Chitinases of <Emphasis Type="Italic">Coffea arabica</Emphasis> genotypes resistant to orange rust <Emphasis Type="Italic">Hemileia vastatrix</Emphasis>

Two Coffea arabica — Hemileia vastatrix incompatible interactions (I₁: coffee cv. Caturra — rust race VI and I₂: coffee cv S4 Agaro — rust race II) and a compatible interaction (coffee cv. Caturra — rust race II) were compared in relation to the infection process and chitinase activity. In the two incompatible interactions the fungus ceased growth in the early infection stages, while in the compatible interaction no fungus growth inhibition was observed. A high constitutive level of chitinase activity was detected in the intercellular fluid of healthy leaves. Upon infection, chitinase isoforms were more abundant in incompatible interactions than in the compatible interaction. Immunodetection showed that class I chitinases are particularly relevant in the incompatible interactions and might participate in the defence response of the coffee plants.     

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

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

Histology of somatic embryogenesis in <Emphasis Type="Italic">Coffea arabica</Emphasis> L.

The objective of this study was to characterize the histodifferentiation of somatic embryogenesis obtained from leaf explants of C. arabica. Therefore, we histologically analyzed the respective stages of the process: leaf segments at 0, 4, 7, 15 and 30 days of cultivation, Type 1 primary calli (primary calli with embryogenic competence) and 2 (primary calli with no embryogenic competence), embryogenic calli, globular, torpedo and cotyledonary embryos, and mature zygotic embryos. Callus formation occurred after seven days of culture, with successive divisions of procambium cell. In this cultivation phase, it was found that Type 1 primary calli are basically formed by parenchymal cells with reduced intercellular spacing, whereas Type 2 primary calli are predominantly composed of parenchymal cells with ample intercellular spaces and embryogenic calli composed entirely of meristematic cells. After 330 days, it was evident from the differentiation of somatic embryogenesis that there was formation of globular somatic embryos, consisting of a characteristic protoderm surrounding the fundamental meristem. With the maturation of these propagules after 360 days, torpedo-stage somatic embryos arose, in which tissue polarization and early differentiation of procambial strands were verified. After 390 days, cotyledonary somatic embryos were obtained, where the onset of vessel elements differentiation was verified, a characteristic also observed in mature zygotic embryos. We concluded that somatic embryogenesis obtained from C. arabica leaves initiates from procambium cell divisions that, in the course of cultivation, produce mature somatic embryos suitable for regenerating whole plants.     

Introgression of a leaf rust resistance gene from <Emphasis Type="Italic">Aegilops caudata</Emphasis> to bread wheat

Rusts are the most important biotic constraints limiting wheat productivity worldwide. Deployment of cultivars with broad spectrum rust resistance is the only environmentally viable option to combat these diseases. Identification and introgression of novel sources of resistance is a continuous process to combat the ever evolving pathogens. The germplasm of nonprogenitor Aegilops species with substantial amount of variability has been exploited to a limited extent. In the present investigation introgression, inheritance and molecular mapping of a leaf rust resistance gene of Ae. caudata (CC) acc. pau3556 in cultivated wheat were undertaken. An F(2) population derived from the cross of Triticum aestivum cv. WL711 - Ae. caudata introgression line T291-2 with wheat cultivar PBW343 segregated for a single dominant leaf rust resistance gene at the seedling and adult plant stages. Progeny testing in F(3) confirmed the introgression of a single gene for leaf rust resistance. Bulked segregant analysis using polymorphic D-genome-specific SSR markers and the cosegregation of the 5DS anchored markers (Xcfd18, Xcfd78, Xfd81 and Xcfd189) with the rust resistance in the F(2) population mapped the leaf rust resistance gene (LrAC) on the short arm of wheat chromosome 5D. Genetic complementation and the linked molecular markers revealed that LrAC is a novel homoeoallele of an orthologue Lr57 already introgressed from the 5M chromosome of Ae. geniculata on 5DS of wheat.     

Influence of triacontanol on somatic embryogenesis in <Emphasis Type="Italic">Coffea arabica</Emphasis> L. and <Emphasis Type="Italic">Coffea canephora</Emphasis> P. ex Fr.

Summary A highly reproducible method for regeneration of Coffea arabica and C. canephora plants via direct somatic embryogenesis from cultured leaf and stem segments of regenerated plants was developed. Embryogenesis was influenced by the presence of triacontanol (TRIA) in the medium. TRIA incorporated at 4.55 and 11.38 μM in half-strength MS basal medium containing 1.1 μM 6-benzyladenine (BA) and 2.28 μM indole-3-acetic acid (IAA) induced direct somatic embryogenesis in both species. A maximum of 260±31.8 and 59.2±12.8 somatic embryos per culture were induced from in vitro leaf explants of C. arabica and C. canephora, respectively. TRIA also induced embryo formation from in vitro stem segment callus tissues along with multiplication of primary embryos into secondary embryos. By using TRIA, it was possible to obtain somatic embryogenesis in C. arabica and C. canephora.     

Genetic mapping of a barley leaf rust resistance gene <Emphasis Type="Italic">Rph26</Emphasis> introgressed from <Emphasis Type="Italic">Hordeum bulbosum</Emphasis>

Key message

The quantitative barley leaf rust resistance gene, Rph26, was fine mapped within a H. bulbosum introgression on barley chromosome 1HL. This provides the tools for pyramiding with other resistance genes.

Abstract

A novel quantitative resistance gene, Rph26, effective against barley leaf rust (Puccinia hordei) was introgressed from Hordeum bulbosum into the barley (Hordeum vulgare) cultivar ‘Emir’. The effect of Rph26 was to reduce the observed symptoms of leaf rust infection (uredinium number and infection type). In addition, this resistance also increased the fungal latency period and reduced the fungal biomass within infected leaves. The resulting introgression line 200A12, containing Rph26, was backcrossed to its barley parental cultivar ‘Emir’ to create an F₂ population focused on detecting interspecific recombination within the introgressed segment. A total of 1368 individuals from this F₂ population were genotyped with flanking markers at either end of the 1HL introgression, resulting in the identification of 19 genotypes, which had undergone interspecific recombination within the original introgression. F₃ seeds that were homozygous for the introgressions of reduced size were selected from each F₂ recombinant and were used for subsequent genotyping and phenotyping. Rph26 was genetically mapped to the proximal end of the introgressed segment located at the distal end of chromosome 1HL. Molecular markers closely linked to Rph26 were identified and will enable this disease resistance gene to be combined with other sources of quantitative resistance to maximize the effectiveness and durability of leaf rust resistance in barley breeding. Heterozygous genotypes containing a single copy of Rph26 had an intermediate phenotype when compared with the homozygous resistant and susceptible genotypes, indicating an incompletely dominant inheritance.

     

Molecular mapping of leaf rust resistance gene <Emphasis Type="Italic">Rph14</Emphasis> in <Emphasis Type="Italic">Hordeum vulgare</Emphasis>

An incompletely dominant gene conferring resistance to Puccinia hordei, Rph14, identified previously in an accession of Hordeum vulgare, confers resistance to all known pathotypes of P. hordei in Australia. Knowledge of the chromosomal location of Rph14 and the identification of DNA markers closely linked to it will facilitate combining it with other important leaf rust resistance genes to achieve long lasting resistance. The inheritance of Rph14 was confirmed using 146 and 106 F₃ lines derived from the crosses ‘Baudin’/‘PI 584760’ (Rph14) and ‘Ricardo’/‘PI 584760’ (Rph14), respectively. Bulk segregant analysis on DNA from the parental genotypes and resistant and susceptible DNA bulks using DArT markers located Rph14 to the short arm of chromosome 2H. DArT marker bPb-1664 was identified as having the closest genetic association with Rph14. PCR based marker analysis identified a single SSR marker, Bmag692, linked closely to Rph14 at a map distance of 2.1 and 3.8 cm in the ‘Baudin’/‘PI 584760’and ‘Ricardo’/‘PI 584760’ populations, respectively.     

Comparison of the <Emphasis Type="Italic">Coffea canephora</Emphasis> and <Emphasis Type="Italic">C. arabica</Emphasis> karyotype based on chromosomal DNA content

Nuclear genome size has been measured in various plants, seeing that knowledge of the DNA content is useful for taxonomic and evolutive studies, plant breeding programs and genome sequencing projects. Besides the nuclear DNA content, tools and protocols to quantify the chromosomal DNA content have been also applied, expanding the data about genomic structure. This study was conducted in order to calculate the Coffea canephora and Coffea arabica chromosomal DNA content, associating cytogenetic methodologies with flow cytometry (FCM) and image cytometry (ICM) tools. FCM analysis showed that the mean nuclear DNA content of C. canephora and C. arabica is 2C = 1.41 and 2.62 pg, respectively. The cytogenetic methodology provided prometaphase and metaphase cells exhibiting adequate chromosomes for the ICM measurements and karyogram assembly. Based on cytogenetic, FCM and ICM results; it was possible to calculate the chromosomal DNA content of the two species. The 1C chromosomal DNA content of C. canephora ranged from 0.09 (chromosome 1) to 0.05 pg (chromosome 11) and C. arabica from 0.09 (chromosome 1) to 0.03 pg (chromosome 22). The methodology presented in this study was suitable for DNA content measuring of each chromosome of C. canephora and C. arabica. The cytogenetic characterization and chromosomal DNA content analyses evidenced that C. arabica is a true allotetraploid originated from a cross between Coffea diploid species. Besides, the same analyses also reinforce that C. canephora is a possible progenitor of C. arabica.     

Direct somatic embryogenesis from <Emphasis Type="Italic">Coffea arabica</Emphasis> L. and <Emphasis Type="Italic">Coffea canephora</Emphasis> P ex Fr. under the influence of ethylene action inhibitor-silver nitrate

For the first time direct somatic embryogenesis from hypocotyl explants of in vitro regenerated plantlets of C. arabica and C. canephora was achieved on modified MS medium containing 10 – 70 μM silver nitrate supplemented with 1.1 μM N⁶ benzyladenine and 2.85 μM indole-3-acetic acid. A maximum of 144.1±7.3 and 68.7±3.3 embryos per explant were produced at 40 μM silver nitrate in C. canephora and C. arabica respectively. Only yellow friable embryogenic callus obtained from the cut edges of most of leaf explants of both C. arabica and C. canephora at all concentrations of silver nitrate were tried in this experiment. Formation of secondary embryos from stage I primary embryos (small yellow, round, globular embryos) was more (28.23±1.3) at 60 μM silver nitrate in C. canephora, while 40 μM silver nitrate supported more of secondary embryo formation in C. arabica (40.5±1.2). When stage II (green globular round matured embryos) and stage III primary embryos (tubular stage embryos) were used, secondary embryo formation was very small and many of these embryos developed into plantlets and some of them even rooted. By using these protocols within 45 – 60 days it is possible to get secondary embryos from primary embryos and direct somatic embryos from hypocotyls of in vitro plantlets in both these Coffea species.     

Impact of the Coffea canephora gene introgression on beverage quality of <Emphasis Type="Italic">C. arabica</Emphasis>

Lines of Coffea arabica derived from the Timor Hybrid (hybrid between C. arabica and C. canephora) are resistant to coffee leaf rust (Hemileia vastatrix) and to the nematode Meloidogyne exigua. The introgression of C. canephora resistance genes is suspected of causing a drop in beverage quality. Coffee samples from pure lines, compared in a Trial 1, and from F1 hybrids and parental lines from a half-diallel trial in a Trial 2, were studied for beverage quality, chemical composition and amount of introgressed genetic material. Chemical analyses (caffeine, chlorogenic acids, fat, trigonelline, sucrose) were carried out with near-infrared spectrometry by reflectance of green coffee. The number of amplified fragment length polymorphic (AFLP) markers introgressed from the Timor Hybrid varied from 1 to 37 for the lines studied. There were significant differences between lines for all of the biochemical compounds analysed and for the acidity and the overall standard of the beverage. Two lines (T17927, T17924) were significantly poorer than the controls for sucrose and beverage acidity. T17924 also had more chlorogenic acids and was poorer for the overall standard. However, two highly introgressed lines, T17934 and T17931 (25 and 30 AFLP markers, respectively), did not differ from the non-introgressed controls. There were no correlations between the number of AFLP markers and the chemical contents or beverage attributes. Significant correlations were found between the performance of the parents and their general combining ability for beverage quality. It was concluded that it should be possible to find lines with both the desired resistance genes and good beverage quality. Selection can avoid accompanying the introgression of resistance genes with a drop in beverage quality.     

Transgenic coffee fruits from <Emphasis Type="Italic">Coffea arabica</Emphasis> genetically modified by bombardment

The genetic modification of Coffea arabica fruits is an important tool for the investigation of physiological characteristics and functional validation of genes related to coffee bean quality traits. In this work, plants of C. arabica cultivar Catuaí Vermelho were successfully genetically modified by bombardment of embryogenic calli. Calli were obtained from 90% of the leaf explants cultivated in a callogenesis-inducing medium modified with 20 μM 2,4-dichlorophenoxyacetic acid (2,4-D). The resulting calli were bombarded with the pBI426 vector containing a uidA and nptII gene fusion that was driven by the double CaMV35s promoter. Kanamycin-selected embryos were positive for β-glucuronidase (GUS) activity in histochemical assays and for target gene amplification by polymerase chain reaction. Integration of the nptII gene was confirmed by Southern blot and showed a low copy number (one to three) of insertions. Transformed plants showed normal development and settled fruits. GUS expression was assessed in the flower and fruit organs demonstrating the capacity of the double CaMV35s promoter to drive long-term stable expression of uidA in C. arabica fruit tissues. Moreover, we obtained a T₁ progeny presenting 3:1 Mendelian segregation of the uidA gene. This investigation is the first to report exogenous gene expression in coffee fruits and transgenic inheritance in C. arabica plants.     

Characterisation of a new stripe rust resistance gene <Emphasis Type="Italic">Yr47</Emphasis> and its genetic association with the leaf rust resistance gene <Emphasis Type="Italic">Lr52</Emphasis>

Two Iranian common wheat landraces AUS28183 and AUS28187 from the Watkins collection showed high levels of seedling resistance against Australian pathotypes of leaf rust and stripe rust pathogens. Chi-squared analyses of rust response segregation among F₃ populations derived from crosses of AUS28183 and AUS28187 with a susceptible genotype AUS27229 revealed monogenic inheritance of leaf rust and stripe rust resistance. As both genotypes produced similar leaf rust and stripe rust infection types, they were assumed to carry the same genes. The genes were temporarily named as LrW1 and YrW1. Molecular mapping placed LrW1 and YrW1 in the short arm of chromosome 5B, about 10 and 15 cM proximal to the SSR marker gwm234, respectively, and the marker cfb309 mapped 8–12 cM proximal to YrW1. LrW1 mapped 3–6 cM distal to YrW1 in two F₃ populations. AUS28183 corresponded to the accession V336 of the Watkins collection which was the original source of Lr52. Based on the genomic location and accession records, LrW1 was concluded to be Lr52. Because no other seedling stripe rust resistance gene has previously been mapped in chromosome 5BS, YrW1 was permanently named as Yr47. A combination of flanking markers gwm234 and cfb309 with phenotypic assays could be used to ascertain the presence of Lr52 and Yr47 in segregating populations. This investigation characterised a valuable source of dual leaf rust and stripe rust resistance for deployment in new wheat cultivars. Transfer of Lr52 and Yr47 into current Australian wheat backgrounds is in progress.     

Molecular mapping of stripe rust resistance gene <Emphasis Type="Italic">YrHu</Emphasis> derived from <Emphasis Type="Italic">Psathyrostachys huashanica</Emphasis>

Wheat stripe rust is a destructive disease that affects most wheat-growing areas worldwide. Resistance genes from related species and genera add to the genetic diversity available to wheat breeding programs. The stripe rust-resistant introgression line H9020-17-25-6-4 was developed from a cross of resistant Psathyrostachys huashanica with the susceptible wheat cultivar 7182. H9020-17-25-6-4 is resistant to all existing Chinese stripe rust races, including the three most widely virulent races, CYR32, CYR33, and V26. We attempted to characterize this new line by genomic in situ hybridization (GISH) and genetic analysis. GISH using P. huashanica genomic DNA as a probe indicated that the translocated segment was too small to be detected. Genetic analysis involving F₁, F₂, and F_2:3 materials derived from a cross of Mingxian 169 and H9020-17-25-6-4 indicated that a single dominant gene from H9020-17-25-6-4, temporarily designated YrHu, conferred resistance to CYR29 and CYR33. A genetic map consisting of four simple sequence repeat, two sequence-tagged site (STS), and two sequence-related amplified polymorphism markers was constructed. YrHu was located on the short arm of chromosome 3A and was about 0.7 and 1.5 cM proximal to EST-STS markers BG604577 and BE489244, respectively. Both the gene and the closely linked markers could be used in marker-assisted selection.     

Genetic mapping of stem rust resistance gene <Emphasis Type="Italic">Sr13</Emphasis> in tetraploid wheat (<Emphasis Type="Italic">Triticum turgidum</Emphasis> ssp. <Emphasis Type="Italic">durum</Emphasis> L.)

Wheat stem rust caused by Puccinia graminis f. sp. tritici, can cause significant yield losses. To combat the disease, breeders have deployed resistance genes both individually and in combinations to increase resistance durability. A new race, TTKSK (Ug99), identified in Uganda in 1999 is virulent on most of the resistance genes currently deployed, and is rapidly spreading to other regions of the world. It is therefore important to identify, map, and deploy resistance genes that are still effective against TTKSK. One of these resistance genes, Sr13, was previously assigned to the long arm of chromosome 6A, but its precise map location was not known. In this study, the genome location of Sr13 was determined in four tetraploid wheat (T. turgidum ssp. durum) mapping populations involving the TTKSK resistant varieties Kronos, Kofa, Medora and Sceptre. Our results showed that resistance was linked to common molecular markers in all four populations, suggesting that these durum lines carry the same resistance gene. Based on its chromosome location and infection types against different races of stem rust, this gene is postulated to be Sr13. Sr13 was mapped within a 1.2–2.8 cM interval (depending on the mapping population) between EST markers CD926040 and BE471213, which corresponds to a 285-kb region in rice chromosome 2, and a 3.1-Mb region in Brachypodium chromosome 3. These maps will be the foundation for developing high-density maps, identifying diagnostic markers, and positional cloning of Sr13.     

<Emphasis Type="Italic">Pm37</Emphasis>, a new broadly effective powdery mildew resistance gene from <Emphasis Type="Italic">Triticum </Emphasis><Emphasis Type="Italic">timopheevii</Emphasis>

Powdery mildew is an important foliar disease in wheat, especially in areas with a cool or maritime climate. A dominant powdery mildew resistance gene transferred to the hexaploid germplasm line NC99BGTAG11 from T. timopheevii subsp. armeniacum was mapped distally on the long arm of chromosome 7A. Differential reactions were observed between the resistance gene in NC99BGTAG11 and the alleles of the Pm1 locus that is also located on chromosome arm 7AL. Observed segregation in F_2:3 lines from the cross NC99BGTAG11 × Axminster (Pm1a) demonstrate that germplasm line NC99BGTAG11 carries a novel powdery mildew resistance gene, which is now designated as Pm37. This new gene is highly effective against all powdery mildew isolates tested so far. Analyses of the population with molecular markers indicate that Pm37 is located 16 cM proximal to the Pm1 complex. Simple sequence repeat (SSR) markers Xgwm332 and Xwmc790 were located 0.5 cM proximal and distal, respectively, to Pm37. In order to identify new markers in the region, wheat expressed sequence tags (ESTs) located in the distal 10% of 7AL that were orthologous to sequences from chromosome 6 of rice were targeted. The two new EST-derived STS markers were located distal to Pm37 and one marker was closely linked to the Pm1a region. These new markers can be used in marker-assisted selection schemes to develop wheat cultivars with pyramids of powdery mildew resistance genes, including combinations of Pm37 in coupling linkage with alleles of the Pm1 locus.     

Inheritance and mapping of powdery mildew resistance gene <Emphasis Type="Italic">Pm43</Emphasis> introgressed from <Emphasis Type="Italic">Thinopyrum</Emphasis><Emphasis Type="Italic">intermedium</Emphasis> into wheat

Powdery mildew resistance from Thinopyrum intermedium was introgressed into common wheat (Triticum aestivum L.). Genetic analysis of the F₁, F₂, F₃ and BC₁ populations from powdery mildew resistant line CH5025 revealed that resistance was controlled by a single dominant allele. The gene responsible for powdery mildew resistance was mapped by the linkage analysis of a segregating F₂ population. The resistance gene was linked to five co-dominant genomic SSR markers (Xcfd233, Xwmc41, Xbarc11, Xgwm539 and Xwmc175) and their most likely order was Xcfd233–Xwmc41–Pm43–Xbarc11–Xgwm539–Xwmc175 at 2.6, 2.3, 4.2, 3.5 and 7.0 cM, respectively. Using the Chinese Spring nullisomic-tetrasomic and ditelosomic lines, the polymorphic markers and the resistance gene were assigned to chromosome 2DL. As no powdery mildew resistance gene was previously assigned to chromosome 2DL, this new resistance gene was designated Pm43. Pm43, together with the identified closely linked markers, could be useful in marker-assisted selection for pyramiding powdery mildew resistance genes. Runli He and Zhijian Chang contributed equally to this work.     

SNP in the <Emphasis Type="Italic">Coffea arabica</Emphasis> genome associated with coffee quality

Association analysis was performed at the whole genome level to identify loci affecting the caffeine and trigonelline content of Coffea arabica beans. DNA extracted from extreme phenotypes was bulked (high and low caffeine, and high and low trigonelline) based on biochemical analysis of the germplasm collection. Sequencing and mapping using the combined reference genomes of C. canephora and C. eugenioides (CC and CE) identified 1351 non-synonymous SNPs that distinguished the low- and high-caffeine bulks. Gene annotation analysis with Blast2GO revealed that these SNPs corresponding to 908 genes with 56 unique KEGG pathways and 49 unique enzymes. Based on KEGG pathway-based analysis, 40 caffeine-associated SNPs were discovered, among which nine SNPs were tightly associated with genes encoding enzymes involved in the conversion of substrates (i.e. SAM, xanthine and IMP) which participate in the caffeine biosynthesic pathways. Likewise, 1060 non-synonymous SNPs were found to distinguish the low- and high-trigonelline bulks. They were associated with 719 genes involved in 61 unique KEGG pathways and 51 unique enzymes. The KEGG pathway-based analysis revealed 24 trigonelline-associated SNPs tightly linked to genes encoding enzymes involved in the conversion of substrates (i.e. SAM, L-tryptophan) which participate in the trigonelline biosynthesis pathways. These SNPs could be useful targets for further functional validation and subsequent application in arabica quality breeding.