Determination of the frequencies of restorer- and maintainer-alleles involved in CMS1 and CMS2 in German chive varieties

There are two cytoplasmic male sterility (CMS)‐systems in chives (Allium schoenoprasum L.), which can be employed in hybrid breeding. However, the probability for selection of maintainer genotypes from German open pollinated varieties is not known. Therefore, the allelic frequencies of the restorer genes X and T involved in CMS¹ were determined in 12 German commercial chive varieties by test crossing single plants to male sterile, temperature‐insensitive genotypes [(S₁)xxT] for segregation analyses of offspring. Temperature sensitive genotypes [(S₁)xxT_] are able to produce pollen at higher temperatures, and should therefore be excluded from hybrid breeding to avoid self‐pollination of the maternal parent. The mean value of the frequency of the non‐restoring allele x in the populations examined was 0.62. The mean value of the allele t, which is responsible for the temperature insensitivity, was 0.9. As a consequence of these allelic frequencies about one‐third of all plants of the chive varieties examined were designated CMS₁ maintainer genotypes, leading to the production of temperature insensitive male sterile lines. The incidence of CMS₂ maintainers in the German varieties examined was nearly four times lower than CMS₁ maintainers. The mean value of the frequency of the non‐restoring allele _st2 involved in the CMS₂‐system was 0.29.     

A simple method to control the seed purity of japonica hybrid rice varieties using PCR-based markers

Cytoplasmic male sterility (CMS) by the cms‐bo cytoplasm and its restoration by the nuclear restorer gene, Rf‐1, are used for seed production of japonica hybrid rice varieties. To produce pure hybrid seeds, a prerequisite is to properly manage the seed purity of parental lines, especially CMS lines. In this study, three dominant polymerase chain reaction (PCR)‐based markers (M1, M2 and M3) were developed to detect mutual contamination in seed batches of CMS lines, maintainer lines, restorer lines and hybrids. M1 detected the mitochondrial sequence that was present in the cytoplasm of common japonica varieties and absent in the cms‐bo cytoplasm. M2 and M3 detected the chromosomal sequence related to the Rf‐1 allele in restorer lines and the rf‐1 allele in common japonica varieties, respectively. By the strategic use of these markers, japonica hybrids and their parental lines could be efficiently distinguished from each other. Furthermore, sensitivity tests for the three markers with a series of crude DNA samples prepared from polished grains demonstrated that these markers could detect one contaminating grain among 500 or 1000 grains. Therefore, the bulk PCR analyses with the markers developed here probably make it possible to control the seed purity of japonica hybrids properly by selecting appropriate seed batches of their parental lines quickly and efficiently.     

Conversion of the genic male sterility (GMS) system of bell pepper (Capsicum annuum L.) to cytoplasmic male sterility (CMS)

Non‐pungent bell pepper (Capsicum annuum L.) lacks the cytoplasmic male sterility (CMS) nuclear restorer allele, Rf, and CMS cannot be employed in its F₁ hybrid seed production. To demonstrate that the genic male sterility (GMS) system in non‐pungent bell pepper can be converted to the CMS male sterility system, the conversion of GMS to CMS for non‐pungent bell pepper line GC3 was conducted by introgression of S‐type cytoplasm and the Rf allele from tropical pungent donors. After morphological traits were evaluated, two lines from BC₁F₁ containing S‐type cytoplasm and four lines from BC₂F₂ containing Rf allele, phenotypically similar to GC3, were obtained and could be employed as CMS male sterile lines and restorer lines for non‐pungent bell pepper. Four molecular markers potentially linked to traits of interest were also evaluated in BC₁F₁ and BC₁F₂ populations. This is the first time that GMS has been successfully converted to CMS in bell pepper, a significant contribution for bell pepper hybrid seed production.     

Development of a molecular CAPS marker for the self-incompatibility locus in Brassica napus and identification of different S alleles

Using primers annealing to S locus sequences the cleaved amplified polymorphic sequences (CAPS) method was applied to develop a marker and to characterize different alleles at the self‐incompatibility locus in Brassica napus. A segregating F₂ population from a cross of a self‐incompatible (SI) and a self‐compatible parent, as well as seven SI lines representing four different S alleles were used. Several primers specific to the S locus in B. oleracea and B. campestris, chosen from the literature, allow polymerase chain reaction (PCR) amplification of genomic DNA. However, only one primer pair amplified a single specific and reproducible PCR fragment of the expected length in B. napus. Digestion with restriction endonucleases revealed polymorphisms for two CAPS markers absolutely linked to the S locus. Using the codominant marker efMboI it was possible to discriminate all three F₂ genotypes. With this marker and an additional marker using another primer pair it was possible to distinguish between three of the four different S alleles and five of the seven SI lines, respectively.     

Random amplified polymorphic DNA variation within and among meadow bromegrass progeny types

Ten meadow bromegrass genotypes tested as half‐sib (polycross, PX; open‐pollinated, OP) and selfed (S₁) progenies were surveyed using random amplified polymorphic DNA. Fourteen primers, which produced 32 markers, were selected to determine the genotypes of 360 individuals from the three progeny tests. Analysis of molecular variance was performed in each progeny test, and genetic distances between genotypes and progeny types were determined. Among genotype genetic variation in half‐sib (PX and OP) progenies was similar. Genetic variation among genotypes for S₁ progenies (30.5%) was about twice that found in the half‐sib progenies. Variation between individual S₁ progenies ranged from 15.7% to 50.1%, while in the half‐sib progenies, the range was 6.9‐24.1%. Based on average distances between progeny types for a given genotype, OP and PX were closer to each other than to the S₁. An analysis of variance of the molecular marker frequency occurrence was performed for 12 plants within each progeny type of each genotype. Marker frequencies, expressed in percentages, ranged from 10.7% to 84.3%. No significant differences were found for genotype and progeny type‐genotype interaction suggesting that all genotypes behave in a similar manner across the different progeny types.     

Identification of S-alleles using polymerase chain reaction-cleaved amplified polymorphic sequence of the S-locus receptor kinase in inbreeding lines of Brassica oleracea

Identification and DNA polymorphism of the S‐locus receptor kinasegene (SRK) was analysed by pollen tube tests, polymerase chain reaction‐cleaved amplified polymorphic sequence (PCR‐CAPS) and nucleotide sequencing. SRK‐specific primers that can distinguish class and class II S haplotypes amplified single DNA fragments of 900‐1050 bp. The DNA fragments of 22 inbred lines amplified with a class SRK‐specific primer pair determined seven types with HinfI and EcoRII. In addition, the DNA fragments of 17 inbred lines amplified with a class II SRK‐specific primer pair determined three types with Hinf1. Nucleotide sequencing of the DNA fragments amplified from 10S haplotypes showed that exons of the 3′‐end in SRK are highly conserved, and that there is much variation of the introns, which produced polymorphism of the band pattern in PCR‐CAPS profiles. The S haplotypes of the plants were determined by restriction analysis of PCR products and agreed with results based on pollen tube growth tests. The PCR‐CAPS analysis using specific primer pairs of SRK is considered to be useful for S allele identification in breeding programmes.     

Molecular characterization of cytoplasmic male sterility conditioned by <Emphasis Type="Italic">Gossypium harknessii</Emphasis> cytoplasm (CMS-D2) in upland cotton

Cytoplasmic male sterility (CMS) is a maternally inherited trait that fails to produce functional pollen grains. The CMS system is widely employed to facilitate the utilization of heterosis in major crops. However, little is known about the CMS associated genes in Upland cotton (Gossypium hirsutum). The objective of this study was to compare CMS cotton (CMS-D2) with the cytoplasm from G. harknessii and its isogenic maintainer line with the normal fertile Upland cotton cytoplasm to identify CMS-D2 specific gene(s) and to develop CMS-specific sequence characterized amplified region (SCAR) markers. Based on Southern blot analysis using 10 mitochondrial gene-specific probes (cob, cox2, atp6, atp9, nad3, cox3, atpA, cox1, nad6 and nad9), three probes (cox3, atpA, and nad6) revealed restriction fragment length polymorphisms (RFLP) between the CMS-D2 and its isogenic maintainer line. RT-PCR confirmed that the three genes were differentially expressed between the two lines. These results indicated that there existed structural and expression variations in the three genes when the mitochondrial D2 genome was transferred into Upland cotton. Genome walking and rapid amplification of cDNA ends (RACE) were further performed to analyze the sequences of these genes and their flanking regions. For cox3 and nad6, there was only one different nucleotide each in the gene regions between the two lines. Also some nucleotides upstream of the ATG codon were different. For atpA, the sequences downstream the atpA were significantly different between the two cytoplasmic lines. Furthermore, two nucleotides at the -4 and -5 position from ATG codon were also changed between the two cytoplasms (i.e., CG→AA), and this mutation also exists in RNA sequences. Interestingly, nine nucleotides (ATGCAACTA) were also inserted at the location of 899 bp upstream of ATG codon in the CMS line. The results suggest that the abnormal sequence and expression of atpA gene is associated with CMS expression in Upland cotton. According to the significant different sequences downstream the atpA gene, a CMS-D2 specific SCAR marker was developed. The CMS-specific PCR bands were verified for 10 cultivars containing either normal- or CMS-D2cytoplasm. This will allow quick and reliable identification of the cytoplasmic types of individual plants at the seedling stage, and assessment of the purity of F₁ seed lots.     

SSR and SCAR markers linked to the fertility-restoring gene for a D2-type cytoplasmic male-sterile line in wheat

‘Yi 4060’ is an elite restorer line of a non‐photoperiod‐sensitive D²‐type cytoplasmic male‐sterile (CMS) line of wheat. Random amplified polymorphic DNA (RAPD) and simple sequence repeat (SSR) markers were employed to map one major fertility‐restoring gene (D²Rf₁) in ‘Yi 4060′. The sterile and fertile DNA pools were established from individuals in BC₆, based on bulked segregant analysis. One RAPD marker E09, linked to D²Rf₁, was converted to a SCAR marker and designated as E09‐SCAR₈₆₅. The genetic distance between E09‐SCAR₈₆₅ and D²Rf₁ is 9.5 cM. Two SSR markers, Xgwm11 and Xgwm18, were also linked to a D²Rf₁ and co‐segregated with E09‐SCAR₈₆₅. The three molecular markers are useful in marker‐assisted breeding of the elite restorer lines for D² ‐type CMS lines in wheat.     

Identification of Rfd1, a novel restorer-of-fertility locus for cytoplasmic male-sterility caused by DCGMS cytoplasm and development of simple PCR markers linked to the Rfd1 locus in radish (Raphanus sativus L.)

Previously, novel cytoplasmic male-sterility (CMS) caused by DCGMS cytoplasm was discovered in radish (Raphanus sativus L.) introduced from Uzbekistan. We performed extensive progeny tests and identified two fertility restorer lines (‘R171’ and ‘R121’) for this new CMS. Two F₁ hybrid populations were self-pollinated and backcrossed to produce F₂ and BC populations. Inheritance patterns of male-sterility in segregating populations varied depending on paternal lines. Segregation of male-sterility in F₂ populations originating from the cross between MS19 and R121 showed that a single locus was involved in fertility restoration. However, populations originating from the cross between MS15 and R171 showed the involvement of more than one restorer-of-fertility genes. The single fertility restorer locus identified in the cross between MS19 and R121 was designated Rfd1 locus. Bulked segregant analysis was performed using RAPD and AFLP, which identified one marker each. Both RAPD and AFLP markers were converted into simple PCR-based co-dominant markers after their isolated flanking sequences were analyzed. Indels 773-bp and 67-bp in length were identified between two Rfd1 allele-linked flanking sequences of the RAPD and AFLP fragments, respectively, then utilized to develop simple PCR markers. In addition, we prove that the newly identified Rfd1 locus is independent of the Rfo locus, another radish fertility restorer for CMS caused by Ogura cytoplasm.     

Identification of S-alleles in almond using multiplex PCR

The S-genotypes of eight almond (Prunus dulcis Miller (D.A. Webb)) cultivars from different geographical origins and of nine new selections from the CEBAS-CSIC (Murcia, Spain) breeding program were determined using single and multiplex PCR with different sets of specific oligonucleotide primers. The results of PCR using the AS1II- and AmyC5R-specific primers showed amplification in a single reaction of 10 different self-incompatibility alleles and of the self-compatibility allele S _f. However, the amplified fragments of the S _f allele were of similar sizes to those amplified from the S ₃ self-incompatibility allele. For this reason, a specific PCR primer CEBASf was designed from the intron sequence of S _f. A multiplex-PCR reaction using the AS1II, CEBASf and AmyC5R primers permitted unequivocal identification of the 10 self-incompatibility alleles and of the self-compatibility allele. Multiplex PCR opens the possibility to identify new S-alleles using different sets of primers. The applications of these PCR markers in the almond-breeding programs are discussed.     

Limitations of PCR‐based molecular markers to identify male‐sterile and maintainer plants from Indian onion (Allium cepa L.) populations

The main aim of this study was to validate PCR markers for determining cytoplasm and genotypes at the Ms locus in short‐day onion. Three cytoplasmic (OSN, MKFR and accD) and four nuclear (OPT, jnurf13, AcSKP1 and AcPMS1) markers were employed. Sel. 121‐1 had 100% S cytoplasm, whereas Sel. 121‐2, ‘Pusa Red’ and ‘Pusa Madhavi’ had 88%, 33% and 17% S cytoplasm, respectively. ‘Early Grano’ and ‘Pusa Riddhi’ did not possess S cytoplasm. Observations in 33 commercial varieties revealed two with sterile (S) cytoplasm. Nuclear markers were not found in linkage disequilibrium with the Ms locus, and the constitution of Ms alleles by OPT was different from other three markers, which were in conformity with each other. The other three markers predicted that most of the plants should be homozygous recessive. Anther colour also did not confirm the sterility status. It can be concluded that accD may be used for cytoplasm determination based on the ease of its use. For the Ms locus tagging, more markers are needed to be evaluated to isolate maintainer lines from open‐pollinated populations.     

Haplotype analysis of CMS-associated DNA markers in sweet peppers

Cytoplasmic male sterility (CMS)/restorer-of-fertility (Rf) is an economical and efficient system to produce F₁ hybrid seeds. Although the CMS/Rf system has been used to produce hybrid seeds of hot peppers, this system has never been used for sweet pepper seed production, presumably due to the inability to select stable restorer lines during the breeding process. To test the feasibility of the CMS/Rf system in sweet pepper breeding, we investigated the distribution of haplotypes of previously developed, CMS-associated markers (orf456, ψ atp6-2, CRF-SCAR, OPP13-CAPS, PR-CAPS, and PR-SNP) in 27 commercial sweet pepper F₁ hybrids and 12 breeding lines. When CMS-associated cytoplasmic markers orf456 and ψ atp6-2 were applied, male sterile cytoplasm was not detected in commercial sweet pepper cultivars. When nuclear haplotype markers linked to Rf were applied, all sweet pepper cultivars showed haplotype 3, haplotype 1, and the rf genotype for OPP13-CAPS, PR-CAPS, and CRF-SCAR, respectively. In contrast, we were able to detect male sterile cytoplasm in some breeding lines, and we were also able to detect polymorphisms for PR-CAPS between stable and unstable maintainer lines. The 17T7-SNP also showed polymorphisms between unstable and stable maintainer (or restorer) lines. In conclusion, we expect that it will be possible to develop stable A, B, and C sweet pepper lines using CMS-associated markers and that this will eventually lead to successful implementation of the CMS/Rf system to produce F₁ hybrid sweet pepper seeds.     

Development of SCAR and CAPS markers linked to a recessive male sterility gene in lettuce (<Emphasis Type="Italic">Lactuca sativa</Emphasis> L.)

Genetic male sterility (GMS) has been a useful system for the production of hybrid varieties in self-pollinated plants. We obtained a GMS line developed from a spontaneous mutation in lettuce (Lactuca sativa L.). Genetic analysis in our previous study revealed that the sterility was controlled by a recessive gene which was named ms-S. For simple and quick screening of individuals showing male sterility, we attempted molecular mapping of the ms-S locus using an amplified fragment length polymorphism (AFLP) technique. From the examination of 4,096 AFLP primer combinations, 63 AFLP markers were found to be linked to the gene and nine of them were successfully converted into sequence characterized amplified region (SCAR) markers and cleaved amplified polymorphic sequence (CAPS) markers. Linkage analysis indicated that these nine markers were closely linked to the ms-S gene and all were located on the same side of the gene. The minimum genetic distance between the ms-S gene and a marker was 3.1 cM. These results provide additional information for map-based cloning of the ms-S gene and will be of great help for lettuce breeding using GMS to produce F₁ hybrids.     

Identification of a major QTL for adult plant resistance to coffee leaf rust (Hemileia vastatrix) in the natural Timor hybrid (Coffea arabica x C. canephora)

Most of the commercial varieties of coffee (Coffea arabica L.) derived from the Timor hybrid (TH) have been shown to contain major genes for coffee leaf rust (CLR) resistance. To identify markers tightly linked to such genes, an F₂ mapping population derived from a cross between ‘Caturra’ (susceptible variety) and the TH‐derived DI.200 line (highly resistant) was generated. Using expressed sequence information and a bioinformatics approach, both targeted region amplified polymorphism (TRAPs) markers and simple sequence repeat (SSR) markers were identified. Phenotypic evaluations in the field and under controlled conditions confirmed the existence of one quantitative trait locus for CLR resistance. Four candidate SSR markers were associated with high CLR resistance. They spanning a region of 2.5 cM designated Q_{CLR_4} located within chromosome 4 of the international C. canephora map. The presence of this region was confirmed in a set of elite lines and commercial varieties. The Q_{CLR_4} region corresponds to a new and genetically independent S_H locus that could potentially be useful in gene pyramiding with other genes to enhance rust resistance in TH derivatives.     

A unique introgression from Moricandia arvensis confers male fertility upon two different cytoplasmic male-sterile lines of Brassica juncea

A Brassica juncea line carrying an introgression from Moricandia arvensis restored male fertility to two cytoplasmic male‐sterile (CMS) B. juncea lines carrying either M. arvensis or Diplotaxis catholica cytoplasm. Genetics of fertility restoration was studied in the F₁, F₂, F₃ and backcross generations of the cross between CMS and fertility‐restorer lines. No male‐sterile plants were found in F1‐F₃ generations of the cross between CMS [M. arvensis] B. juncea and the restorer. However, a 1: 1 segregation for male sterility and fertility was observed when the F1 was pollinated with non‐restorer pollen from a euplasmic line. These results clearly show that restoration is mono‐genic and gametophytic. In CMS lines carrying D. catholica cytoplasm, the restorer conferred male fertility to the F1 and showed 3: 1 and 1: 1 segregations for male fertility and sterility in F₂ and BC1 generations, respectively, indicating a monogenic, sporophytic mode of fertility restoration. The results were also supported by pollen stainability in the F1 which was about 65% in M. arvensis‐based CMS and >90% in D. catholica‐based CMS. The above results are discussed in the light of previous molecular studies which showed association between CMS and atpA in both systems.     

Development of a cytoplasmic male‐sterile line NJCMS4A for hybrid soybean production

Cytoplasmic male‐sterile (CMS) lines are being used to produce hybrid seeds. Thus far, four CMS sources in soybean [Glycine max (L.) Merr.] have been reported in China. However, they are not sufficient or efficient in meeting the requirements of commercial soybean hybrid seed production. In this study, 33 varieties were tested for CMS using 45 crosses among 37 landraces and 17 annual wild soybean accessions (Glycine soja Sieb. et Zucc.). The cross of N23661 × N23658 showed partial to complete male sterility in backcross generations, while the corresponding reciprocal cross showed normal male fertility. Thus, the cytoplasm of N23661 is male‐sterile, the continuously backcrossed line is a male‐sterile line (designated NJCMS4A), and N23658 is its maintainer (designated NJCM4B). The male fertility of NJCMS4A was restored by another accession, Nansheng9403. Accordingly, NJCMS4A along with its maintainer and restorer composes a complete set of three lines for producing hybrid soybean. Using mitochondrial markers and sequence analyses, NJCMS4A is a CMS line with its cytoplasm not identical to the four previously reported CMS sources in soybean.     

Improved discrimination of self-incompatibility S-RNase alleles in cherry and high throughput genotyping by automated sizing of first intron polymerase chain reaction products

A novel polymerase chain reaction (PCR) approach to determine and confirm the self‐incompatibility (S) genotype of cherries is reported. The method involves PCR amplification with a new pair of consensus primers that immediately flank the first intron of cherry S‐RNases, one of which is fluorescently labelled. Fluorescent amplification products range from 234 to c. 460 bp and can be sized accurately on an automated sequencer. Thirteen S alleles reported in sweet cherry can be distinguished, except for S₂ and S₇, which have an amplification product of exactly the same size. S₁₃, which is also amplified, gives a microsatellite‐like trace which shows minor intra‐allelic length variation. This method gives fast and accurate results and should be especially useful for medium/high‐throughput genotyping of wild and cultivated cherries.     

Development of chloroplast‐specific microsatellite markers for molecular characterization of alloplasmic lines and phylogenetic analysis in wheat

Wheat (Triticum aestivum L.) is strictly a self‐pollinated crop, where hybrid breeding requires well‐characterized cytoplasmic male sterile (CMS) lines. The CMS has mostly been developed by substituting nuclear genome of wheat into the cytoplasm from wild relatives. Molecular characterization of 90 genotypes including 82 CMS lines originating from five different species, namely Aegilops speltoides, Ae. kotschyi, Ae. variabilis, Triticum araraticum and T. timopheevii, and eight popular varieties was carried out. Consequently, a set of 25 microsatellite markers specific to chloroplast (cpSSRs) were designed and successfully validated for specificity of amplification. A total of 15 cpSSRs (60%) were found polymorphic, of which three cpSSRs (TaCM7, TaCM8 and TaCM11) in genic region and twelve cpSSRs were located in intergenic region. Phylogenetic analysis of genotypes using cpSSRs revealed two major groups well in accordance with respective origin. A set of cpSSRs and phylogeny of CMS belonging to different origins developed, which will be helpful for the improvement in CMS system in wheat. The genic cpSSRs can be used for the allele mining and evolutionary studies.     

Random amplified polymorphic DNA variation among German and Dutch asparagus cultivars

DNA polymorphism among nine cultivars of Asparagus officinalis L. was measured using random amplified polymorphic DNA (RAPD). Of 69 reproducible amplification products from 12 arbitrary decamer primers, 49 RAPD markers were polymorphic and could be used to distinguish six German and three Dutch asparagus cultivars. Even with very small sample sizes, genetic similarity measurements based on the RAPD data allowed accurate grouping of the nine cultivars into distinct clusters, with the exception of two individuals which clustered to closely related varieties. Two German cultivars showed high genetic similarity and were distinct from the remaining German varieties. The German and Dutch cultivars were clearly separated by a relatively large genetic distance.     

Development of SCAR markers for identification of stem rust resistance gene Sr31 in the homozygous or heterozygous condition in bread wheat

The stem rust resistance gene Sr31, transferred from rye (Secale cereale) into wheat (Triticum aestivum L.) imparts resistance to all the virulent pathotypes of stem rust (Puccinia graminis f. sp. tritici) found in India. Wheat genotypes including carriers and non‐carriers of the Sr31 gene were analysed using arbitrary primed polymerase chain reaction (AP‐PCR). AP‐PCR markers viz. SS30.2_580(H) associated with the Sr31 gene and SS26.1₁₁₀₀ associated with the allele for susceptibility were identified. Linkage between the markers and phenotypes was confirmed by analysing an F₂ population obtained from a cross between a resistant and a susceptible genotype. The markers were tightly linked to the respective alleles. Both the AP‐PCR markers were converted into sequence characterized amplified region (SCAR) markers, viz. SCSS30.2₅₇₆ and SCSS26.1₁₁₀₀ respectively. The markers were validated in two more segregating populations and 49 wheat genotypes. Using both markers it was possible to distinguish the homozygous from the heterozygous carriers of the Sr31 gene in the F₂ generation. The markers developed in this study can be used for pyramiding of the Sr31 gene with other rust resistance genes and in marker‐assisted selection.