Map-based cloning of the fertility restoration locus <Emphasis Type="Italic">Rfm1</Emphasis> in cultivated barley (<Emphasis Type="Italic">Hordeum vulgare</Emphasis>)

Hybridization technology has proven valuable in enhancing yields in many crops, but was only recently adopted in the small grain cereals. Hybrid varieties in barley (Hordeum vulgare) rely on the cytoplasmic male sterility (CMS) system msm1 derived from Hordeum vulgare ssp. spontaneum. The major restorer gene described for the msm1 system is known as Rfm1 and maps to the top of chromosome 6H. To gain further insight into mechanisms underlying male fertility restoration in barley, we used a map-based cloning approach to identify the nuclear gene involved in the restoration mechanism of this hybridization system. Taking advantage of the available genomic resources in barley in combination with a custom-made non-gridded BAC library developed from a restorer line, we cloned and sequenced the Rfm1 restorer locus. The characterization and annotation of the nucleotide sequence for the Rfm1 restorer allele allowed for the identification of the candidate gene for Rfm1. The Rfm1 locus carries a tandem repeat of a gene encoding a pentatricopeptide repeat (PPR) protein. Surprisingly, Rfm1 belongs to the PLS-DYW subfamily of PPR genes known for their involvement in RNA editing in plants organelles, but that to date have not been identified as restorer genes.     

Development of InDel markers for the restorer gene <Emphasis Type="Italic">Rf1</Emphasis> and assessment of their utility for marker-assisted selection in cotton

The cytoplasmic male sterility (CMS) system is ideal for exploiting heterosis in crops such as cotton. However, CMS-D2, which is based on Gossypium harknessii cytoplasm, is still not widely used for cotton production. In this study, we developed an efficient marker-assisted selection method based on insertion/deletion (InDel) markers that can identify restorer lines carrying Rf1. Whole-genome resequencing was first completed for restorer [N(Rf1Rf1)] and maintainer [N(rf1rf1)] lines with normal fertile cytoplasm (N). Comparisons with the TM-1 reference genome sequence resulted in the identification of 292,065 and 183,657 InDels for the restorer and maintainer lines, respectively. Most of the InDels in the restorer line were distributed on Chromosome_D05, which carries Rf1. Of the 12 InDel markers near the Rf1 target region that were further validated, four co-dominant markers (i.e., InDel-1891, InDel-3434, InDel-7525, and InDel-9356) co-segregated with Rf1, as verified by a segregation analysis in an F₂ population. We subsequently used InDel-1891 to determine the allele status at the Rf1 locus in a backcross scheme for transferring Rf1. In this study, we developed new markers to increase the marker density in the Rf1 target region, which will be useful for the fine mapping of Rf1. The development of convenient and inexpensive co-segregating InDel markers will facilitate the marker-assisted selection of restorer lines carrying Rf1.     

Expression of the <Emphasis Type="Italic">Ga1-s</Emphasis> gametophyte factor in <Emphasis Type="Italic">shrunken2</Emphasis> sweet corn

Outcrossing is an important problem in specialty maize (Zea mays L.) that can be prevented by using gametophyte factors, such as Ga1-s, which preserve maize plants from pollen contamination. Our objective was to check if the gametophyte factor Ga1-s can protect sweet corn homozygous for sh2 in an efficient and stable way. We combined Ga1-s and sh2 by crossing two popcorn and three sweet corn inbred lines, respectively, in a North Carolina Design II, followed by an ear-to-row breeding program with selection for sh2 phenotype and absence of outcrossing. The released inbred lines homozygous for Ga1-s and sh2 were used for obtaining five hybrids that were evaluated for outcrossing and agronomic performance. Our results show that the gametophyte factor Ga1-s effectively protects the sh2 plants and that this effect was stable across environments. However, the agronomic performance of these inbred lines must be improved. Popcorn donors and sweet corn receptors of Ga1-s were unevenly represented in the released Ga1-s / sh2 inbred lines, suggesting that the viability of sh2 is affected by the genotypes involved. Therefore, breeders should pay attention to the choice of donors of Ga1-s that favors the viability of sh2.     

Hybrids of <Emphasis Type="Italic">Passiflora</Emphasis>: <Emphasis Type="Italic">P. gardneri</Emphasis> versus <Emphasis Type="Italic">P. gibertii,</Emphasis> confirmation of paternity,morphological and cytogenetic characterization

Two new varieties of interspecific hybrids of Passiflora have been developed from the cross between P. gardneri versus P. gibertii, both registered under the Passiflora Society International. Twelve putative hybrids were analyzed. Hybridization was confirmed using RAPD and SSR markers. Primer UBC11 (5′-CCGGCCTTAC-3′) generated informative bands. Primer SSR Pe75 has amplified species-specific fragments and a heterozygote status was observed with two parent bands 300 and 350 bp. The molecular markers generated have been analyzed for the presence or absence of specific informative bands. Based on the morphological characterization, we have identified two hybrid varieties: P. ‘Gabriela’ and P. ‘Bella’. P. ‘Gabriela’ produced flowers in bluish tones, bluish petals on the adaxial and abaxial faces, light blue sepals on the adaxial and light green on the abaxial faces, corona with the base of filaments in intense lilac color and white apex. P. ‘Bella’ produced flowers in lilac tones, intense lilac petals on the adaxial and abaxial faces, dark lilac sepals with whitish edges on the adaxial and light green on the abaxial faces, corona with the base of filaments in intense lilac color and white apex. The cytogenetic analysis verified that the hybrids have the same chromosomal number as the parents (2n = 18); the formation of bivalents between the homeologous chromosomes (n = 9) was observad, leading to regular meiosis, which allows the sexual reproduction and use of these hybrids in breeding programs.     

Fine mapping of <Emphasis Type="Italic">Rf3</Emphasis> and <Emphasis Type="Italic">Rf4</Emphasis> fertility restorer loci of WA-CMS of rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.) and validation of the developed marker system for identification of restorer lines

The Wild Abortive (WA) system is the major cytoplasmic male sterility (CMS) source for hybrid rice production in indica rice and its fertility restoration is reported to be controlled by two major loci viz. Rf3 on chromosome 1 and Rf4 on chromosome 10. With the availability of the rice genome sequence, an attempt was made to fine map, develop candidate gene based markers for Rf3 and Rf4 and validate the developed marker system in a set of known restorer lines. Using polymorphic markers developed from microsatellite markers and candidate gene based markers from Rf3 and Rf4 loci, local linkage maps were constructed in two mapping populations of ~1,500 F₂ progeny from KRH2 (IR58025A/KMR3R) and DRRH2 (IR68897A/DR714-1-2R) hybrids. QTLs and their interactions for fertility restoration in Rf3 and Rf4 loci were identified. The identified QTL in both mapping populations together explained 66–72 % of the phenotypic variance of the trait suggesting their utility in developing a marker system for identification of fertility restorers for WA-CMS. Sequence comparison of the two candidate genes from the Rf3 and Rf4 regions in male sterile (A) and restorer (R) lines showed 2–3 bp indels and a few substitutions in the Rf3 region and indels of 327 and 106 bp in the Rf4 region respectively. The marker system identified in the present study was validated in 212 restorers and 34 maintainers along with earlier reported markers for fertility restoration of WA-CMS. Together DRCG-RF4-14 and DRCG-RF4-8 for the Rf4 locus and DRRM-RF3-5/DRRM-RF3-10 for the Rf3 locus showed a maximum efficiency of 92 % for identification of restorers.     

Sources of resistance to <Emphasis Type="Italic">Phytophthora</Emphasis> root rot within the genus <Emphasis Type="Italic">Beta</Emphasis>

Phytophthora root rot caused by Phytophthora drechsleri Tucker is one of the most devastating sugar beet diseases in tropical areas. To identify genetic resources resistant to this disease, an aggressive isolate of P. drechsleri was selected. Then, a screening method was optimized based on the standard scoring scales of 1–9 (1: no symptoms, 9: complete plant death). Finally, 19 sugar beet lines, three cultivars, and 14 accessions of the wild species Beta vulgaris subsp. maritima, B. macrocarpa, B. procumbens, and B. webbiana were evaluated for resistance to the most aggressive isolate of P. drechsleri by using the optimized method (inoculum included 20 g of rice seed together with superficial wound creation). The isolates of P. drechsleri had significant variation in aggressiveness, and Kv10 was the most aggressive isolate on the susceptible variety Rasoul. The lines O.T.201-15, SP85303-0 (resistant check), and S2-24.P.107 had the lowest disease index with scores of 3.09, 3.13, and 3.27 respectively; they were categorized into the resistant group. The interaction between isolates and genotypes was not significant, which indicated the same response of each genotype to different isolates. Investigating the resistance of different generations of sugar beet revealed that progeny selection would be an effective method for increasing the resistance level of breeding materials to P. drechsleri. Among the wild species, the accession 9402 belonging to B. macrocarpa and the accession 7234 of B. vulgaris subsp. maritima had the lowest disease index (2.29 and 2.60, respectively) and were categorized into the resistant group.     

Obtainment of an intergeneric hybrid between <Emphasis Type="Italic">Forsythia</Emphasis> and <Emphasis Type="Italic">Abeliophyllum</Emphasis>

Forsythia suspensa and F. ‘Courtaneur’ were used as female parents to cross with Abeliophyllum distichum in 2011 and an intergeneric hybrid of F. suspensa × A. distichum was obtained, though with very low seed set. The morphological characteristics, flower fragrance and volatile organic compounds of flowers were analysed. The intergeneric hybrid had intermediate morphological characteristics of both parents and flower fragrance and was confirmed as a true intergeneric hybrid by amplified fragment length polymorphism (AFLP) markers. Compared with its mother parent (F. suspensa), flowers of the intergeneric hybrid are pale yellow with delicate fragrance. Volatile organic compounds of flowers were retrieved by purge-and-trap techniques, and determined by gas chromatography and mass spectrometry (GC–MS). The main volatile organic components of F. suspensa were isoprenoids, while the main volatile organic components of A. distichum and the hybrid of F. suspensa × A. distichum were aliphatics. To determine the time and the site of intergeneric hybridizing barriers occured, the pollen tubes’ behavior after pollination was observed under fluorescence microscopy. It was found that significant pre-fertilization incompatibility existed in intergeneric crossing combinations [F. ‘Courtaneur’ (Pin) × A. distichum (Thrum) and F. suspensa (Pin) × A. distichum (Thrum)], and only a few pollen tubes of A. distichum penetrated into the ovaries of Forsythia. In our research, an intergeneric hybrid between Forsythia and Abeliophyllum was obtained for the first time, which will provide a solid foundation for expanding the flower color range of Forsythia and breeding fragrant-flowered cultivars.     

Hybridization and heterosis in the Plicatula group of <Emphasis Type="Italic">Paspalum</Emphasis>

Most forage cultivars released for the genus Paspalum belong to a section named Plicatula. The species of Plicatula are mostly apomictic and consequently the genetic diversity is locked for their genetic improvement. The objectives were to evaluate the crossability, hybrid fertility, heterosis, and genetic distances between apomictic accessions and a sexual genotype of species of Plicatula group of Paspalum. Crosses were made using 22 apomictic tetraploid accessions belonging to 12 different species as pollen donors, and a sexual tetraploid genotype induced by colchicine from a sexual diploid accession of P. plicatulum. Crossability varied between 0 and 16% among crosses. Viable hybrid offspring were recovered from 15 out of 22 crosses. The most successful crosses involved P. guenoarum, P. plicatulum, P. chaseanum, and P. oteroi. Fertility of the sampled hybrids varied between 1.6% for the cross involving P. lenticulare, and 40.1% for an intraspecific cross (P. plicatulum, accession Hojs388). The genetic distance between parents was estimated using amplified fragment-length polymorphism, and it varied between 0.34 and 0.53. There was no correlation between genetic distances and crossability or fertility of the hybrids. Hybrids from the most numerous families were classified for mode of reproduction using flow cytometric seed analysis. The ratio between sexual and apomictic hybrids varied between 0.6:1 and 1.6:1. A selected group of apomictic hybrids were evaluated for several agronomic traits in the field. Heterosis was observed for frost tolerance and cattle preference. The results indicated that gene transfer via hybridization is possible among several species of Plicatula. Superior hybrids for specific traits can be generated and fixed by apomixis.     

Resistance to <Emphasis Type="Italic">Cucumber green mottle mosaic virus</Emphasis> in <Emphasis Type="Italic">Cucumis sativus</Emphasis>

Cucumber green mottle mosaic virus (CGMMV) is a severe threat for cucumber production worldwide. At present, there are no cultivars available in the market which show an effective resistance or tolerance to CGMMV infection, only wild Cucumis species were reported as resistant. Germplasm accessions of Cucumis sativus, as well as C. anguria and C. metuliferus, were mechanically infected with the European and Asian strains of CGMMV and screened for resistance, by scoring symptom severity, and conventional RT-PCR. The viral loads of both CGMMV strains were determined in a selected number of genotypes using quantitative RT-PCR. Severe symptoms were found following inoculation in C. metuliferus and in 44 C. sativus accessions, including C. sativus var. hardwickii. Ten C. sativus accessions, including C. sativus var. sikkimensis, showed intermediate symptoms and only 2 C. sativus accessions showed mild symptoms. C. anguria was resistant to both strains of CGMMV because no symptoms were expressed and the virus was not detected in systemic leaves. High amounts of virus were found in plants showing severe symptoms, whereas low viral amounts found in those with mild symptoms. In addition, the viral amounts detected in plants which showed intermediate symptoms at 23 and 33 dpi, were significantly higher in plants inoculated with the Asian CGMMV strain than those with the European strain. This difference was statistically significant. Also, the amounts of virus detected over time in plants did not change significantly. Finally, the two newly identified partially resistant C. sativus accessions may well be candidates for breeding programs and reduce the losses produced by CGMMV with resistant commercial cultivars.     

Identification of genetic sources with attenuated <Emphasis Type="Italic">Tomato chlorosis virus</Emphasis>-induced symptoms in <Emphasis Type="Italic">Solanum</Emphasis> (section <Emphasis Type="Italic">Lycopersicon</Emphasis>) germplasm

The whitefly-transmitted Tomato chlorosis virus (ToCV) (genus Crinivirus) is associated with yield and quality losses in field and greenhouse-grown tomatoes (Solanum lycopersicum) in South America. Therefore, the search for sources of ToCV resistance/tolerance is a major breeding priority for this region. A germplasm of 33 Solanum (Lycopersicon) accessions (comprising cultivated and wild species) was evaluated for ToCV reaction in multi-year assays conducted under natural and experimental whitefly vector exposure in Uruguay and Brazil. Reaction to ToCV was assessed employing a symptom severity scale and systemic virus infection was evaluated via RT-PCR and/or molecular hybridization assays. A subgroup of accessions was also evaluated for whitefly reaction in two free-choice bioassays carried out in Uruguay (with Trialeurodes vaporariorum) and Brazil (with Bemisia tabaci Middle-East-Asia-Minor1—MEAM1?=?biotype B). The most stable sources of ToCV tolerance were identified in Solanum habrochaites PI 127827 (mild symptoms and low viral titers) and S. lycopersicum ‘LT05’ (mild symptoms but with high viral titers). These two accessions were efficiently colonized by both whitefly species, thus excluding the potential involvement of vector-resistance mechanisms. Other promising breeding sources were Solanum peruvianum (sensu lato) ‘CGO 6711’ (mild symptoms and low virus titers), Solanum chilense LA1967 (mild symptoms, but with high levels of B. tabaci MEAM1 oviposition) and Solanum pennellii LA0716 (intermediate symptoms and low level of B. tabaci MEAM1 oviposition). Additional studies are necessary to elucidate the genetic basis of the tolerance/resistance identified in this set of Solanum (Lycopersicon) accessions.     

Molecular breeding for resistance to black rot [<Emphasis Type="Italic">Xanthomonas campestris</Emphasis> pv. <Emphasis Type="Italic">campestris</Emphasis> (Pammel) Dowson] in <Emphasis Type="Italic">Brassicas</Emphasis>: recent advances

The Brassicas are affected by several diseases, of which black rot, Xanthomonas campestris pv. campestris (Pam.) Dowson (Xcc), is one of the most widespread and devastating worldwide. The black rot bacteria causes systemic infection in the susceptible plants and penetrate the plants through the hydathodes or wounds. Typical disease symptoms are ‘V’ shaped necrotic lesions appearing from the leaf margins with blackened veins. Periodic outbreaks of the black rot pathogen have occurred worldwide, especially in the continental regions, where high temperatures and humidity favor the incidence of disease occurrence causing huge yield loss. The challenge to control the losses in vegetable brassicas production is made more difficult by the adverse climatic changes and evolution of new pathogenic races. The development of black rot resistant hybrids/varieties is the most reliable long term practical solution for effective disease control. Identification of new resistant genetic resources, tightly linked markers with resistance loci and QTL mapping would facilitate the breeding programme for black rot resistance. Information regarding genetics of resistance and mapping of resistance genes/QTLs will accelerate the marker assisted resistance breeding in brassica crops against Xcc. In future we need to identify the race specific candidate genes for and their validation through transgenics and gene expression. Moreover, it is imperative to identify functional markers for resistance genes through identification of R gene families and their relationship with resistance expression. This comprehensive review will help the researchers working in this area to understand the dynamics of black resistance breeding and to formulate future breeding strategies.     

Genetic mapping of the <Emphasis Type="Italic">qGCR6</Emphasis> locus affecting glossiness of cooked rice

A japonica variety, Koshihikari, is known to have favorable eating quality. Two rice backcross inbred lines (BILs) developed from Koshihikari exhibited significantly different glossiness of cooked rice (GCR), an eating quality trait measured using the Toyo-taste meter. Genetic analysis indicated that the genetic composition of these two BILs differed only on the short arm of chromosome 6, which led to the identification of the qGCR6 locus. Through high-resolution genetic mapping, the qGCR6 locus was further delimited to a 43.9 kb chromosomal region containing ten putative genes. The DNA marker SNP2175, which tightly links to qGCR6, was developed and can be used in marker-assisted breeding programs.     

Broadening the genetic base of <Emphasis Type="Italic">Brassica napus</Emphasis> canola by interspecific crosses with different variants of <Emphasis Type="Italic">B. oleracea</Emphasis>

Broadening the genetic base of the C genome of Brassica napus canola by use of B. oleracea is important. In this study, the prospect of developing B. napus canola lines from B. napus?×?B. oleracea var. alboglabra, botrytis, italica and capitata crosses and the effect of backcrossing the F₁’s to B. napus were investigated. The efficiency of the production of the F₁’s varied depending on the B. oleracea variant used in the cross. Fertility of the F₁ plants was low—produced, on average, about 0.7 F₂ seeds per self-pollination and similar number of BC₁ seeds on backcrossing to B. napus. The F₃ population showed greater fertility than the BC₁F₂; however, this difference diminished with the advancement of generation. The advanced generation populations, whether derived from F₂ or BC₁, showed similar fertility and produced similar size silique with similar number of seeds per silique. Progeny of all F₁’s and BC₁’s stabilized into B. napus, although B. oleracea plant was expected, especially in the progeny of F₁ (ACC) owing to elimination of the A chromosomes during meiosis. Segregation distortion for erucic acid alleles occurred in both F₂ and BC₁ resulting significantly fewer zero-erucic plants than expected; however, plants with?≤?15% erucic acid frequently yielded zero-erucic progeny. No consistent correlation between parent and progeny generation was found for seed glucosinolate content; however, selection for this trait was effective and B. napus canola lines were obtained from all crosses. Silique length showed positive correlation with seed set; the advanced generation populations, whether derived from F₂ or BC₁, were similar for these traits. SSR marker analysis showed that genetically diverse canola lines can be developed by using different variants of B. oleracea in B. napus?×?B. oleracea interspecific crosses.     

Identifying apomixis in matroclinal progeny from an interspecific crossing between <Emphasis Type="Italic">Iris domestica</Emphasis> and three different colors of <Emphasis Type="Italic">Iris dichotoma</Emphasis>

In a previous investigation on the reciprocal difference of interspecific hybridization between three different flower colors of Iris dichotoma and Iris domestica in the F₁ offspring from crosses where I. domestica was a maternal parent were similar in morphological and cytological characters to their maternal parent. This could be evidence of apomixis; however, matroclinal progeny with complete morphological similarity to the maternal parent could be attributed to the heterozygosity for these characters in the pollen parent. The F₁ plants were investigated in order to identify apomixis in I. domestica. Four matroclinal plants were randomly selected from each F₁ population produced from Iris domestica × Iris dichotoma that had three different colors of flowers and were allowed to self-pollinate to establish an F₂ population. All of the F₂ plants had no segregation to I. domestica in their morphological characters. In addition, 13 reciprocal F₁ plants were detected by 25,719 single nucleotide polymorphism (SNP) markers. When I. dichotoma plants with three different flower colors were used as maternal parents, all the progenies were genuine hybrids. When I. domestica were used as maternal parents, all the F₁ plants were apomictic progenies. Apomixis of I. domestica was successfully identified and SNP markers identified F₁ hybrids derived from six interspecific crosses between I. dichotoma and I. domestica, which provides a reference for authenticating offspring identity during Iris cross breeding in the future.     

Pollination and in vitro germination of seeds for interspecific hybridization of <Emphasis Type="Italic">Psidium guajava</Emphasis> and <Emphasis Type="Italic">Psidium cattleianum</Emphasis>

The genus Psidium includes important fruit crops. However, there are very few studies focusing on its reproductive biology, which limits the establishment of breeding programs. The present work investigated the reproductive biology of Psidium guajava and Psidium cattleianum in terms of compatibility of crossings between these two species aiming at interspecific hybridization because the latter species is an important source of resistance against the nematode Meloidogyne enterolobii. Several types of crosses were performed to understand the reproductive biology of these species, including the compatibility of intra- and interspecific crossings, using assisted in vivo germination of pollen grains on the stigma. In addition, the in vitro germination of both Psidium species was studied at different stages of fruit development to rescue young seeds to improve the chances of obtaining the hybrids. No fruits of 270 pollinations were obtained on guava buds at the pre-anthesis stage, regardless of the source of the pollen grain and the cultivar used as female genotype. Microscopic analyzes demonstrated the germination of pollen grains and pollen tube growth at crosses between guava cv. ‘Pedro Sato’ (P. guajava) and Psidium cattleianum. High germination percentages of Psidium cattleianum seeds were obtained in MS medium without sucrose or containing 15 g/L of this carbohydrate.     

Development of RAPD and ISSR derived SCAR markers linked to <Emphasis Type="Italic">Xca1Bo</Emphasis> gene conferring resistance to black rot disease in cauliflower (<Emphasis Type="Italic">Brassica oleracea</Emphasis> var. <Emphasis Type="Italic">botrytis</Emphasis> L.)

Black rot caused by Xanthomonas campestris pv. campestris (Xcc) (Pam.) is the most devastating disease of cauliflower (Brassica oleracea var. botrytis L.; 2n = 2x = 18), taking a heavy toll of the crop. In this study, a random amplified polymorphic DNA (RAPD) and inter simple sequence repeat (ISSR) derived sequence characterized amplified region (SCAR) markers linked to the black rot resistance locus Xca1bo were developed and evaluated as a screening tool for resistance. The RAPD marker OPO-04₈₃₃ and ISSR marker ISSR-11₆₃₅ were identified as closely linked at 1.6 cM distance to the black rot resistance locus Xca1bo. Both the markers OPO-04₈₃₃ and ISSR-11₆₃₅ were cloned, sequenced and converted into SCAR markers and validated in 17 cauliflower breeding lines having different genetic backgrounds. These SCAR markers (ScOPO-04₈₃₃ and ScPKPS-11₆₃₅) amplified common locus and showed 100% accuracy in differentiating resistant and susceptible plants of cauliflower breeding lines. The SCAR markers ScOPO-04₈₃₃ and ScPKPS-11₆₃₅ are the first genetic markers found to be linked to the black rot resistance locus Xca1bo in cauliflower. These markers will be very useful in black rot resistance marker assisted breeding.     

Effects of introgressions from <Emphasis Type="Italic">Festuca pratensis</Emphasis> on winter hardiness of <Emphasis Type="Italic">Lolium perenne</Emphasis>

Previous studies reported that some genotypes with introgressed Festuca chromosome segment(s) in Lolium genome showed enhanced winter hardiness compared to Lolium. The aim of this study was to search comprehensively for the Festuca pratensis chromosome regions affecting winter hardiness-related traits when introgressed into the Lolium perenne genome. Association between F. pratensis introgression and winter hardiness-related traits (fall and winter hardiness indexes, early-spring dry matter yield, and freezing tolerance) were screened in the diploid introgression populations (n = 203) that had some F. pratensis chromosome segments introgressed. Eighty-four intron markers corresponding to unique rice genes randomly distributed across the genome were used for genotyping. Winter hardiness of almost all plants in the introgression populations was lower than that of the F. pratensis and triploid hybrid parents, but the average was higher than that of L. perenne. A significant positive effect of F. pratensis introgression on early-spring dry matter yield was detected on chromosome 7. This quantitative trait locus (QTL) was confirmed by linkage analysis using a backcross population with F. pratensis introgression in the target region of chromosome 7. However, the contribution of the newly identified QTL was rather small (6.7–9.6%), suggesting that superior winter hardiness of F. pratensis compared to L. perenne is conferred by multiple small-effect QTLs. We also detected a previously unreported negative effect of Festuca introgression on winter hardiness. Newly obtained QTL information in this study would contribute to the design of Festuca/Lolium hybrid breeding.     

Transformation of <Emphasis Type="Italic">Campanula</Emphasis> by wild type <Emphasis Type="Italic">Agrobacterium rhizogenes</Emphasis>

Compact growth is an important quality criterion in horticulture. Many Campanula species and cultivars exhibit elongated growth which is suppressed by chemical retardation and cultural practice during production to accommodate to the consumer’s desire. The production of compact plants via transformation with wild type Agrobacterium rhizogenes is an approach with great potential to produce plants that are non-GMO. Efficient transformation and regeneration procedures vary widely among both plant genera and species. Here we present a transformation protocol for Campanula. Hairy roots were produced on 26–90% of the petioles that were used for transformation of C. portenschlagiana (Cp), a C. takesimana × C. punctata hybrid (Chybr) and C. glomerata (Cg). Isolated hairy roots grew autonomously and vigorously without added hormones. The Cg hairy roots produced chlorophyll and generated plantlets in response to treatments with cytokinin (42 µM 2iP) and auxin (0.67 µM NAA). In contrast, regeneration attempts of transformed Cp and Chybr roots lead neither to the production of chlorophyll nor to the regeneration of shoots. Agropine A. rhizogenes strains integrate split T-DNA in T_L- and T_R-DNA fragments into the plant genome. In this study, regenerated plants of Cg did not contain T_R-DNA, indicating that a selective pressure against this T-DNA fragment may exist in Campanula.     

Identification of the <Emphasis Type="Italic">S</Emphasis> locus core sequences determining self-incompatibility and <Emphasis Type="Italic">S</Emphasis> multigene family from draft genome sequences of radish (<Emphasis Type="Italic">Raphanus sativus</Emphasis> L.)

The S core and its flanking sequences were identified from two independent draft genome sequences of radish (Raphanus sativus L.). After gap-filling with PCR, the S core regions and full-length S receptor kinase (SRK) genes from two radish genomes were obtained. Phylogenetic analysis of the SRK genes clearly showed that one S core region belonged to the class I S haplotypes, but the other was included in the class II S haplotypes. Three sequences showing homology with known transposable elements were identified in the core regions, and one intact copia-type long terminal repeat (LTR)-retrotransposon containing a 4125-bp open reading frame (ORF) was identified in the class I S haplotype. A total of 61 genes showing homology with the SRK genes were identified from two draft genome sequences. Among them, the RsKD1 showed the highest homology with the SRK genes. There was 90% nucleotide sequence identity between the RsKD1 and RsSRK1 genes in the kinase domains. The phylogenetic tree of SRK genes and 13 most closely related homologs showed that all homologs were more closely related to the class II SRK genes than to the class I SRKs. Physical mapping of radish SRK-homologous genes and their B. rapa orthologs showed that two radish homologs and their B. rapa orthologs were tightly linked to the SRK genes in radish and B. rapa genomes. Sequence information about multiple SRK-homologs identified in this study would be helpful for designing reliable primer pairs for faithful PCR amplification of the SRK alleles, leading to improvement of the S haplotyping system in radish breeding programs.     

Resistance screening of breeding lines and commercial tomato cultivars for <Emphasis Type="Italic">Meloidogyne incognita</Emphasis> and <Emphasis Type="Italic">M. javanica</Emphasis> populations (Nematoda) from Ethiopia

Soil and root samples were collected from major tomato growing areas of Ethiopia during the 2012/2013 growing season to identify root-knot nematode problems. DNA-based and isozyme techniques revealed that Meloidogyne incognita and M. javanica were the predominant Meloidogyne species across the sampled areas. The aggressiveness of different populations of these species was assessed on tomato cultivars Marmande and Moneymaker. The two most aggressive populations of each species were selected and further tested on 33 tomato genotypes. The resistance screening and mechanism of resistance was performed after inoculation with 100 freshly hatched (<24 h) second-stage juveniles (J2). Eight weeks after inoculation the number of egg masses produced on each cultivar was assessed. For the resistance mechanism study, J2 penetration and their subsequent development inside the tomato roots were examined at 1, 2, 4 and 6 weeks after inoculation. On both cultivars Marmande and Moneymaker all M. incognita and M. javanica populations formed a high number of egg masses indicating highly aggressive behaviour. Populations from ‘Jittu’ and ‘Babile’ for M. incognita and ‘Jittu’ and ‘Koka’ for M. javanica were selected as most aggressive. None of the 33 tomato genotypes were immune for these M. incognita and M. javanica populations. However, several tomato genotypes were found to have a significant effect on the number of egg masses produced indicating possible resistance. For M. javanica populations there were more plants from cultivars or breeding lines on which no egg masses were found compared to M. incognita populations. The lowest number of egg masses for both populations of M. incognita was produced on cultivars Bridget40, Galilea, and Irma while for M. javanica it was on Assila, Eden, Galilea, Tisey, CLN-2366A, CLN-2366B and CLN-2366C. Tomato genotypes, time (weeks after inoculation) and their interaction were significant sources of variation for J2 penetration and their subsequent development inside the tomato roots. Differential penetration was found in breeding lines such as CLN-2366A, CLN-2366B and CLN-2366C, but many of the selected tomato genotypes resistance for the tested M. incognita and M. javanica populations were expressed by delayed nematode development. Therefore, developing a simple screening technique to be used by local farmers or extension workers is crucial to facilitate selection of a suitable cultivar.