the drug treatment even for as long as 16 weeks. There was no change in testosterone levels in these males and the progeny examined from 2 of the recovered males were healthy and fertile, with normal testicular weight and testicular histology. Strikingly, a more rapid recovery, as assessed by mating studies, was observed at the lower dose and longer dosing periods. Insight into possible mechanisms underlying this rapid recovery was obtained at 2 levels. First, histological examination revealed that spermatogenesis was not as severely disrupted at the lower dose and with the longer treatment regimens. Second, gene expression analysis revealed that the more rapid recovery may involve the interplay of ATP-binding cassette efflux and solute carrier influx transporters in the testes.

Discovery of human posterior head 20 (hPH20) and homo sapiens sperm acrosome associated 1 (hSPACA1) immunocontraceptive epitopes and their effects on fertility in male and female mice.

The key goals of immunocontraception research are to obtain full contraceptive effects using vaccines administered to both males and females. Current research concerning human anti-sperm contraceptive vaccines is focused on delineating infertility-related epitopes to avoid autoimmune disease. We constructed phage-display peptide libraries to select epitope peptides derived from human posterior head 20 (hPH20) and homo sapiens sperm acrosome associated 1 (hSPACA1) using sera collected from infertile women harbouring anti-sperm antibodies. Following five rounds of selection, positive colonies were reconfirmed for reactivity with the immunoinfertile sera. We biopanned and analysed the chemical properties of four epitope peptides, named P82, Sa6, Sa37 and Sa76. Synthetic peptides were made and coupled to either bovine serum albumin (BSA) or ovalbumin. We used the BSA-conjugated peptides to immunise BALB/c mice and examined the effects on fertility in female and male mice. The synthetic peptides generated a sperm-specific antibody response in female and male mice that caused a contraceptive state. The immunocontraceptive effect was reversible and, with the disappearance of peptide-specific antibodies, there was complete restoration of fertility. Vaccinations using P82, Sa6 and Sa76 peptides resulted in no apparent side effects. Thus, it is efficient and practical to identify epitope peptide candidates by phage display. These peptides may find clinical application in the specific diagnosis and treatment of male and female infertility and contraceptive vaccine development.

Characterization of fertility restoration in alloplasmic lines derived from hybridization of self-fertilized offspring of barley-wheat (Hordeum vulgare L. × Triticum aestivum L.) amphiploid with common wheat varieties Saratovskaya 29 and Pyrotrix 28

The problems of fertility restoration in the progeny of barley-wheat hybrids (H. vulgare × T. aestivum) are explained by incompatibility between the cytoplasm of cultivated barley and the nuclear genome of common wheat. Appropriate models for studying these problems are alloplasmic lines that combine the cytoplasm of barley and the nuclear genome of wheat. In this work, the differences of fertility restoration in alloplasmic common wheat lines (H. vulgare)-T. aestivum were studied depending on the influence of wheat varieties Saratovskaya 29 (Sar29) and Pyrotrix 28 (Pyr28) used to produce these lines. The alloplasmic lines were created using hybrids between the 48-chromosome offspring (Amph1) of the barley-wheat amphiploid H. vulgare (ya-319) × T. aestivum (Sar29) and these wheat varieties. Backcrossing of the Amph1 (2n = 48) × Sar29 hybrid with the wheat variety Sar29 resulted in the complete sterility in the (H. vulgare)-Sar29 line, which suggests the incompatibility of the nuclear genome of the common wheat variety Sar29 with the cytoplasm of H. vulgare. Crossing of Amph1 (2n = 48) with Pyr28 resulted in the restoration of self-fertility in the hybrid with 2n = 44. In the alloplasmic lines (2n = 42) formed based on plants of the self-fertilized generations of this hybrid, the barley chromosomes were eliminated, and recombination between the nuclear genomes of the parental wheat varieties Sar29 and Pyr28 took place. Alloplasmic recombinant lines (H. vulgare)-T. aestivum with different levels of fertility were isolated. As was shown by the SSR analysis, differences in the fertility between these lines are determined by differences in the content of the genetic material from the wheat varieties Sar29 and Pyr28. The complete restoration of fertility in these alloplasmic recombinant lines is accompanied by the formation of a nuclear genome in which the genetic material of Pyr28 significantly prevails. The conclusion is made that the common wheat variety Pyrotrix 28 is a carrier of a gene (or genes), which determines the restoration of common wheat fertility on the cytoplasm of cultivated barley.

Heteroplasmic and homoplasmic states of mitochondrial and chloroplast DNA regions in progenies of distant common wheat hybrids of different origins

The states of 18S/5S mitochondrial (mt) repeat and some chloroplast (cp) DNA regions have been investigated in alloplasmic lines of common wheat carrying cytoplasm of the barley species Hordeum marinum subsp. gussoneanum Hudson and H. vulgare L. and in the progenies of the reciprocal hybrids between Triticum aestivum L. and Secale cereale L. The heteroplasmic state of the 18/5S mt repeat, which was a result of a biparental mtDNA transmission, has been observed in rye × wheat hybrids and in their progeny carrying rye cytoplasm. For the first time, the heteroplasmic state of cpDNA associated with the heteroplasmy of 18S/5S mt repeat has been detected in cereals with the use of alloplasmic wheat lines. The transition of mt- and cpDNA heteroplasmy and barley cpDNA homoplasmy to wheat homoplasmy is associated with complete restoration of fertility and elimination of the barley chromosome from the newly developing nuclear wheat genome of alloplasmic lines.

English

Polima cytoplasmic male sterility (pol CMS) has been widely used for exploiting heterosis in rapeseed breeding. The dominant restorer gene of pol CMS (Rfp ) is found in the nucleus and is a key component of hybrid production by achieving F1 progeny with complete fertility restoration. To identify the molecular markers associated with the Rfp gene, a near isogenic line (NIL) population of 2,000 individuals segregating for the Rfp locus was generated by crossing and backcrossing for 12 times. This NIL population was used to screen Rfp markers by AFLP technique. Based on the sequence information of AFLP markers that have been identified in previous research, we identified a homologous region of Rfp locus in chromosome 1 of Arabidopsis . Then, six sequenced Brassica rapa BAC clones corresponding to this target region were chosen to design microsatellite (SSR) primer pairs. Twenty-two SSR markers were designed and one of them, KBrDP1, was verified in the 2, 000 NILs population and proved to be strongly linked to Rfp locus. The genetic distance between KBrDP1 and Rfp was 0.2 cM. KBrDP1 marker was found located on linkage group N9 of a published DH mapping population. This SSR marker was useful in marker assisted selection breeding of the elite pol CMS restorer lines in rapeseed.

A novel Rf gene controlling fertility restoration of Ogura male sterility by RNA processing of orf138 found in Japanese wild radish and its STS markers

To reveal the molecular and genetic mechanism of fertility restoration in Ogura male sterility in Japanese wild radish (Raphanus sativus var. hortensis f. raphanistroides), we investigated fertility restoration of a plant that lacks the dominant type of orf687, a previously identified fertility restorer gene. A total of 100 F2 plants were made from the cross between a male-sterile strain with the Ogura cytoplasm, 'MS-Gensuke', and a Japanese wild radish plant. Segregation of pollen fertility in the F2 plants led us to assume that 2 dominant complementary genes controlled the fertility restoration of the plants. However, the fertility of 27 of 59 male-fertile plants was not completely restored, resulting in a group of plants with partial male fertility. Northern blot analysis of the CMS-associated gene orf138 indicated that one restorer allele (termed Rft) was involved in the processing of orf138 RNA. Rapid amplification of cDNA ends (RACE) and subsequent Northern blot analysis confirmed that the orf138 transcript lost a 5' part of the coding region of the orf138 gene in the restored plants. The accumulation of ORF138 protein was significantly reduced by Rft, but trace amounts of the protein were recognized in both partially male-fertile and male-sterile plants with Rft. The relationship of pollen fertility and segregation of co-dominant sequence tagged site (STS) markers in the F2 generation suggested that the penetrance of Rft was so low that Rft needs suitable conditions to function sufficiently for the complete restoration of fertility.

Identification of AFLP and SCAR markers linked to the male fertility restorer gene of pol CMS (Brassica napus L.)

The pol cytoplasmic male-sterility system has been widely used as a component for utilization of heterosis in Brassica napus and offers an attractive system for study on nuclear–mitochondrial interactions in plants. Genetic analyses have indicated that one dominant gene, Rfp, was required to achieve complete fertility restoration. As a first step toward cloning of this restorer gene, we attempted molecular mapping of the Rfp locus using the amplified fragment length polymorphism (AFLP) technique combined with bulked segregant analysis (BSA) method. A BC1 population segregating for Rfp gene was used for tagging. From the survey of 1,024 AFLP primer combinations, 13 linked AFLP markers were obtained and five of them were successfully converted into sequence characterized amplified region (SCAR) markers. A population of 193 plants was screened using these markers and the closest AFLP markers flanking Rfp were at the distances of 2.0 and 5.3 cM away, respectively. Further the AFLP or SCAR markers linked to the Rfp gene were integrated to one doubled-haploid (DH) population derived from the cross Quantum × No.2127-17 available in our laboratory, and Rfp gene was mapped on N18, which was the same as the previous report. These molecular markers will facilitate the marker-assisted selection (MAS) of pol CMS restorer lines.