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.     

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.     

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.     

Mapping of new resistance (<Emphasis Type="Italic">Vr2</Emphasis>, <Emphasis Type="Italic">Rm1</Emphasis>) and ornamental (<Emphasis Type="Italic">Di2</Emphasis>, <Emphasis Type="Italic">pl</Emphasis>) Mendelian trait loci in peach

Peach powdery mildew is one of the major diseases of the peach. Various sources of resistance to PPM have thus been identified, including the single dominant locus Vr2 carried by the peach rootstock ‘Pamirskij 5’. To map Vr2, a linkage map based on microsatellite markers was constructed from the F₂ progeny (WP²) derived from the cross ‘Weeping Flower Peach’ × ‘Pamirskij 5’. Self-pollinations of the parents were also performed. Under greenhouse conditions, all progenies were scored after artificial inoculations in two classes of reactions to PPM (resistant/susceptible). In addition to Vr2, WP² segregated for three other traits from ‘Weeping Flower Peach’: Rm1 for green peach aphid resistance, Di2 for double-flower and pl for weeping-growth habit. With their genomic locations unknown or underdocumented, all were phenotyped as Mendelian characters and mapped: Vr2 mapped at the top of LG8, at 3.3 cM, close to the CPSCT018 marker; Rm1 mapped at the bottom of LG1, at a position of 116.5 cM, cosegregating with the UDAp-467 marker and in the same region as Rm2 from ‘Rubira’^®; Di2 mapped at 28.8 cM on LG6, close to the MA027a marker; and pl mapped at 44.1 cM on LG3 between the MA039a and SSRLG3_16m46 markers. Furthermore, this study revealed, for the first time, a pseudo-linkage between two traits of the peach: Vr2 and the Gr locus, which controls the red/green color of foliage. The present work therefore constitutes a significant preliminary step for implementing marker-assisted selection for the four major traits targeted in this study.     

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.     

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.     

Complementary DNA (cDNA) cloning and functional verification of resistance to head smut disease (<Emphasis Type="Italic">Sphacelotheca reiliana</Emphasis>) of an NBS–LRR gene <Emphasis Type="Italic">ZmNL</Emphasis> in maize (<Emphasis Type="Italic">Zea mays</Emphasis>)

The nucleotide-binding site (NBS)-leucine-rich repeat (LRR) gene family comprises the largest number of known disease resistance (R) genes and is one of the largest gene families in plants. In the present study, the full-length cDNA of ZmNL (GenBank Accession Number KF765443) was isolated using Rapid Amplification of cDNA Ends. The nucleotide sequence of ZmNL contains an open reading frame of 3156 bp that encodes the ZmNL protein, which is comprised of 1051 amino acid residues. This putative protein has high homology to other known resistance proteins (84% to Triticum aestivum LR10) and belongs to the CC–NBS–LRR type R gene family. The ZmNL gene was introduced into the maize inbred line of Huangzao4 which was highly susceptible to head smut under the control of the maize ubiquitin promoter by Agrobacterium-mediated transformation. The head smut disease incidence of 3 T₂ transgenic lines was significantly reduced (by 18.38–29.40%) compared with the wild type, which indicated that the overexpression of ZmNL gene in maize enhanced the resistance to the fungus Sporisorium reilianum (Kühn) Clint of these plants.     

Resistance to the cabbage root fly, <Emphasis Type="Italic">Delia radicum</Emphasis> (Diptera,Anthomyiidae), of turnip varieties (<Emphasis Type="Italic">Brassica rapa</Emphasis> subsp. <Emphasis Type="Italic">rapa</Emphasis>)

The cabbage root fly Delia radicum L. (Diptera: Anthomyiidae) is one of the major pests of many Brassica crops in the temperate areas of Europe and North America. At present, turnip (B. rapa ssp. rapa L.) varieties resistant to the pest does not exist. With the aim to fill this gap, a no-choice tolerance test of 56 accessions among turnips, turnip tops and turnip greens was performed under controlled conditions by introducing D. radicum eggs. Plant survival, leaf and root conditions, pupae number and weight significantly varied among plant accessions. Ten putatively resistant and ten susceptible accessions (control group) were selected from this first screening, transplanted in the field and exposed to natural infestation to detect antibiosis and antixenosis mechanisms. Both in the laboratory and in the field, pupae number significantly varied within accessions and between resistant and susceptible group, although pupal weight did not, indicating the absence of antibiosis effect on this early stage. In the field, the number of galleries was significantly lower in the resistant group in comparison with the control. Resistant accessions had smaller size, and a smaller, white and mostly buried root. Within the resistant and susceptible accessions, larger plants harboured more pupae, however purple roots were those most preferred, and the hosted pupae weighed most. Three accessions from the resistant group (MBGBR0178, MBGBR0570 and MBGBR0371) stand out for resistance to D. radicum possibly through antixenosis mechanisms.     

Generation of interspecific hybrids between <Emphasis Type="Italic">Trifolium vesiculosum</Emphasis> and <Emphasis Type="Italic">T. alexandrinum</Emphasis> using embryo rescue

Interspecific hybrids were developed between Trifolium alexandrinum cultivar Wardan × Trifolium vesiculosum and T. alexandrinum cultivar BL1 × T. vesiculosum through embryo rescue, as the crosses failed to set seed under natural conditions. Trifolium vesiculosum was used as a donor/male parent in this study as it is reported to possess tolerance to stem rot and high forage yield. Fertilization in crossed florets of the crosses was manifested from the recovery of swollen ovaries (< 7.80%) and confirmed from the presence of one degenerated ovule in most (> 93.00%) of the swollen ovaries. The hybrid embryos at various developmental stages (heart, torpedo and cotyledonary) were rescued at a frequency of 2.56% from Wardan × T. vesiculosum and 6.12% from BL1 × T. vesiculosum. Differentiation occurred only in the cotyledonary stage embryos, resulting in 17 putative interspecific hybrid plantlets. The assessment of plantlet hybridity through SSR markers (for the alleles inherited from the donor parent), micromorphological leaf traits (leaf texture and stomata) and morphological characters (plant height, leaflet length and width) confirmed production of two interspecific hybrids designated as AV1 and BV3 representing both the crosses. AV1 displayed moderate resistance and BV3 was resistant to stem rot.     

Genetic effect of <Emphasis Type="Italic">Striga</Emphasis> resistance in sorghum genotypes

Striga is an important parasitic weed causing substantial economic losses in cereal and legume crop production in sub-Saharan Africa. Integrated Striga management approaches such as a combined use of Striga resistant varieties and Fusarium oxysporum f.sp. strigae (FOS), a biocontrol agent of Striga, are an option to control the parasite and to boost sorghum productivity. Understanding host gene action influencing Striga resistance, with or without FOS treatment, is key to developing improved sorghum varieties with durable resistance and high yield. The objective of this study was to determine the gene action and inheritance of Striga resistance using genetically diverse populations of sorghum involving FOS treatment. Twelve sorghum parents selected for Striga resistance, FOS compatibility or superior agronomic performances were crossed using a bi-parental mating scheme. The selected male and female parents and their F₁ progenies, backcross derivatives and the F₂ segregants were field evaluated at three locations in Tanzania known for their severe Striga infestations using a lattice experimental design with two replications. The following data were collected and subjected to generation mean analysis (GMA): days-to-50% flowering (DFL), seed yield per plant (SYP) and number of Striga per plant (SN). GMA showed the preponderance of additive genetic action contributing to the total genetic variation in the evaluated sorghum populations. The additive genetic effect for DFL, SYP and SN, with and without FOS treatments, ranged from 72.02 to 86.65% and 41.49 to 95.44%, 75.62 to 91.42% and 71.83 to 91.89%, and 77.35 to 93.56% and 72.86 to 95.84%, in that order. The contribution of non-additive genetic effects was minimal and varied among generations. FOS application reduced DFL and SN and improved SYP in most of the tested sorghum populations. DFL of sorghum populations was reduced by a mean of 8 days under FOS treatment compared to the untreated control in families such as 675 × 654, AS435 × AS426 and 1563 × AS436. FOS treatment improved SYP with a mean of 6.44 g plant^?1 in 3424 × 3993 and 3984 × 672. The numbers of Striga plants were reduced with a mean of 16 plants due to FOS treatment in the crosses of 675 × 654, 1563 × AS436, 4567 × AS424, and 3984 × 672. The study demonstrated that additive genes were predominantly responsible for the inheritance of Striga resistance in sorghum. Pure line cultivar development targeting reduced DFL, SN and high SYP in the selected populations may provide enhanced response to selection for integrated Striga management (ISM) programme.     

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.     

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.     

Fine mapping of <Emphasis Type="Italic">Ren3</Emphasis> reveals two loci mediating hypersensitive response against <Emphasis Type="Italic">Erysiphe necator</Emphasis> in grapevine

Grapevine (Vitis vinifera L.) is economically very important for the production of wine, table grapes and raisins. However, grapevine is threatened by a brought range of pathogens. A destructive disease worldwide is powdery mildew caused by the ascomycete Erysiphe necator. In the grapevine cultivar `Regent’ a resistance locus against E. necator, Ren3, was previously reported. It spans an interval of approximately seven Mb on chromosome 15. We attempted to delimit this interval to facilitate its further molecular analysis. New simple sequence repeat markers targeted to the Ren3 region were designed. They were applied for fine mapping in the cross populations of ‘Regent’ × ‘Lemberger’ and ‘Regent’ × ‘Cabernet Sauvignon’ that segregate for E. necator resistance. Complementarily we scored E. necator infection levels of ‘Regent’ × ‘Lemberger’ progeny at different time points over the course of the vegetation period in 2015 and 2016. Subsequent QTL analysis revealed a maximum LOD value that shifted during the season from marker GF15-10 located at 2.2 Mb to marker GF15-53 located at 3.5 Mb and to marker ScORA7* located at 9.4 Mb on chromosome 15 (positions according to the grapevine reference genome of PN40024). To investigate the Ren3-encoded resistance mechanism we performed detached leaf infection assays for microscopic studies. These revealed that Ren3 carrying individuals react with a hypersensitive response. Results of detached leaf assays on recombinants in the Ren3 locus indicate that not only one, but two distinct genetic regions on chromosome 15 mediate hypersensitive response against E. necator.     

The sporophytic self-incompatibility mating system is conserved in <Emphasis Type="Italic">Olea europaea</Emphasis> subsp. <Emphasis Type="Italic">cuspidata and O. e. europaea</Emphasis>

Fruit setting after self-pollination, crosses and free-pollination appears to be erratic in the cultivated olive tree [Olea europaea subsp europaea L. (O. e. europaea L.)] because of a lack of a suitable model to enable prediction of rates. The same lack of prediction also applies to the wild taxon Olea europaea subsp cuspidata (O. e. cuspidata). Because of their close phylogenetic relationships, we hypothesize that O. e. cuspidata and cultivated olive share the same self-incompatibility system. We used data recently published in a wide study involving four O. e. cuspidata accessions and four olive cultivars. Because the olive varieties have been deciphered for their S-allele pair, that infer determinants present in the stigma and pistil, and that coat the pollen, we deciphered the S-alleles carried by three of the O. e. cuspidata accessions. Data are too scarce and the number of accessions too small to speculate on the O. e. cuspidata genetic population structure. The working hypothesis is confirmed. This study and data from the Italian team will enable us to embark on a large-scale hybridization program between the two subsp. to obtain a wide range of progenies for screening for responses to cold, diseases and pests.     

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.     

Fine mapping the <Emphasis Type="Italic">BjPl1</Emphasis> gene for purple leaf color in B2 of <Emphasis Type="Italic">Brassica juncea</Emphasis> L. through comparative mapping and whole-genome re-sequencing

Purple plants with higher anthocyanin content have attracted increasing attention in recent years due to their advantageous biological functions and nutritional value. A spontaneous mutant with purple leaves, designated 1280-1, was discovered in Brassica juncea line 1280. A previous genetic analysis indicated that the purple leaf trait in 1280-1 was controlled by a dominant gene (BjPl1). In the present study, an analysis of total anthocyanin content further indicated that the purple leaf trait was controlled by a complete dominance gene. According to a survey of 426 primers available from public resources, BjPl1 was assigned to linkage group B2 of B. juncea. In the early stage of this research, based on comparative mapping in Brassica, two simple sequence repeat (SSR) markers developed from A2 of B. rapa delimited the BjPl1 gene to a 0.7-cM genetic interval in the corresponding linkage map. According to information on the B. juncea genome released recently, the location of BjPl1 was further narrowed to a 225-kb interval (17.74–17.97 Mb). Within the target region, whole-genome re-sequencing identified two candidate regions (17.74–17.78 Mb and 17.93–17.96 Mb). Through Blast analysis of the two candidate intervals, four homologous anthocyanin biosynthetic genes were identified and localized to a 17.93–17.96 Mb interval of B2 (approximately 27 kb), which might include BjPl1. This work lays the foundation for the isolation of BjPl1 and will further improve our understanding of the molecular mechanisms of the anthocyanin metabolic pathway in Brassica.     

Development and trait evaluation of chromosome single-segment substitution lines of <Emphasis Type="Italic">O. meridionalis</Emphasis> in the background of <Emphasis Type="Italic">O. sativa</Emphasis>

O. meridionalis is a wild species belonging to AA genome in the Oryza genus, which has a lot of beneficial genes for improvement of cultivated rice. In the present study, 99 chromosome single-segment substitution lines (SSSLs) were developed carrying donor segments of O. meridionalis in the genetic background of an indica cultivar, Huajingxian 74 (HJX74). The total lengths of the 99 substituted segments in the SSSLs were 1580.16 cM, with an average length of 15.11 cM per substituted segment, covering 873.94 cM and 54.98% of O.meridionalis genome. Phenotypic investigations of the SSSLs showed that three SSSLs had red pericarp, awn and showed seed shattering, respectively, indicating that these genes of O. meridionalis responsible for these traits have been transferred to the SSSLs. And wide variations were observed in seven quantitative traits including heading date and yield-related traits in 82 SSSLs.At P ≤ 0.001, 77 SSSLs showed significant differences compared with HJX74 in at least one trait either in the fall of 2014 or spring of 2015, and a total of 28 stable QTLs were detected in 24 SSSLs in both seasons. These results suggest that the SSSLs library of O. meridionalis developed in this study offers a good germplasm platform for the identification and transformation of beneficial genes of O. meridionalis, and facilitates the conservation of gene resources of O. meridionalis in vivo for long periods.     

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.     

Wild <Emphasis Type="Italic">Coffea arabica</Emphasis> resistant to <Emphasis Type="Italic">Meloidogyne paranaensis</Emphasis> and genetic parameters for resistance

Arabica coffee production is based on highly productive cultivars; however, these cultivars are susceptible to infestation by several biotic agents, including root-knot nematodes. Collections of wild Coffea arabica germplasm represent an important source of genetic variability for resistant cultivar development. In this study, 1046 plants derived from 71 wild coffee trees were evaluated with respect to Meloidogyne paranaensis resistance. In addition to information on plants reactions, we also evaluated the genetic parameters related to resistance. Progenies from the five most promising plants were also evaluated regarding resistance to M. incognita and M. exigua. The yield potential of selected plants was estimated through analysis of data for fruits harvested in 4 different years. Forty-seven plants were considered resistant based on reproduction factor values. The estimated heritability was high for all analyzed variables leading to substantial selection gain, mainly at the progeny mean level. On the basis of heritabilities and genetic correlations, we conclude that selection could be performed based on values of the gall and egg mass index. However, higher genetic gain could be obtained based on nematode count variables. A second experiment confirmed the reactions of the selected five plants to M. paranaensis, and multiple resistance was detected in three of them. The resistant accessions also have yield potential.