S-genotyping of old apple cultivars from the Carpathian basin: methodological, breeding and evolutionary aspects

Apple exhibits self-incompatibility controlled by the multiallelic S-locus. Twenty-three old apple cultivars were S-genotyped using three different approaches (allele-specific polymerase chain reaction (PCR) + cleaved amplified polymorphic sequences (CAPS), consensus PCR + sequencing and consensus PCR + CAPS) to compare the robustness and reliability of these techniques and characterise genotypes from the Carpathian basin that might be useful in resistance breeding. Best results were obtained using the ASPF3 and ASPR3S consensus primer pair that detected 96% of all alleles carried by the 23 cultivars tested. Flow cytometry analysis was also needed to control the completeness of the genotypes as was seen in case of a tetraploid cultivar with only three assigned S-alleles. The genetic disparity between the old Carpathian basin and modern apple cultivars was indicated by differences in allele frequency data (S ₉, S ₂₄ and S ₂₆) as well as single nucleotide polymorphisms in S ₁, S ₂, S ₇ S ₂₄ and S ₂₆ and indels in S ₂₀ and S ₂₆ alleles. An alignment of partial genomic sequences indicated trans-specific and trans-generic evolution of S-ribonuclease alleles in the Maloideae subfamily (S ₂₆ and S ₂₈) and a possibly recent introgression event (S ₁) between Malus × domestica and Malus sylvestris. These data suggest that the genome of old cultivars from the Carpathian basin was enriched by several Malus taxa and are free from the consequences of modern breeding. These cultivars may contribute to the widening of the genetic basis of cultivated apple and prevent genetic erosion in future commercial cultivars.     

Molecular cloning of the self-incompatibility genes S1 and S3 from almond (Prunus dulcis cv. Ferragnès)

Almond (Prunus dulcis) displays gametophytic self-incompatibility. In the work reported here, we cloned two novel S-RNase genes from almond cultivar Ferragnès (genotype S1S3) using PCR. The S1-RNase gene has the same coding region as the Sb gene cloned from almond cultivated in the USA; however, their introns are different in sequence. S1 was cloned and sequenced from six different cultivars originating in Europe. The full-length of the S3-RNase gene was cloned using two primers corresponding to the start and stop codons contexts. Two introns are present in the S3 gene, unique among the S-RNase genes. Sequence-specific PCR was performed to confirm that the two cloned genes co-segregate with the S-locus using progenies of a controlled cross between Tuono (S1Sf) and Ferragnès (S1S3). Based on the structural differences of S- and S-like RNase genes, we discuss the evolutionary relationship between the two groups of RNase genes. Received: 18 February 2001 / Accepted: 26 June 2001     

Determination of self-locompatibility genotypes of Korean apple cultivars based on S-RNase PCR

To prevent self-fertilization, apple has a gametophytic self-incompatibility mechanism, part of a widespread intraspecific system, that is controlled by a multi-allelic locus. This attribute has been exploited in breeding programs for new cultivars. Likewise, many apple orchards depend on artificial pollination. Therefore, molecular analysis and early identification of the self-incompatibility (S) genotype could greatly improve breeding schemes and pollen donors selection. Here, we PCR-amplified the S-RNase PCR fragments from a total of 14 cultivars and parents, using new primers (ASPF3+ASPR3) common to 23 S-alleles in apple. The S-genotypes were determined for the following: ‘Hongro’ (S₁S₃), ‘Gamhong’ (S₁S₉), ‘Saenara’ (S₁S₃), ‘Chukwang’ (S₃S₉), ‘Hwahong’ (S₃S₉), ‘Seokwang’ (S₃S₃), ‘Hwarang’ (S₁S₉), ‘Sunhong’ (S₃S₉), ‘S.E.B.’ (S₁S₁₉), ‘S.G.D.’ (S₂S₃), and ‘Mollie’s Delicious’ (S₃S₇). We also confirmed the characteristics of the S-genotypes for eight Korean apple cultivars by PCR-Southern blot analysis, using seven S-RNases as probes.     

Molecular characterization of S-RNase genes and S-genotypes in the Japanese apricot (Prunus mume Sieb. et Zucc.)

Three partial S-RNase genes, MSRN-1, MSRN-2, and MSRN-3, in the Japanese apricot (Prunus mume Sieb. et Zucc.) were isolated from the three cultivars Nankou, Gyokuei, and Kairyouuchidaume, respectively. The structural characteristics revealed that S-RNase genes from the Japanese apricot were in the T2/SRNase-type S-RNase family with five conserved regions (C1, C2, C3, RC4, and C5) and one variable region (RHV) as reported in the other rosaceous plants. In the phylogenetic tree of T2/S SRNase-type RNases, three S-RNase genes of the Japanese apricot did not form a species-specific subgroup but the Prunus subfamily did. At least seven S-allelic genes were present in the Japanese apricot, and S-genotypes of six representative cultivars, including Nankou, Gyokuei, Kairyouuchidaume, Baigou, Kagajizou, and Oushuku were first established in this study as S ₁ S ₇, S ₂ S ₆, S ₃ S ₄, S ₃ S ₆, S ₃ S ₆ and S ₁ S ₅, respectively. An extended elucidation of the S-genotype would contribute to a more efficient breeding program of the Japanese apricot. Received: 5 September 2000 / Revision accepted: 22 December 2000     

Molecular characterization of newS-RNases (‘S31’ and ‘S32’) in apple (Malus ×domestica Borkh.)’) in apple (Malus ×domestica Borkh.)

Apple exhibits gametophytic self-incompatibility (GSI) that is controlled by the multiallelic S-locus. This S-locus encodes polymorphicS ribonuclease (S-RNase) for the pistil-part 5 determinant. Information aboutS-genotypes is important when selecting pollen donors for fruit production and breeding of new cultivars. We determined the 5-genotypes of ‘Charden’ (S₂S₃S₄), ‘Winesap’ (S₁S₂₈), ‘York Imperial’ (S₂S₃₁), ‘Stark Earliblaze’ (S₁S₂₈), and ‘Burgundy’ (S₂₀S₃₂), byS-RNase sequencing and S-allele-specific PCR analysis. Two newS-RNases, S₃₁ and S₃₂, were also identified from ‘York Imperial’ and ‘Burgundy’, respectively. These newS-alleles contained the conserved eight cysteine residues and two histidine residues essential for RNase activity. Whereas S₃₁ showed high similarity to S₂₀ (94%), S₃₂ exhibited 58% (to S₂₄) to 76% (to S₂₅) similarity in the exon regions. We designed newS-allele-specific primers for amplifying S₃₁- and S₃₂-RNasc-specific fragments; these can serve as specific gene markers. We also rearranged the apple S-allele numbers containing those newS-RNases. They should be useful, along with anS-RNase-based PCR system, in determining S-genotypes and analyzing new alleles from apple cultivars.     

Self-incompatibility genotypes in almond re-evaluated by PCR,stylar ribonucleases,sequencing analysis and controlled pollinations

As part of the almond breeding programme at IRTA, we investigated the S genotypes of several cultivars using a combination of RNase zymograms, testcrosses, pollen-tube growth analysis and molecular identification by PCR analysis. For some of the cultivars examined, discrepancies appeared between their S alleles as reported in the literature and those found in this investigation, leading to a re-evaluation of their S genotypes. Analysis of the stylar ribonucleases (RNases), which are known to correlate with S alleles, of cvs. Achaak, Ardechoise, Desmayo Largueta, Ferrastar, Gabaix, Garbí, Glorieta, Languedoc, Primorskiy and Texas revealed inconsistencies with respect to the S₅ and S₁₀ alleles. However, PCR with the conserved primer pair AS1II/AmyC5R failed to detect any of these inconsistencies. When the S alleles from Desmayo Largueta, Gabaix, Primorskiy and Texas were sequenced, Texas and Primorskiy were found to carry the reported S₅ allele, while Desmayo Largueta and Gabaix carried a new allele, which has been tentatively denoted as S₂₅ This new S allele, previously reported to be S₁₀, was also identified in Achaak, Ardechoise and Ferrastar. The proposed new S genotypes are Achaak (S₂S₂₅), Ardechoise (S₁S₂₅), Desmayo Largueta (S₁S₂₅), Ferrastar (S₂S₂₅) and Gabaix (S₁₀S₂₅). The S alleles of Garbí, Glorieta, Languedoc, Texas and Primorskiy remain as reported in the literature. Testcrosses in the field and laboratory confirmed the new S genotypes. One cultivar (Gabaix) could be assigned to the existing cross-incompatibility group O of unique genotypes, and two new groups were established (XVI and XVII) consisting of two cultivars each. The clarification of these S alleles will be useful in almond breeding programmes and for planning new commercial orchards in the future.     

Differential plant growth and osmotic effects of two maize (<Emphasis Type="Italic">Zea mays</Emphasis> L<Emphasis Type="Italic">.</Emphasis>) cultivars to exogenous glycinebetaine application under drought stress

We investigated the effects of exogenous glycinebetaine (GB) and drought stress (DS) on grain yield (GY) and production of dry matter (DM) and osmolytes in two maize (Zea mays L.) cultivars i.e. Shaandan 9 (S₉) and Shaandan 911 (S₉₁₁) during the entire growing period. Drought stress substantially reduced DM and GY but increased free proline, endogenous GB, soluble sugar and K⁺ concentrations in leaves of both cultivars. The DM production, GY, drought index (DI) and concentrations of these osmolytes were greater for S₉ than those for S₉₁₁ under DS. The significant differences in these parameters suggested that S₉ was more drought-tolerant as compared to S₉₁₁. Additionally, foliar application of GB increased the concentrations of all osmolytes measured, DM and GY of both cultivars under DS. These positive responses of exogenous GB spray were more pronounced in S₉₁₁ as compared to those in S₉. Further correlation analysis involving a number of parameters indicated that maize production was tighterly correlated with accumulation of the osmolytes measured during DS rather than well-watered controls. Accordingly, this study demonstrated the notion of an anti-drought role of exogenous GB by osmoregulation under DS, particularly in this drought sensitive cultivar. Thus, exogenous GB application might be firstly used with drought sensitive species/cultivars when exposed to DS.     

Photosynthesis and the Enzymes of Photosynthetic Carbon Reduction Cycle in Mulberry During Water Stress and Recovery

Net photosynthetic rate (P _N), stomatal conductance (g _S), transpiration rate (E), intercellular CO₂ concentration (C _i), leaf water potential (_w), leaf area, chlorophyll (Chl) content, and the activities of photosynthetic carbon reduction cycle (PCR) enzymes in two mulberry (Morus alba L.) cultivars (drought tolerant Anantha and drought sensitive M-5) were studied during water stress and recovery. During water stress, P _N, g _S, and E declined whereas C _i increased. P _N, g _S, and E were less affected in Anantha than in M-5, which indicates tolerance nature of Anantha over M-5. Activities of ribulose-5-phosphate kinase, NAD- and NADP-glyceraldehyde-3-phosphate dehydrogenases, and 3-phosphoglycerate kinase decreased with increasing stress in both the cultivars. The enzyme activities less affected in tolerant (Anantha) than in sensitive cultivar (M-5) were restored after re-watering to almost initial values in both the cultivars. Re-watering of the plants led to an almost complete recovery of P _N, E, and g _S, indicating that a short-term stress brings about reversible effect in these two cultivars of mulberry.     

Characterization of three new S-alleles and development of an S-allele-specific PCR system for rapidly identifying the S-genotype in apple cultivars

Apple (Malus domestica Borkh), a member of the Rosaceae, shows gametophytic self-incompatibility (GSI) controlled by polymorphic S-alleles. Identifying the S-genotypes of apple cultivars can be applied on correct assignment of apple cultivars to cross-compatibility groups, which is important for the efficient production of apple fruit. This study characterized three new S-alleles (designated S ₄₄ , S ₄₅ , and S ₄₆ ) in apple and developed an efficient analysis method that can be used to characterize S-genotypes by utilizing allele-specific polymerase chain reaction rapidly. Nineteen allele-specific primers were selectively designed to identify different alleles. Using this method, S-genotypes of 157 apple cultivars were identified.     

Cloning and characterization of genomic DNA sequences of four self-incompatibility alleles in sweet cherry (<Emphasis Type="Italic">Prunus avium</Emphasis> L.)

Gametophytic self-incompatibility (GSI) in sweet cherry is determined by a locus S with multiple alleles. In the style, the S-locus codifies for an allele-specific ribonuclease (S-RNase) that is involved in the rejection of pollen that carries the same S allele. In this work we report the cloning and genomic DNA sequence analysis including the 5 flanking regions of four S-RNases of sweet cherry (Prunus avium L., Rosaceae). DNA from the cultivars Ferrovia, Pico Colorado, Taleguera Brillante and Vittoria was amplified through PCR using primers designed in the conserved sequences of sweet cherry S-RNases. Two alleles were amplified for each cultivar and three of them correspond to three new S-alleles named S ₂₃ , S ₂₄ and S ₂₅ present in 'Pico Colorado', 'Vittoria' and 'Taleguera Brillante' respectively. To confirm the identity of the amplified fragments, the genomic DNA of these three putative S-RNases and the allele S ₁₂ amplified in the cultivar Ferrovia were cloned and sequenced. The nucleotide and deduced amino-acid sequences obtained contained the structural features of rosaceous S-RNases. The isolation of the 5-flanking sequences of these four S-RNases revealed a conserved putative TATA box and high similarity among them downstream from that sequence. However, similarity was low compared with the 5-flanking regions of S-RNases from the Maloideae. S ₆ - and S ₂₄ -RNase sequences are highly similar, and most amino-acid substitutions among these two RNases occur outside the rosaceous hypervariable region (RHV), but within another highly variable region. The confirmation of the different specificity of these two S-RNases would help elucidate which regions of the S-RNase sequences play a role in S-pollen specific recognition.Communicated by H.F. Linskens     

Genomic characterization of self-incompatibility ribonucleases (S-RNases) in European pear cultivars and development of PCR detection for 20 alleles

Sexual self-incompatibility in European pear (Pyrus communis L.) is controlled by a single locus (S-locus) encoding a polymorphic stylar ribonuclease (S-RNase) that is responsible for the female function in pollen–pistil recognition. In this study, genomic DNA sequences corresponding to five new S-RNase alleles (named S ₂₀ , S ₂₁ , S ₂₂ , S ₂₃ , and S ₂₄ ) and to S _m were characterized in European pear cultivars. Re-sequencing S _q from ‘General Le Clerc’ showed this S-RNase to encode the same protein as S ₁₂ . Based on these findings, a polymerase chain reaction (PCR)-based method was developed for the molecular typing of cultivars bearing 20 S-RNases (S ₁ –S ₁₄ , S _m , and S ₂₀ –S ₂₄ ) using consensus and allele-specific primers. Genomic PCR with consensus primers amplified product sizes characteristic of the S-RNases S ₁ , S ₂ , S ₄ , S ₁₀ , S ₁₃ , and S ₂₀ . However, the allele groups S ₃ /S ₁₂ , S ₆ /S ₈ /S ₁₁ /S ₂₂ and S ₅ /S ₇ /S ₉ /S ₁₄ /S _m /S ₂₁ /S ₂₃ /S ₂₄ amplified PCR products of similar size. To discriminate between alleles within these groups, primers to specifically amplify each S-RNase were developed. Application of this approach in 19 cultivars with published S-alleles allowed re-evaluation of one of the alleles of ‘Passe Crassane,’ ‘Conference,’ and ‘Condo.’ Finally, this method was used to assign S-genotypes to 37 cultivars. Test crosses confirmed molecular results. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Identification of S-genotypes in Chinese cherry cultivars (Prunus pseudocerasus Lindl.)

Self-incompatibility has been studied extensively at the molecular level in Solanaceae, Rosaceae, and Scrophulariaceae, all of which exhibit gametophytic self-incompatibility. In the present study, we successfully isolated nine S-RNase alleles from cultivars of Chinese cherry by PCR amplification from genomic DNA and stylar cDNA combining with cleaved amplified polymorphic sequence marker. Analysis of amino acid sequences revealed five novel S-alleles, S ₂ , S ₄ , S ₆ , S ₈ , and S ₉ , with respective accession numbers in the NCBI database of EF541168, EF541173, EF541172, FJ628598, and FJ628599. Results showed that “Dongtang” and “Yinzhu” contained six S-alleles (S ₁ , S ₃ , S ₅ , S ₇ , S ₈ , and S ₉ ); “Taishanganying” contained four S-alleles (S ₁ , S ₂ , S ₄ , and S ₆ ); “Daiba”, “Dayingzui”, and “Xiaomizi” contained four S-alleles (S ₁ , S ₂ , S ₅ , and S ₈ ); “Laiyangduanzhi”, “Shuangquanchangba”, and “Daqingye” contained three S-alleles (S ₁ , S ₂ , and S ₈ ). It is interesting that different cultivars collected from the same place hold the same S-genotypes. Moreover, pollination tests and pollen tube growth assays showed that nine cultivars were self-compatible. Chinese cherry presented in this article are naturally polyploidy, which is a very useful material for the study of self-compatibility, and much of this information will be valuable for further work on self-(in)compatibility of fruit tree in Rosaceae.     

Development of SCAR markers linked to self-incompatibility in <Emphasis Type="Italic">Brassica napus</Emphasis> L.

‘SI1300’ is a self-incompatible Brassica napus line generated by introgressing an S haplotype from B. rapa ‘Xishuibai’ into a rapeseed cultivar ‘Huayou No. 1’. Five S-locus specific primer pairs were employed to develop cleaved amplified polymorphic sequences (CAPS) markers linked the S haplotype of ‘SI1300’. Two segregating populations (F₂ and BC₁) from the cross between ‘SI1300’ and self-compatible European spring cultivar ‘Defender’, were generated to verify the molecular markers. CAPS analysis revealed no desirable polymorphism between self-incompatible and self-compatible plants. Twenty primer pairs were designed based on the homology-based candidate gene method, and six dominant sequence characterized amplified region (SCAR) markers linked with the S-locus were developed. Of the six markers, three were derived from the SRK and SP11 alleles of class II B. rapa S haplotypes and linked with S haplotype of ‘SI1300’. The other three markers were designed from the SLG-A10 and co-segregated with S haplotype of ‘Defender’. We successfully combined two pairs of them and characterized two multiplex PCR markers which could discriminate the homozygous and heterozygous genotypes. These markers were further validated in 24 F₃ and 22 BC₁F₂ lines of ‘SI1300 × Defender’ and another two segregating populations from the cross ‘SI1300 × Yu No. 9’. Nucleotide sequences of fragments linked with S-locus of ‘SI1300’ showed 99% identity to B. rapa class II S-60 haplotype, and fragments from ‘Defender’ were 97% and 94% identical to SLG and SRK of B. rapa class I S-47 haplotype, respectively. ‘SI1300’ was considered to carry two class II S haplotypes and the S haplotype on the A-genome derived from B. rapa ‘Xishuibai’ determines the SI phenotype, while ‘Defender’ carry a class I S haplotype derived from B. rapa and a class II S haplotype from B. oleracea. SCAR markers developed in this study will be helpful for improving SI lines and accelerating marker-assisted selection process in rapeseed SI hybrid breeding program.     

Introgression molecular analysis of a leaf rust resistance gene from <Emphasis Type="Italic">Coffea liberica</Emphasis> into <Emphasis Type="Italic">C. arabica</Emphasis> L.

Leaf rust caused by the fungus Hemileia vastatrix is the most devastating disease of arabica coffee (Coffea arabica). Therefore, developing leaf rust-resistant varieties has been a breeding objective of the highest priority in many countries. The purpose of the present work was to gain insight into the mechanism of introgression into C. arabica of a leaf rust resistance gene from C. liberica (i.e. S_H3 resistance factor) and to identify associated molecular markers. An F₂ progeny (i.e. 101 individuals) derived from a cross between Matari, an arabica accession and liberica-introgressed line S.288, was evaluated for resistance against three different races of H. vastatrix. The progeny segregated for the S_H3 gene in a 3:1 ratio, as expected for a single dominant gene. Amplified fragment length polymorphism analysis of a population subset using 80 different primer combinations revealed that at least half of the total polymorphism observed in the population is associated with introgression of C. liberica chromosome fragments. Furthermore, 15 primer combinations generating candidate marker bands associated with the S_H3 resistance gene were used to analyse the whole F₂ population. A total of 34 marker bands originating from S.288 and attributable to introgression were scored. None exhibited segregation distortion. Linkage analysis revealed only three distinct introgressed fragments corresponding to a total length of 52.8 cM. Twenty-one markers were strongly associated (LOD score >14) with the S_H3 gene and were grouped together in a single linkage group of 6.3 cM. The results are discussed in relation to the efficient use of genetic resources in arabica breeding.     

Introgression of group 4 and 7 chromosomes of <Emphasis Type="Italic">Ae</Emphasis>. <Emphasis Type="Italic">peregrina</Emphasis> in wheat enhances grain iron and zinc density

Dietary deficiency of iron and zinc micronutrients affects more than two billion people worldwide. Breeding for micronutrient-dense crops is the most sustainable and cost-effective approach for alleviation of micronutrient malnutrition. Three accessions of Aegilops peregrina (Hack.) Maire & Weill (2n = 28, U^PU^PS^PS^P), selected for high grain iron and zinc concentration were crossed with Triticum aestivum L. cv. Chinese Spring (Ph ^I ). The sterile F₁ hybrids were backcrossed with elite wheat cultivars to get fertile BC₂F₂ derivatives. Some of the fertile BC₂F₂ derivatives showed nearly 100% increase in grain iron and more than 200% increase in grain zinc concentration compared to the recipient wheat cultivars. The development of derivatives with significantly higher grain micronutrients, high thousand-grain weight and harvest index suggests that the enhanced micronutrient concentration is due to the distinct genetic system of Ae. peregrina and not to the concentration effect. Genomic in situ hybridization, comparison of introgressed chromosomes with the standard karyotype of Ae. peregrina and simple sequence repeat marker analysis revealed the introgression of 7S^P chromosomes in five selected derivatives, 7U^P in four, group 4 and 4S^P in three and a translocated 5U^P of Ae. peregrina in one of the selected derivatives. Molecular marker analysis using the introgressed chromosome markers indicated that two of the BC₂F₃ progenies were stabilized as disomic addition lines. It could, therefore, be concluded that the group 4 and 7 chromosomes of Ae. peregrina carry the genes for high grain iron and zinc concentration.     

Physical size of the <Emphasis Type="Italic">S</Emphasis> locus region defined by genetic recombination and genome sequencing in <Emphasis Type="Italic">Ipomoea trifida</Emphasis>, Convolvulaceae

Sporophytic self-incompatibility (SSI) in the genus Ipomoea (Convolvulaceae) is controlled by a single polymorphic S locus. We have previously analyzed genomic sequences of an approximately 300 kb region spanning the S locus of the S ₁ haplotype and characterized the genomic structure around this locus. Here, we further define the physical size of the S locus region by mapping recombination breakpoints, based on sequence analysis of PCR fragments amplified from the genomic DNA of recombinants. From the recombination analysis, the S locus of the S ₁ haplotype was delimited to a 0.23 cM region of the linkage map, which corresponds to a maximum physical size of 212 kb. To analyze differences in genomic organization between S haplotypes, fosmid contigs spanning approximately 67 kb of the S ₁₀ haplotype were sequenced. Comparison with the S ₁ genomic sequence revealed that the S haplotype-specific divergent regions (SDRs) spanned 50.7 and 34.5 kb in the S ₁ and S ₁₀ haplotypes, respectively and that their flanking regions showed a high sequence similarity. In the sequenced region of the S ₁₀ haplotype, five of the 12 predicted open reading frames (ORFs) were found to be located in the divergent region and showed co-linear organization of genes between the two S haplotypes. Based on the size of the SDRs, the physical size of the S locus was estimated to fall within the range 34–50 kb in Ipomoea.     

Three QTLs for <Emphasis Type="Italic">Botrytis cinerea</Emphasis> resistance in tomato

Tomato (Solanum lycopersicum) is susceptible to grey mold (Botrytis cinerea). Partial resistance to this fungus was identified in accessions of wild relatives of tomato such as S. habrochaites LYC4. In order to identify loci involved in quantitative resistance (QTLs) to B. cinerea, a population of 174 F₂ plants was made originating from a cross between S. lycopersicum cv. Moneymaker and S. habrochaites LYC4. The population was genotyped and tested for susceptibility to grey mold using a stem bioassay. Rbcq1, a QTL reducing lesion growth (LG) and Rbcq2, a QTL reducing disease incidence (DI) were identified. Rbcq1 is located on Chromosome 1 and explained 12% of the total phenotypic variation while Rbcq2 is located on Chromosome 2 and explained 15% of the total phenotypic variation. Both QTL effects were confirmed by assessing disease resistance in two BC₂S₁ progenies segregating for either of the two QTLs. One additional QTL, Rbcq4 on Chromosome 4 reducing DI, was identified in one of the BC₂S₁ progenies. F₂ individuals, homozygous for the Rbcq2 and Rbcq4 alleles of S. habrochaites showed a reduction of DI by 48%. QTLs from S. habrochaites LYC4 offer good perspectives for breeding B. cinerea resistant tomato cultivars. Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.     

Development of sequence characterized DNA markers linked to leaf rust (<Emphasis Type="Italic">Hemileia vastatrix</Emphasis>) resistance in coffee (<Emphasis Type="Italic">Coffea arabica</Emphasis> L.)

Coffee leaf rust due to Hemileia vastatrix is one of the most serious diseases in Arabica coffee (Coffea arabica). A resistance gene (S_H3) has been transferred from C. liberica into C. arabica. The present work aimed at developing sequence-characterized genetic markers for leaf rust resistance. Linkage between markers and leaf rust resistance was tested by analysing two segregating populations, one F₂ population of 101 individuals and one backcross (BC₂) population of 43 individuals, derived from a cross between a susceptible and a SH3-introgressed resistant genotype. A total of ten sequence-characterized genetic markers closely associated with the S_H3 leaf rust resistance gene were generated. These included simple sequence repeats (SSR) markers, sequence-characterised amplified regions (SCAR) markers resulting from the conversion of amplified fragment length polymorphism (AFLP) markers previously identified and SCAR markers derived from end-sequences of bacterial artificial chromosome (BAC) clones. Those BAC clones were identified by screening of C. arabica genomic BAC library using a cloned AFLP-marker as probe. The markers we developed are easy and inexpensive to run, requiring one PCR step followed by gel separation. While three markers were linked in repulsion with the S_H3 gene, seven markers were clustered in coupling around the S_H3 gene. Notably, two markers appeared to co-segregate perfectly with the S_H3 gene in the two plant populations analyzed. These markers are suitable for marker-assisted selection for leaf rust resistance and to facilitate pyramiding of the S_H3 gene with other leaf rust resistance genes.     

A simple chromosomal marker can reliably distinguishes <Emphasis Type="Italic">Poncirus</Emphasis> from <Emphasis Type="Italic">Citrus</Emphasis> species

Several chromosome types have been recognized in Citrus and related genera by chromomycin A₃ (CMA) banding patterns and fluorescent in situ hybridization (FISH). They can be used to characterize cultivars and species or as markers in hybridization and backcrossing experiments. In the present work, characterization of six cultivars of P. trifoliata (“Barnes”, “Fawcett”, “Flying Dragon”, “Pomeroy”, “Rubidoux”, “USDA”) and one P. trifoliata × C. limonia hybrid was performed by sequential analyses of CMA banding and FISH using 5S and 45S rDNA as probes. All six cultivars showed a similar CMA⁺ banding pattern with the karyotype formula 4B + 8D + 6F. The capital letters indicate chromosomal types: B, a chromosome with one telomeric and one proximal band; D, with only one telomeric band; F, without bands. In situ hybridization labeling was also similar among cultivars. Three chromosome pairs displayed a closely linked set of 5S and 45S rDNA sites, two of them co-located with the proximal band of the B type chromosomes (B/5S-45S) and the third one co-located with the terminal band of a D pair (D/5S-45S). The B/5S-45S chromosome has never been found in any citrus accessions investigated so far. Therefore, this B chromosome can be used as a marker to recognize the intergeneric Poncirus × Citrus hybrids. The intergeneric hybrid analyzed here displayed the karyotype formula 4B + 8D + 6F, with two chromosome types B/5S-45S and two D/5S-45S. The karyotype formula and the presence of two B/5S-45S chromosomes clearly indicate that the plant investigated is a symmetric hybrid. It also demonstrates the suitability of karyotype analyses to differentiate zygotic embryos or somatic cell fusions involving trifoliate orange germplasm. During the submission of this paper, we analyzed 25 other citrus cultivars with the same methodology and we found that the chromosome marker reported here can indeed distinguish Poncirus trifoliata from grapefruits, pummelos, and one variegated access of Citrus, besides the previously reported access of limes, limons, citrons, and sweet-oranges. However, among 14 mandarin cultivars, two of them displayed a single B/5S-45S chromosome, whereas in Citrus hystrix D.C., a far related species belonging to the Papeda subgenus, this chromosome type was found in homozygosis. Since these two mandarin cultivars are probably of hybrid origin, we assume that for almost all commercial cultivars and species of the subgenus Citrus this B type chromosome is a useful genetic marker.