Developing and validating microsatellite markers in elephant grass (<Emphasis Type="Italic">Pennisetum purpureum</Emphasis> S.)

Elephant grass [Pennisetum purpureum S.; syn. Cenchrus purpureus (Schumach.) Morrone] is an important global forage crop and is recognized for high yields of herbage with good nutritive value. It also has high biomass potential to be utilized as a biofuel feedstock. Whereas several previous genetic studies adapted simple sequence repeat (SSR) markers from pearl millet [Pennisetum glaucum (L.) R.Br.] for investigations in elephant grass, the present study developed SSR markers from 3536 DNA sequences derived from 16 elephant grass entries. A total of 3866 SSRs were identified including 1028 monomeric, 2019 dimeric, 735 trimeric, 49 tetrameric, 20 pentameric and 15 hexameric repeat motifs. Three hundred and seven sequences contained more than one repeated motif, and 154 SSRs were present in compound formation. Susequenctly,  four elephant grass and two pearl millet genotypes were chosen to validate 727 SSR markers. Of these, 628 markers produced visually detectable amplification products, including 73 (11.6%) polymorphic ones across all six genotypes. Polymorphism between the four elephant grass genotypes was revealed by 316 (50.6%) markers with diversity index values ranging from 0.75 to 0.38. Dimeric SSRs had the highest polymorphic rate (48.7%). These validated SSR markers had 58.6% (368 of 628) transferability rate to pearl millet. The availability of these polymorphic SSR markers will support advanced genetic studies in P. purpureum and its relatives.     

Phenotypic and genotypic screening for rust resistance in common bean germplasm in Uganda

Rust caused by Uromyces appendiculatus (Pers., Pers.) Unger is one of the major foliar diseases of common bean (Phaseolus vulgaris) in Uganda. The use of host resistance remains the best option in managing this disease. The objective of this study was to identify sources of broad-spectrum rust resistance in common bean germplasm including landraces, commercial cultivars and introduced genotypes using a combination of phenotypic and genotypic screening with 22 simple sequence repeat (SSR) markers located on chromosome Pv04. A total of 138 genotypes were field screened from 2014 and 2015 using an alpha lattice design. The variance and correlation of disease incidence, area under the disease progression curve (AUDPC) and total grain yield were computed using GenStat. The polymorphism information content of the genotypes was determined, and the association of the markers and the disease resistance traits were analyzed using PowerMarker and TASSEL respectively. Resistance of each genotype was compared to the presence and absence of amplified markers. There were highly significant differences (P < 0.001) among the genotypes for disease incidence, AUDPC and total grain yield and a strong correlation (P < 0.001) between disease incidence and AUDPC in both years. The SSR markers, BARC_PV_SSR04725, bean_ssr_0778 and bean_ssr_2892 were associated (P ≤ 0.05) with rust resistance. Fifteen 15 genotypes which included the landraces Nabufumbo, and Kapchorwa white, and the commercial cultivar NABE 2 were identified as new sources of rust resistance that would be useful in future bean breeding programmes in Uganda.     

Associating chemical analysis to molecular markers for the valorization of <Emphasis Type="Italic">Citrus aurantium</Emphasis> leaves: a useful starting point for marker-assisted selection

Variation in metabolite composition and content is often observed in citrus, however, it is poorly understood to what extent this variation has a genetic basis. C. aurantium genotypes originating from Tunisia were evaluated to detect genomic (SSR markers) and chemotypic polymorphisms and to discover possible associations between them. A total of fifteen highly polymorphic SSR markers were selected to screen the genetic variability of the most widespread sour orange genotypes. Targeted secondary metabolite profiling analysis generated twenty-one compounds differentially accumulated in the leaves of sour orange genotypes. PCA analysis revealed that genomic and chemotypic data generated similar pattern of clustering, highlighting the intra-specific variability in C. aurantium species. Both data were integrated, leading to the identification of associated SSR alleles with secondary metabolites. Based on results, a relatively high correlation (r = 0.381; p < 0.0001) between chemotypic patterns and genetic markers was identified. Associations between traits of interest for phenolic compounds and genetic markers were tested using statistical methods including three linear model approaches. These results consolidate the presence of a chemical fingerprint that may be suitable for assessing identity and quality of a particular genotype which will be very useful for citrus breeding programs.     

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.     

Overcoming unilateral incompatibility in crosses with wild allotetraploid potato species <Emphasis Type="Italic">Solanum stoloniferum</Emphasis> Schldtl. &amp; Bouchet

The germplasm of valuable for breeding wild allotetraploid potato species Solanum stoloniferum is rarely used because of pre- and postzygotic reproductive barriers with cultivated potatoes. One of the factors that complicate crosses between S. stoloniferum and S. tuberosum is unilateral incompatibility (UI). Here, we present the results of application of S. verrucosum and S _v S _v -lines for overcoming UI in crosses with S. stoloniferum and of generating male fertile hybrids derived from this species. S _v S _v -lines are F2 S. tuberosum dihaploid × S. verrucosum that are male fertile and have D/γ-type cytoplasm. Since they are homozygous for S _v gene from S. verrucosum, they were expected to have the same ability for elimination of prezygotic incompatibility as this species. Three accessions of S. verrucosum and seven S _v S _v -lines were pollinated by 26 accessions of S. stoloniferum. The crosses with S. verrucosum failed or had low efficacy (1.5–2.4 seeds per pollination). On the other hand, use of S _v S _v -lines was more efficient: 15.8 seeds per pollination. In spite of low percentage of germination (1.9%), 40 seedlings of interspecific hybrids were produced. The experiment on hybridization between S _v S _v -lines and S. stoloniferum has been reproduced with the accession PI 205522 of the wild species, which had DNA markers of PVY and LB resistance genes and W/γ cytoplasm: 950 hybrid seeds and 12 viable seedlings were produced. The genome of the seedlings was doubled by colchicine treatment, which generated hexaploids that formed highly fertile pollen and set seeds from self-pollination. We were able to cross them as females with the variety Katahdin.     

Stability of <Emphasis Type="Italic">Solanum aethiopicum</Emphasis> Shum accessions under varied water deficit stress levels and identification of pertinent breeding traits for resistance to water shortage

Drought is a major constraint to productivity of Solanum aethiopicum ‘Shum’ group due to loss in market and nutrient value of stressed plants. This study evaluated S. aethiopicum Shum group accessions to identify genotypes (G) that excel across moisture deficit stress levels (WLs). A split-plot arrangement composed of four WLs and twenty accessions of S. aethiopicum as main plot and sub-plot factors, respectively, was implemented in a screenhouse, and repeated for two experiments. In each experiment, there was a highly significant effect of at least two WLs on mean performance among at least two accessions for most of the traits at p < 0.05. Further, very highly significant WL × G interactions were obtained for leaf relative water content (LRWC), leaves per plant (LPP) and plant height (PH), and non-significant for leaf blade length and leaf blade width. The order of priority as breeding traits for stability superiority across WLs was suggested as LRWC > PH > LPP. Consequently, based on LRWC, the most superiorly stable accessions were identified as accession 160 followed by accessions 145, 137, 108P and 184G while the least stable ones were identified as accessions 163G, 141, 163 and 108. The broad sense heritability (H ²) for each of the three recommended traits for drought resistance breeding was above 0.9 thus supportive for a good response to selection. Drought stress negatively affected the performance of S. aethiopicum Shum group but the exhibited variation allowed for selection of superiorly stable genotypes.     

Genetic mapping of resistance to <Emphasis Type="Italic">Puccinia hordei</Emphasis> in three barley doubled-haploid populations

The success of breeding for barley leaf rust (BLR) resistance relies on regular discovery, characterization and mapping of new resistance sources. Greenhouse and field studies revealed that the barley cultivars Baronesse, Patty and RAH1995 carry good levels of adult plant resistance (APR) to BLR. Doubled haploid populations [(Baronesse/Stirling (B/S), Patty/Tallon (P/T) and RAH1995/Baudin (R/B)] were investigated in this study to understand inheritance and map resistance to BLR. The seedlings of two populations (B/S and R/B) segregated for leaf rust response that conformed to a single gene ratio (\({\text{X}}_{1:1}^{2}\) = 0.12, P > 0.7 for B/S and \({\text{X}}_{1:1}^{2}\) = 0.34, P > 0.5 for R/B) whereas seedlings of third population (P/T) segregated for two-gene ratio (\({\text{X}}_{1:1}^{2}\) = 0.17, P > 0.6) when tested in greenhouse. It was concluded that the single gene in Baudin and one of the two genes in Tallon is likely Rph12, whereas gene responsible for seedling resistance in Stirling is Rph9.am (allele of Rph12). The second seedling gene in Tallon is uncharacterized. In the field, APR was noted in lines that were susceptible as seedlings. A range of disease responses (CI 5–90) was observed in all three populations. Marker trait association analysis detected three QTLs each in populations B/S (QRph.sun-2H.1, QRph.sun-5H.1 and QRph.sun-6H.1) and R/B (QRph.sun-1H, QRph.sun-2H.2, QRph.sun-3H and QRph.sun-6H.2), and four QTLs in population P/T (QRph.sun-6H.2, QRph.sun-1H.2, QRph.sun-5H.2 and QRph.sun-7H) that significantly contributed to low leaf rust disease coefficients. High frequency of QRph. sun-5H.1, QRph. sun-6H.1, QRph. sun-1H.1, QRph. sun-2H.2, QRph. sun-6H.2, QRph. sun-7H (based on presence of the marker, closely associated to the respective QTLs) was observed in international commercial barley germplasm and hence providing an opportunity for rapid integration into breeding programmes. The identified candidate markers closely linked to these QTLs will assist in selecting and assembling new APR gene combinations; expectantly this will help in achieving good levels of durable resistance for controlling BLR.     

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.     

Performance of cowpea varieties under <Emphasis Type="Italic">Striga gesnerioides</Emphasis> (Willd.) Vatke infestation using biplot analysis

Striga gesnerioides (Willd) Vatke, is a major destructive parasitic weed of cowpea (Vigna unguiculata (L.) Walp.) which causes substantial yield reduction in West and Central Africa. The presence of different virulent races within the parasite population contributes to significant genotype × environment interaction, and complicates breeding for durable resistance to Striga. A 3-year study was conducted at three locations in the dry savanna agro-ecology of Nigeria, where Striga gesnerioides is endemic. The primary objective of the study was to identify cowpea genotypes with high yield under Striga infestation and yield stability across test environments and to access suitability of the test environment. Data collected on grain yield and yield components were subjected to analysis of variance (ANOVA). Means from ANOVA were subjected to the genotype main effect plus genotype × environment (GGE) biplot analysis to examine the multi-environment trial data and rank genotypes according to the environments. Genotypes, environment, and genotypes × environment interaction mean squares were significant for grain yield and yield components, and number of emerged Striga plants. The environment accounted for 35.01%, whereas the genotype × environment interaction accounted for 9.10% of the variation in grain yield. The GGE biplot identified UAM09 1046-6-1 (V7), and UAM09 1046-6-2 (V8), as ideal genotypes suggesting that these genotypes performed relatively well in all study environments and could be regarded as adapted to a wide range of locations. Tilla was the most repeatable and ideal location for selecting widely adapted genotypes for resistance to S. gesnerioides.     

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.     

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.     

Interspecific hybridization in <Emphasis Type="Italic">Sarcococca</Emphasis> supported by analysis of ploidy level,genome size and genetic relationships

Knowledge of ploidy level differences, genome size and genetic relationships between species facilitates interspecific hybridization in ornamentals. For Sarcococca (Buxaceae) only limited (cyto)genetic information is available. The aim of this study was to determine the genome size and chromosome number and to unravel the genetic relationships of a breeder’s collection using AFLP marker analysis. Based on these results, interspecific crosses were made and the efficiency and hybrid status was verified. Two groups of diploid plants (2n = 2x = 24) were observed, with either a genome size of 4.11–4.20 or 7.25–9.63 pg/2C. All the tetraploid genotypes (2n = 4x = 48) had genome sizes ranging from 7.91 to 8.18 pg/2C. In crosses between parents with equal ploidy level and genome size a higher crossing efficiency (on average 58% of the hybridizations resulting in fruits) and more true hybrids (on average 96% of the offspring) were obtained compared to crosses between plants with different genome size and ploidy level (on average 23% fruits and 24% hybrids, respectively). In none of the cross combinations, the ploidy level or genome size was found to be a complete hybridization barrier, although unilateral incongruity was found in some cross combinations. Distant genetic relationships did not hamper the hybridization within Sarcococca genotypes. Our findings will contribute to a more efficient breeding program and a faster achievement of hybrids with an added value.     

Further QTL mapping for yield component traits using introgression lines in rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.) under drought field environments

To better understand the underlying mechanisms of agronomic traits related to drought resistance and discover candidate genes or chromosome segments for drought-tolerant rice breeding, a fundamental introgression population, BC₃, derived from the backcross of local upland rice cv. Haogelao (donor parent) and super yield lowland rice cv. Shennong265 (recurrent parent) had been constructed before 2006. Previous quantitative trait locus (QTL) mapping results using 180 and 94 BC₃F_6,7 rice introgression lines (ILs) with 187 and 130 simple sequence repeat (SSR) markers for agronomy and physiology traits under drought in the field have been reported in 2009 and 2012, respectively. In this report, we conducted further QTL mapping for grain yield component traits under water-stressed (WS) and well-watered (WW) field conditions during 3 years (2012, 2013 and 2014). We used 62 SSR markers, 41 of which were newly screened, and 492 BC₄F_2,4 core lines derived from the fourth backcross between D123, an elite drought-tolerant IL (BC₃F₇), and Shennong265. Under WS conditions, a total of 19 QTLs were detected, all of which were associated with the new SSRs. Each QTL was only identified in 1 year and one site except for qPL-12-1 and qPL-5, which additively increased panicle length under drought stress. qPL-12-1 was detected in 2013 between new marker RM1337 and old marker RM3455 (34.39 cM) and was a major QTL with high reliability and 15.36% phenotypic variance. qPL-5 was a minor QTL detected in 2013 and 2014 between new marker RM5693 and old marker RM3476. Two QTLs for plant height (qPHL-3-1 and qPHP-12) were detected under both WS and WW conditions in 1 year and one site. qPHL-3-1, a major QTL from Shennong265 for decreasing plant height of leaf located on chromosome 3 between two new markers, explained 22.57% of phenotypic variation with high reliability under WS conditions. On the contrary, qPHP-12 was a minor QTL for increasing plant height of panicle from Haogelao on chromosome 12. Except for these two QTLs, all other 17 QTLs mapped under WS conditions were not mapped under WW conditions; thus, they were all related to drought tolerance. Thirteen QTLs mapped from Haogelao under WS conditions showed improved drought tolerance. However, a major QTL for delayed heading date from Shennong265, qDHD-12, enhanced drought tolerance, was located on chromosome 12 between new marker RM1337 and old marker RM3455 (11.11 cM), explained 21.84% of phenotypic variance and showed a negative additive effect (shortening delay days under WS compared with WW). Importantly, chromosome 12 was enriched with seven QTLs, five of which, including major qDHD-12, congregated near new marker RM1337. In addition, four of the seven QTLs improved drought resistance and were located between RM1337 and RM3455, including three minor QTLs from Haogelao for thousand kernel weight, tiller number and panicle length, respectively, and the major QTL qDHD-12 from Shennong265. These results strongly suggested that the newly screened RM1337 marker may be used for marker-assisted selection (MAS) in drought-tolerant rice breeding and that there is a pleiotropic gene or cluster of genes linked to drought tolerance. Another major QTL (qTKW-1-2) for increasing thousand kernel weight from Haogelao was also identified under WW conditions. These results are helpful for MAS in rice breeding and drought-resistant gene cloning.     

Estimation of genetic diversity among 34 genotypes in the genus <Emphasis Type="Italic">Cajanus</Emphasis> with contrasting host response to the pod borer and its allied pests

The genetic divergence among 34 genotypes belonging to 12 species of genus Cajanus were carried out using plant pest interaction and DNA marker analysis. Principal component analysis based on average percentage of pod damage caused by pod borer, plume moth, and blue butterfly in the field conditions, and growth of their larva and pupa on an artificial diet in vitro dispersed these genotypes into four coordinates evincing high genetic divergence as expected. DNA marker analysis using 11 pairs of SSR and nine ISSR primers showed higher polymorphism at the species level, and these primers exhibited variation with regard to average band informativeness, resolving power, and PIC value. No single primer was able to distinguish between all the 34 genotypes of Cajanus but nine species specific amplified fragments were generated by five ISSR primers. The pairwise Jaccard’s similarity coefficient and Nei’s genetic distance values revealed a higher level of inter-specific genetic variation in the genus Cajanus. The clustering of genotypes based on Jaccard’s similarity coefficient vis-a-vis Nei’s genetic distance agreed with the sectional classification of the genus Cajanus. Seven cultivars of C. cajan and the genotypes of their wild progenitor C. cajanifolius remained in one cluster, whereas accessions of C. platycarpus and C. scarabaeoides were out grouped. The rest of the genotypes belonging to nine species of Cajanus formed another cluster. The principal coordinate analysis also supported this clustering pattern. Moreover, these findings have good many implications for future breeding endeavors aimed at the introgression of pod borer resistance alleles.     

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.     

Induced expression of selected plant defence related genes in pot azalea, <Emphasis Type="Italic">Rhododendron simsii</Emphasis> hybrid

A set of putative marker genes to study plant defense responses against Polyphagotarsonemus latus, a key pest in the production of Rhododendron simsii hybrids, was selected and validated. Genes belonged to the biosynthetic pathway of phytohormones jasmonic acid (JA) (RsLOX, RsAOS, RsAOC, RsOPR3 and RsJMT) and salicylic acid (SA) (RsPAL and RsICS). Furthermore, RsPPO, a putative marker gene for oxidative stress response was successfully cloned from R. simsii. A CTAB-based extraction protocol was optimized to assure excellent RNA quality for subsequent RT-qPCR analysis. The RT-qPCR protocol was extensively tested and RsRG7 and RsRG14 were selected as reference genes from a geNorm pilot study. Validation of the marker genes was done after application with elicitors [methyl jasmonate (MeJA), coronatine, β-aminobutyric acid and acibenzolar-Smethyl] or wounding. Both 100 μM MeJA and 0.1 μM coronatine had a significant effect on the expression of all marker genes. Foliar application of MeJA on the shoots resulted in a significantly earlier response when compared to root application and subsequent sampling of the shoots. Expression patterns after MeJA treatment were generally the same in six R. simsii genotypes: ‘Nordlicht’, ‘Elien’, ‘Aiko Pink’, ‘Michelle Marie’, ‘Mevrouw Gerard Kint’ and ‘Sachsenstern’. Wounding resulted in the same expression patterns as MeJA treatment except for RsJMT. None of the genotypes showed a significant induction of the latter gene 6 h upon wounding. Findings of these experiments indicated that the tolerant genotype ‘Elien’ has low basal expression levels of RsPPO. This might be the first step towards the breeding of mite-tolerant genotypes.     

Selection of pea genotypes with partial resistance to <Emphasis Type="Italic">Sclerotinia sclerotiorum</Emphasis> across a wide range of temperatures and periods of high relative humidity

Partial resistance to Sclerotinia sclerotiorum based on stem lesion advancement was assessed for nine wild pea genotypes from five geographic origins and two cultivated genotypes, when peas were inoculated and incubated at all combinations of five temperatures (15.6, 18.3, 21.1, 23.9, 29.4°C) and four period(s) of high relative humidity (PHRH; 12, 24, 48, 72 h). PHRH of 12 and 24 h should not be used when screening plants for resistance to S. sclerotiorum regardless of the incubation temperature, since stem lesions are rarely (2.7%) visible at 12 h and there were no significant differences (P ≤ 0.05) in lesion lengths among and within genotypes at all temperatures assessed after 24 h. However, PHRH of 48 and 72 h are recommended for use to assess partial resistance since significant differences in stem lesion length among the genotypes were observed and characterized for these periods. Genotypes with cool (15.6 and 18.3°C) versus warm (23.9 and 29.4°C) temperature partial resistance to S. sclerotiorum were identified, and genotypes PI 240515 and PI 169603 appear to have the best cool and warm temperature partial resistance, respectively, among the genotypes assessed. A temperature of 21.1°C was the optimal temperature favouring lesion advancement for the majority of the genotypes evaluated. PI 169603 demonstrated the best partial resistance to S. sclerotiorum across the widest temperature and PHRH ranges and is recommended to plant breeders as the best single genotype to develop future cultivars with improved partial resistance to S. sclerotiorum based on stem lesion advancement.     

Development of a core set of SSR markers for the characterization of <Emphasis Type="Italic">Gossypium</Emphasis> germplasm

Molecular markers such as simple sequence repeats (SSR) are a useful tool for characterizing genetic diversity of Gossypium germplasm. Genetic profiles by DNA fingerprinting of cotton accessions can only be compared among different collections if a common set of molecular markers are used by different laboratories and/or research projects. Herein, we propose and report a core set of 105 SSR markers with wide genome coverage of at least four evenly distributed markers per chromosome for the 26 tetraploid cotton chromosomes. The core marker set represents the efforts of ten research groups involved in marker development, and have been systematically evaluated for DNA polymorphism on the 12 genotypes belonging to six Gossypium species [known collectively as the cotton marker database (CMD) panel]. A total of 35 marker bins in triplex sets were arranged from the 105 markers that were each labeled with one of the three fluorescent dyes (FAM, HEX, and NED). Results from this study indicated that the core marker set was robust in revealing DNA polymorphism either between and within species. Average value of polymorphism information content (PIC) among the CMD panel was 0.65, and that within the cultivated cotton species Gossypium hirsutum was 0.29. Based on the similarity matrix and phylogenetic analysis of the CMD panel, the core marker set appeared to be sufficient in characterizing the diversity within G. hirsutum and other Gossypium species. The portability of this core marker set would facilitate the systematic characterization and the simultaneous comparison among various research efforts involved in genetic diversity analysis and germplasm resource preservation.     

Identification and fine mapping of molecular markers closely linked to fruit spines size <Emphasis Type="Italic">ss</Emphasis> gene in cucumber (<Emphasis Type="Italic">Cucumis sativus</Emphasis> L.)

Fruit spine size is one of the importantly external quality traits effected the economic value of cucumber fruit. Morphological–cytological observation of the fruit spine size phenotype indicated that large spine formation arises from an increasing of spiny pedestal cell number caused by cell division, and best periods to accurately score fruit spine size trait was 4th day before flowering to 7th day after flowering according the continuous observation. Genetic analysis showed that a single dominant gene determined the fruit spine size trait in cucumber. BC₁ population (189 individuals) of two inbred lines (large spine PI197088 and small spine SA0422) was used for primary mapping of the SS/ss locus with 7 markers covering an interval of 37.1 cM. An F₂ segregating population of 1032 individuals constructed from the same two parents (PI197088 and SA0422) was used to fine mapping of the SS/ss locus. Six new markers linked to the gene were successfully screened for construction of a fine linkage map, in which the SS/ss locus was located in the region flanked by marker SE1 (3 recombinants) and SSR43 (2 recombinants) with a 189 kb physical distance. Markers from this study will be valuable for candidate gene cloning and marker-assisted selection for cucumber breeding.