Inheritance and relationship between key agronomic and quality traits in an interspecific cross between Coffea pseudozanguebariae Bridson and C. canephora Pierre

The inheritance and relationships between four traits of agronomic and quality interest??fructification time, caffeine, and heteroside contents and 100-bean weight??were analyzed in the first backcross hybrids derived from an interspecific cross between Coffea pseudozanguebariae and Coffea canephora. We showed that short vs. long fructification time was governed by one major gene with two co-dominant alleles ft1 and ft2. Absence vs. presence of both caffeine and heteroside was also controlled by one major gene. The allele responsible for the presence of caffeine (caf2) dominated over the absence one (caf1) whereas both alleles controlling heteroside, het1 and het2, were co-dominant. The fructification time and the heteroside content were additive while the caffeine content seemed multiplicative. The 100-bean weight was additive and under a polygenic control. The two genes ft and caf were linked, separated by 30.8?cM, and were independent from the het gene. The relationships between the four traits were not strong enough, except between 100-bean weight and fructification time (r?=?0.43) or caffeine content (r?=?0.41). Recombination occurred between the genes controlling the four traits suggesting that new introgressed Robusta varieties, characterized by short, medium, or long fructification time depending on demand, bigger seeds with low or no caffeine content, and being heteroside-free, could be produced.     

Genetic investigations on the caffeine and chlorogenic acid relationship in an interspecific cross between Coffea liberica dewevrei and C. pseudozanguebariae

The objective of the paper was to identify the number of major loci explaining caffeine content in coffee seeds. Investigations were based on previously published results: (1) Caffeine binds to chlorogenic acids in a 1:1 molecular ratio; (2) Between species, the caffeine content is correlated to the chlorogenic acid content; (3) Only a part of chlorogenic acids is bound to caffeine. Especially, the content ratio between caffeine and chlorogenic acids varied between species. For identifying the number of major loci, a quantitative trait locus (QTL) approach was carried out using an interspecific cross between two highly differentiated species—Coffea liberica dewevrei and Coffea pseudozanguebariae, the latter being a caffeine-free species. As main finding, two QTLs, i.e., RCQ1 and CQA1, were identified allowing us to explain up to 97 % of the caffeine content variance. RCQ1 explained variation of the caffeine/chlorogenic acid ratio and was genetically independent of the second QTL. The latter explained the part of the caffeine content which was dependent on the chlorogenic acid content. The findings also confirmed that only a part of chlorogenic acids were trapped by caffeine, as in wild species.     

Assisted-selection of naturally caffeine-free coffee cultivars—characterization of SNPs from a methyltransferase gene

Breeding of caffeine-free coffee cultivars require tools for an early selection of progenies bearing this later trait. Genes from caffeine synthesis and degradation represent major targets for the development of molecular markers for assisted selection. In this study, we characterized SNPs identified on the caffeine synthase gene from AC1 mutant, a naturally caffeine-free arabica coffee plant. Molecular analysis of normal and mutant sequences indicates the occurrence of SNPs in protein domains, potentially associated with caffeine synthesis in coffee. Progenies F₂, F₁BC₁ and BC from crosses of AC mutants and elite cultivars were evaluated regarding caffeine content in grains and genomic segregation profile of selected SNPs. Genotyping analysis allowed the discrimination between homozygous and heterozygous plants. Quantification of caffeine content indicated a significant variability among progenies and a low frequency of caffeine-free plants. Statistical analyses of genotyping and phenotyping results showed significant association between presence of selected SNPs and reduced caffeine content. Moreover, this association occurs through all evaluated genetic backgrounds and generations, indicating an inheritance stability of both trait and markers. The molecular markers described here represent a successful case of assisted-selection in coffee, indicating their potential use for breeding of caffeine-free cultivars.     

Relationship between parental chromosomic contribution and nuclear DNA content in the coffee interspecific hybrid C. pseudozanguebariae×C. liberica var ‘dewevrei’

 F₁ hybrids were obtained between two coffee species with the same chromosome number (2n=22) but with different nuclear DNA contents [C. pseudozanguebariae (PSE) 2C=1.13 pg and C. liberica var ‘dewevrei’ (DEW) 2C=1.42 pg]. G2 hybrids were obtained by open-pollination of the F₁ hybrids. Genomic in situ hybridisation (GISH) and flow cytometry were used on six F₁ hybrids and seven G2 hybrids to determine their parental chromosomic contribution and their nuclear DNA content (qDNA), respectively. GISH efficiently identified chromosomes from both species. F₁ hybrids had a qDNA intermediate between that of the parental species and contained the expected 11 chromosomes from each species. There was a linear relationship between the number of PSE chromosomes and the nuclear DNA content, which indicates that flow cytometry can be used to give a rough estimate of the parental chromosomic contribution in G2 hybrids. Received: 1 August 1997/Accepted: 25 August 1997     

Untersuchungen über den Transport von Kaffein in der Kaffeepflanze (Coffea arabica)

Summary Caffeine transport in small amounts from leaf to leaf was demonstrated by application of doubly-labelled caffeine. There was no translocation from the subtending leaves into the fruits, but heavy translocation from the pericaprp into the seed tissue could be shown. The results were confirmed by the analysis of grafts between caffeine-containing and caffeine-free species. The importance of the pericarp with respect to the caffeine content of the mature coffee bean is discussed.

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Diese Untersuchungen wurden vom Schweizerischen Nationalfonds für wissenschaftliche Forschung unterstützt. Fräulein Lotti Brugger danken wir für die gewissenhafte Mitarbeit.     

Trigonelline inheritance in the interspecific Coffea pseudozanguebariae×C. liberica var. dewevrei cross

Trigonelline alkaloid is present in coffee beans, and during roasting it gives rise to the major coffee aroma compounds (several alkyl-pyridines and pyrroles). In this study we investigated the genetic inheritance of trigonelline accumulation in green beans in an interspecific cross between a wild east African species, Coffea pseudozanguebariae (PSE) and the west African species C. liberica var. dewevrei (DEW). Trigonelline content was measured by HPLC in both parental species, F₁ hybrids and the reciprocal backcross hybrids (BCDEW and BCPSE). The results showed that, on average, PSE accumulated twice as much trigonelline as DEW. No year effect or interaction (genotype×year) was recorded. Trigonelline showed high heritability (71%), which meant that the genotypic value could be easily estimated from the phenotypic value. However, the fact that this trait was not additive suggested the possibility of nucleo-cytoplasmic inheritance. This hypothesis was confirmed by: (1) similar levels of trigonelline content in the PSE, F₁, BCPSE and BCDEW groups, all having the same maternal cytoplasm, and (2) the location of one nuclear QTL on the G linkage group. Received: 2 May 2000 / Accepted: 19 June 2000     

Relations between and inheritance of chlorogenic acid contents in an interspecific cross between Coffea pseudozanguebariae and Coffea liberica var ‘dewevrei’

 Chlorogenic acids (CGA) are phenolic compounds commonly found in green coffee beans. The main CGA classes are caffeoylquinic acids (CQA), dicaffeoylquinic acids (diCQA), and feruloylquinic acids (FQA). Each contains three isomers differing in the number and identity of the acylating residues. An interspecific cross between Coffea pseudozanguebariae (low CGA content) and C. liberica var ‘dewevrei’ (high CGA content) was investigated for CGA contents in F₁ and back-cross hybrids. Relations within and between CGA classes were studied and confirmed the known biosynthesis pathway. A single major gene was noted for the 3-FQA isomer; absence was dominant. Additivity was found for most other isomers either with or without the transformation of variables. Conversely, most ratios were not additive, due to a curvilinear relation between some isomers. The consequences for breeding both in terms of cup taste improvement and disease resistance are discussed. Received: 20 July 1998 / Accepted: 14 August 1998     

Induced responses of Coffea arabica to attack of Coccus viridis stimulate locomotion of the herbivore

The green scale, Coccus viridis (Green) (Hemiptera: Coccidae), is an insect pest of coffee and several other perennial cultivated plant species. We investigated changes in alkaloid and phenolic contents in coffee plants as a response to herbivory by this insect. Greenhouse‐grown, 11‐month‐old coffee plants were artificially infested with the coccid and compared with control, uninfested plants. Leaf samples were taken at 15, 30, 45, and 60 days after infestation, and high‐performance liquid chromatography was used to identify and quantify alkaloid and phenolic compounds induced by the coccids at each sampling date. Of the compounds investigated, caffeine was the main coffee alkaloid detected in fully developed leaves, and its concentration in infested plants was twice as high as in the control plants. The main coffee phenolics were caffeic and chlorogenic acid, and a significant increase in their concentrations occurred only in plants infested by C. viridis. A positive and significant relationship was found between alkaloid and phenolic concentrations and the infestation level by adults and nymphs of C. viridis. Caffeine and chlorogenic acid applied on coffee leaves stimulated the locomotory activity of the green scale, thus reducing their feeding compared to untreated leaves. This is the first study to show increased levels of coffee alkaloids and phenolics in response to herbivory by scale insects. The elevation of caffeine and chlorogenic acid levels in coffee leaves because of C. viridis infestation seems to affect this generalist insect by stimulating the locomotion of crawlers.     

Occurrence of theobromine synthase genes in purine alkaloid-free species of <Emphasis Type="Italic">Camellia</Emphasis> plants

Caffeine (1,3,7-trimethylxanthine) and theobromine (3,7-dimethylxanthine) are purine alkaloids that are present in high concentrations in plants of some species of Camellia. However, most members of the genus Camellia contain no purine alkaloids. Tracer experiments using [8-¹⁴C]adenine and [8-¹⁴C]theobromine showed that the purine alkaloid pathway is not fully functional in leaves of purine alkaloid-free species. In five species of purine alkaloid-free Camellia plants, sufficient evidence was obtained to show the occurrence of genes that are homologous to caffeine synthase. Recombinant enzymes derived from purine alkaloid-free species showed only theobromine synthase activity. Unlike the caffeine synthase gene, these genes were expressed more strongly in mature tissue than in young tissue. The nucleotide sequence data reported here have been deposited in the GenBank database under the accession numbers AB297451 (CjCS1), AB362882 (CgCS1), AB362883 (CgCS2), AB362884 (CkCS1), AB362885 (ClCS1), and AB362886 (CcCS2).     

Biotechnological applications for the improvement of coffee (Coffea arabica L.)

Summary The important advances in coffee biotechnological techniques which have been made particularly during the last 10yr could benefit the coffee breeder in practice and open new perspectives for the development of new varieties. The molecular phylogeny of Coffea species has been established using DNA sequence data. The molecular markers have revealed an extremely reduced genetic diversity in Coffea arabica L. in comparison to C. canephora. However, wild accessions collected in the Ethiopian highlands appeared to constitute a valuable gene reservoir. A complete genetic linkage map of C. canephora was reported and additional ones are being constructed, particularly on C. arabica. The integration of Molecular Assisted Selection in coffee breeding promises to drastically increase the efficiency of breeding programs. Economically important genes of the caffeine biosynthetic pathway or genes encoding for seed storage proteins have been isolated. The high performance already achieved in the in vitro propagation process by somatic embryogenesis offers the possibility to mass propagate superior hybrids in different countries of both C. arabica (selected F₁ hybrids) and C. canephora (rootstock variety). Pilot productions by somatic embryogenesis currently permit preparation for commercial application. Somaclonal variation was observed. The percentage of the off-types can vary between 3 and 10% depending on the genotype. Seed cryopreservation enables a routine use for long-term conservation of coffee genetic resources. Transgenic plants have been obtained for the C. arabica and C. canephora cultivated species through Agrobacterium-mediated transformation which constitutes the technique now currently used to transfer directly genes in coffee plants.     

Molecular diagnosis of ochratoxinogenicAspergillus species

Toxigenic and non-toxigenic black aspergilli belonging to theAspergillus niger aggregate and toA. carbonarius were compared to each other and to strains of other species by DNA fingerprinting. AFLPs showed a clear separation ofA. niger andA. carbonarius. However, no clear correlation between the genetic similarity of the strains and the ability to produce ochratoxin A (OTA) was detected. Based on AFLP, marker sequences were chosen for the construction of SCAR-PCR primers for the detection ofA. carbonarius. A similar approach was used forA. ochraceus, another fungus of concern regarding ochratoxin A contamination of coffee. Cluster analysis ofA. ochraceus isolates mainly from Brazilian coffee showed a very close genetic similarity. Three species specific primer pairs were developed and one of these was used for the PCR and realtime PCR (RT-PCR) based detection of the mould in green coffee.A. ochraceus was specifically and rapidly detected and quantified in green coffee. A positive correlation between the amount of DNA and OTA content was established.     

Dissection of a major QTL for photoperiod sensitivity in rice: its association with a gene expressed in an age-dependent manner

 A proposed major quantitative trait locus (QTL) for photoperiod sensitivity on chromosome 6 in rice was examined by introducing a chromosomal segment from a sensitive line into an insensitive one. The crossing experiments showed that a range of variation in heading date occurred in the later generations and that the region might contain at least a major gene and two additional recessive genes controlling photoperiod sensitivity. Gene mapping experiments showed that the major gene was Se-1 and that a recessive gene (tentatively named se-pat) was loosely linked to it. The responses to photoperiods were examined among the different genotypes under natural and controlled conditions. The two genes acted additively on the degree of photoperiod sensitivity. However, se-pat plants showed a response to photoperiods that differed from that of the other sensitive lines; a short-day treatment at the seedling stage delayed heading in the former plants, suggesting that the manner of its expression was age-dependent. A recessive gene similar to se-pat seemed to be widely distributed in wild and cultivated rice, suggesting that the gene complex in the region plays a significant role in response to photoperiod. Received: 8 October 1997 / Accepted: 1 April 1998     

Use of various coffee industry residues for the cultivation of Pleurotus ostreatus in solid state fermentation

Studies were carried out to evaluate the feasibility of using coffee industry residues, viz. coffee husk, coffee leaves and spent coffee ground as substrates in solid state fermentation (SSF) to cultivate edible mushrooms Pleurotus. Eight strains of Pleurotus ostreatus and two strains of Pleurotus sajor‐caju were screened on a medium prepared from aqueous extract of coffee husk and agar. Based on best mycelial growth (9.68 mm/day) and biomass production (43.4 mg/plate in 9 days at 24°C), the strain P. ostreatus LPB 09 was selected for detailed studies. SSF was carried out using these substrates under different moisture conditions (45–75%) and spawn rates (2.5–25%). In general, although a 25% spawn rate appeared superior, the 10% spawn rate was recommended for all the three substrates in view of the process economics, as there was not any significant difference in the increase with 10 to 15%. The ideal moisture content for mycelial growth was 60–65% for coffee husk and spent coffee ground, and 60–70% for coffee leaves. The biological efficiency (BE), which is defined as the ratio of the weight of fresh fruiting bodies to the weight of dry substrate, multiplied by 100, and which indicates the fructification ability of the fungus for utilizing the substrate, was best with coffee husk. With coffee husk as the substrate, the first fructification occurred after 20 days of inoculation, and the biological efficiency reached about 97% after 60 days. When coffee leaves were used as the substrate, no fructification was observed even upon prolonged cultivation. With spent ground as the substrate, the first fructification occurred 23 days after inoculation and the biological efficiency reached about 90% in 50 days. There was a significant decrease in the caffeine and tannin contents (61 and 79%, respectively) of coffee husk after 60 days. It was remarkable to observe that caffeine was adsorbed onto the fruiting body (0.157%), indicating that it was not completely degraded by the fungal culture. However, no tannins were found in the fruiting body, indicating that the fungal strain was capable of degrading them. The results showed the feasibility of using coffee husk and spent coffee ground as substrates without any pre‐treatment for the cultivation of edible fungi in SSF, and provided one of the first steps towards an economical utilization of these otherwise unutilized or poorly utilized residues.     

Advances in coffee tissue culture and its practical applications

Genetic improvement of coffee, an important commercial crop, through classical breeding is slow and cumbersome. Biotechnology offers alternative strategies for generating new and improved coffee varieties, including those resistances to environmental extremes, pests, and diseases, low in caffeine, and with uniform fruit maturation. Large improvements in somatic embryogenesis, development of haploids, and scale-up of micropropagation have been accomplished in the last 5 yr. The recent identification of expressed sequence tags (EST) that are differentially expressed during the infestation of coffee plants by coffee leaf miners and the isolation and cloning of the promoter for the N-methyltransferase gene associated with caffeine production open up the possibility of producing varieties of coffee with new traits. This review provides a summary of in vitro biological advances and directions as to how they could be applied to improve the production and quality of coffee.     

Biotechnological management of coffee pulp — isolation,screening, characterization,selection of caffeine-degrading fungi and natural microflora present in coffee pulp and husk

A total of 272 strains of filamentous fungi were isolated from soil, leaves of coffee plants and coffee cherries collected in coffee-growing areas of Mexico on three semi-synthetic culture media containing coffee extract, coffee extract with sucrose and coffee pulp extract. The isolated strains were purified by conventional techniques and identified by microscopic examination. Strains were selected on the basis of their caffeine-degrading ability in well-defined liquid medium containing caffeine. Most of the isolated microorganisms belong to Aspergillus, Penicillium, Trichoderma, Fusarium, and Humicola genera. Five strains belonging to Aspergillus species and two strains belonging to Penicillium species had the ability to degrade almost 100% of the caffeine in liquid medium. A comparative study on the evaluation of natural microflora present in coffee pulp and coffee husk revealed the presence of a wide variety of microorganisms. The percentage distribution of fungi, bacteria and yeast was almost similar in all the samples, except in coffee husk where the fungal population was slightly higher than in the other two samples. The yeast population was predominant when the coffee pulp was lyophilized immediately after pulping. However, there was a wide diversity in the microbial population with respect to selective media containing functional nutritional groups like cellulose, starch and pectin.     

Effect of primary culture medium composition on high frequency somatic embryogenesis in different Coffea species

High frequency somatic embryogenesis can be obtained over a 7–8 month culture period when using current routing coffee tree micropropagation protocols. To reduce this response time and improve the embryo formation yield, eight different media were tested for primary culture. These media differed from the classically used ones by their mineral nitrogen and plant growth regulator concentrations. An increase from 0.66 to 0.75 in the NO₃/NO₃+NH₄ ratio and a 2-fold lower plant growth regulator concentration in the primary culture medium led to substantial improvements in terms of rapidity and embryo/plantlet regeneration frequencies. Embryo development time was reduced by up to 3 months with a 5-fold increase in the number of formed embryos. These results were obtained for the two cultivated coffee tree species, Coffea canephora and C. arabica, and for a wild one, C. heterocalyx, but not for a second wild species, C. sp. Moloundou showing a species-specific␣response. The new conditions described in this paper led to a substantial enhancement that should be particularly helpful for clonal propagation and genetic engineering of cultivated coffee plants.     

CaXMT和TCS1突变体基因串联共表达及体外酶活检测分析

咖啡碱和可可碱是茶叶生物碱的主要组分,且咖啡碱是茶叶重要的滋味物质,随着咖啡碱在食品和药物领域的应用愈发广泛,咖啡碱的生物合成成为新的研究热点.目前市场上的咖啡碱主要靠化学合成,为了探索其生物合成途径,该研究将咖啡黄嘌呤核苷甲基转移酶(coffee xanthosine methyltransferase,CaXMT)基因和茶树咖啡碱合成酶(tea caffeine synthase,TCS1)基因的4个突变体分别串联至同一大肠杆菌表达载体pMAL-c5X,诱导融合蛋白共表达,并进行SDS-PAGE凝胶电泳分析.结果表明:目的蛋白成功表达后,应用超声破碎法制备含有目的蛋白的粗酶液,添加底物黄嘌呤核苷(xanthosine,XR)和甲基供体S-腺苷甲硫氨酸(S-adenosyl-L-methionine,SAM)进行体外酶促反应,将反应产物进行高效液相色谱检测.检测结果显示,pMAL-CaXMT-TM2/3/4的体外酶促反应产物仅有可可碱生成,均未见咖啡碱生成.该研究结果为构建生物合成咖啡碱和可可碱的串联共表达载体奠定了基础,也为进一步研究生物合成咖啡碱和可可碱提供了新思路.     

Intra- and interspecific variations in nuclear parameters of two closely related species of Narcissus L.

Nuclear DNA amount, nuclear area, genome volume and karyotype length were analysed in different populations of two closely related species of Narcissus. There are intra- and interspecific variations in these parameters. 4C DNA amount and karyotype length, on one hand, and 2C DNA amount and telophase nuclear area, on the other, are not correlated. It seems that DNA content and chromosome length are independent parameters. However, 4C DNA content and karyotype volume are correlated, and are also correlated to different density estimations (4C DNA to Kar.length & 2C DNA to telophase area). These facts suggest that the relative length of the chromosomes is genetically controlled and that it is independent of the DNA that they contain. It seems that the interpopulational differences in DNA content are correlated with length changes of small segments in almost all chromosomes.