Molecular analysis of introgressive breeding in coffee (Coffea arabica L.)

Nineteen arabica coffee introgression lines (BC₁F₄) and two accessions derived from a spontaneous interspecific cross (i.e. Timor Hybrid) between Coffea arabica (2n=4x=44) and C. canephora (2n=2x=22) were analysed for the introgression of C. canephora genetic material. The Timor Hybrid-derived genotypes were evaluated by AFLP, using 42 different primer combinations, and compared to 23 accessions of C. arabica and 8 accessions of C. canephora. A total of 1062 polymorphic fragments were scored among the 52 accessions analysed. One hundred and seventy-eight markers consisting of 109 additional bands (i.e. introgressed markers) and 69 missing bands distinguished the group composed of the Timor Hybrid-derived genotypes from the accessions of C. arabica. AFLP therefore seemed to be an extremely efficient technique for DNA marker generation in coffee as well as for the detection of introgression in C. arabica. The genetic diversity observed in the Timor Hybrid-derived genotypes appeared to be approximately double that in C. arabica. Although representing only a small proportion of the genetic diversity available in C. canephora, the Timor Hybrid obviously constitutes a considerable source of genetic diversity for arabica breeding. Analysis of genetic relationships among the Timor Hybrid-derived genotypes suggested that introgression was not restricted to chromosome substitution but also involved chromosome recombinations. Furthermore, the Timor Hybrid-derived genotypes varied considerably in the number of AFLP markers attributable to introgression. In this way, the introgressed markers identified in the analysed arabica coffee introgressed genotypes were estimated to represent from 9% to 29% of the C. canephora genome. Nevertheless, the amount of alien genetic material in the introgression arabica lines remains substantial and should justify the development of adapted breeding strategies. Received: 2 February 1999 / Accepted: 12 May 1999     

Distribution of fungicide-derived copper in soils,litter and vegetation of different aged stands of coffee (Coffea arabica L.) in Kenya

Summary Samples of soils, litter and vegetation, collected from 24, 14 and 4 years old stands of coffee (Coffea arabica L.), which had been routinely treated with copper fungicides, were analysed for copper content. Total soil copper content was found to be a function of (a) stand age and (b) soil depth. Highest levels were encountered in the 0–20 cm zone of the 24 year old stand; soil collected beneath these bushes showed significant contamination to a depth of 80 cm. Soils from the younger stands showed proportionally less copper contamination. Copper levels in litter samples showed increases relative to stand age; litter from the oldest stand was heavily contaminated and leaf litter showed a higher copper content than that of the woody fraction. Tissues from coffee bushes could be divided into 2 groups on the basis of their copper content; those tissues which demonstrated a progressive age-dependent increase (bark, lateral stems and fine roots) and those tissues where copper levels did not reflect the cumulative exposure of the plant to this element (wood, different age classes of foliage).     

Aluminium differentially modifies lipid metabolism from the phosphoinositide pathway in Coffea arabica cells

The effect of aluminium (Al) on phosphoinositide-specific phospholipase C (PLC) and lipid kinase activities was examined in a cellular suspension of coffee. Two main effects were seen when cells were treated with AlCl3. In periods as short as 1 minute, Al-exposed cells increased the activity of PLC and IP3 formation up to two fold. Over longer periods PLC activity was inhibited by more than 50%. The activity of phosphatidylinositol 4-kinase (Pl 4-K), phosphatidylinositol phosphate 5-kinase (PIP 5-K) and diacylglycerol kinase increased when cells were incubated in the presence of different concentrations of AlCl3. The present study reports for the first time that Al may have different effects on the Pl-signaling pathway depending on the time of exposure. Our results strongly support the hypothesis that Al disrupts the metabolism of membrane phospholipids regulating not only PLC but also other enzymes that have key roles in signal-transduction pathways.     

Differential gene expression during somatic embryogenesis in Coffea arabica L., revealed by RT-PCR differential display

Molecular and biochemical studies of somatic embryogenesis may help to shed light on the mechanisms governing this phenomenon. In this article, a differential display analysis approach was employed to investigate the changes taking place during the induction of somatic embryogenesis in leaf explants and suspension cultures of coffee. Cloned fragments show homologies to several proteins reported in databases, but only one has previously been described as regulated during somatic embryogenesis. By a reverse dot blot modification, the expression pattern of such fragments was evaluated.     

Plant regeneration from protoplasts of embryogenic cell suspensions of Coffea arabica L. cv. caturra

Coffee plants were regenerated from protoplasts isolated from embryogenic cell suspension cultures derived from somatic embryos of Coffea arabica L. cv. caturra. Yields of viable protoplasts ranged from 1×10⁵ to 6×10⁵ protoplast/g fresh weight. Protoplast preparations usually contained no contaminating cells, and when present, the number of cells never exceeded 0.1% of the total. Plating efficiencies of protoplast ranged from 1 to 10%. Embryogenic protocolonies obtained after several subcultures in a medium supplemented with 0.5 mg/l each of benzylaminopurine, 2,4-dichlorophenoxyacetic acid and naphtaleneacetic acid, were transferred to a medium lacking plant growth regulators. Well differentiated embryos were formed in selected protocolonies that contained many embryos-like structures. Approximately 70% of the somatic embryos developed into green rooted plantlets which were succesfully transferred to vessels containing sterilized scoria. Plants grown for two months in scoria were finally transferred to greenhouse.Abbreviations B5 medium according to Gamborg et. al.(1968) - BAP 6-benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - NAA naphtaleneacetic acid     

Inter-simple sequence repeat (ISSR) analysis of genetic variation of Chondrus crispus populations from North Atlantic

ISSR analysis was used to investigate genetic variations of 184 haploid and diploid samples from nine North Atlantic Chondrus crispus Stackhouse populations and one outgroup Yellow Sea Chondrus ocellatus Holmes population. Twenty-two of 50 primers were selected and 163 loci were scored for genetic diversity analysis. Genetic diversity varied among populations, percentage of polymorphic bands (PPB) ranged from 27.0 to 55.8%, H (Nei's genetic diversity) ranged from 0.11 to 0.20 and I (Shannon's information index) ranged from 0.16 to 0.30. Estimators PPB, H and I had similar values in intra-population genetic diversity, regardless of calculation methods. Analysis of molecular variance (AMOVA) apportioned inter-population and intra-population variations for C. crispus, showing more genetic variance (56.5%) occurred in intra-population, and 43.5% variation among nine populations. The Mantel test suggested that genetic differentiation between nine C. crispus populations was closely related with geographic distances (R = 0.78, P = 0.002). Results suggest that, on larger distance scale (ca. >1000 km), ISSR analysis is useful for determining genetic differentiations of C. crispus populations including morphologically inseparable haploid and diploid individuals.     

Genetic diversity among geographically separated populations of Nepenthes mirabilis

The distribution of Nepenthes mirabilis ranges from Northeast (NE) to South (S) Thailand. Eleven individuals from NE, S and Suen Jatujak market in Bangkok, Central (C) Thailand, were collected and divided into four populations according to their geographical areas. These four populations were analyzed to determine a genetic diversity profile using thirteen inter-simple sequence repeat markers. The individuals produced 75.18% polymorphic banding profiles. The Shannon’s index was used to estimate genetic diversity. Total genetic diversity (H _T) and inter-population genetic diversity (H _S) were 0.854 and 0.678, respectively. The degree of genetic differentiation (G _ST) of the species populations is 0.206, whereas the gene flow (Nm) among all the various geographical area populations is 1.016. Both the dendrogram and the results of the Shannon’s diversity index suggest great genetic diversity. These results support the broad range of distribution sites of Nepenthes mirabilis, which would require high genetic diversity to adapt to the environmental variations that can be found between NE, C, and S Thailand. ANOVA shows that the genetic diversity in each population is not significantly different (P > 0.05). Mantel tests reveal that geographical distance is an important factor for affecting the genetic distances among populations.     

Somatic embryogenesis and regeneration of plantlets in protoplast cultures from somatic embryos of coffee (Coffea canephora P. ex Fr.)

Somatic embryogenesis was achieved in protoplast cultures of coffee. Protoplasts were isolated from cell suspension-derived somatic embryos of Coffea canephora. After repeated subculture in a medium supplemented with 0.5 mg/l of each of kinetin, 2,4-dichlorophenoxyacetic acid (2,4-D), and naphthaleneacetic acid (NAA), microcalli developed. Transfer of these microcalli to a medium lacking growth regulators resulted in the formation of globular embryos. Upon subculture without growth regulators they grew to well-differentiated embryos, Eventually some of them developed to plantlets which were transferred to the greenhouse for further observation.     

Inter-simple sequence repeat (ISSR) diversity within Monarda fistulosa var. brevis (Lamiaceae) and divergence between var. brevis and var. fistulosa in West Virginia

Inter-simple sequence repeat (ISSR) banding patterns were used to examine genetic diversity within and among populations ofMonarda fistulosa var.brevis, a rare taxon restricted to several populations in limestone glades and barrens in eastern West Virginia and Virginia. More than 34% of the total ISSR diversity in var.brevis occurred among populations, which is high when compared to the few other rare species that have been examined for ISSR variation. Prior studies demonstrated that var.brevis is morphologically distinct from the more widespread var.fistulosa, and that the differences are maintained when the two varieties are grown together in a uniform environment. The present study utilizing ISSR markers indicated that the two varieties are distinct, though quite similar genetically, and this is concordant with prior investigations documenting their morphological and habitat differences. However, the ISSR results suggest that the two varieties have diverged relatively recently and/or there is a low level of gene flow between them.     

Inter-simple sequence repeat (ISSR) amplification for analysis of microsatellite motif frequency and fingerprinting in rice (Oryza sativa L.)

 Inter-simple sequence repeat (ISSR) amplification was used to analyze microsatellite motif frequency in the rice genome and to evaluate genetic diversity among rice cultivars. A total of 32 primers, containing different simple sequence repeat (SSR) motifs, were tested for amplification on a panel of 59 varieties, representative of the diversity of cultivated rice (Oryza sativa L.). The ISSR analysis provided insights into the organization, frequency and levels of polymorphism of different simple sequence repeats in rice. The more common dinucleotide motifs were more amenable to ISSR analysis than the more infrequent tri-, tetra- and penta-nucleotide motifs. The ISSR results suggested that within the dinucleotide class, the poly(GA) motif was more common than the poly(GT) motif and that the frequency and clustering of specific tri- and tetra-nucleotide simple sequence repeats was variable and motif-specific. Furthermore, trinucleotide ISSR markers were found to be less polymorphic than either dinucleotide or certain tetranucleotide ISSR markers, suggesting which motifs would be better targets for microsatellite marker development. The ISSR amplification pattern was used to group the rice genotypes by cluster analysis. These results were compared to surveys of the same varieties for amplified fragment length polymorphism (AFLP), restriction fragment length polymorphism (RFLP) and isozyme markers. The ISSR fingerprint could be used to differentiate the genotypes belonging to either Japonica or Indica sub species of cultivated rice and to dissect finer levels of diversity within each subspecies. A higher percentage of polymorphic bands was produced with the ISSR technique than the AFLP method, based on a similar PCR reaction. Therefore, ISSR amplification proved to be a valuable method for determining genetic variability among rice varieties and for rapidly identifying cultivars. This efficient genetic fingerprinting technique would be useful for characterizing the large numbers of rice accessions held in national and international germplasm centers. Received: 25 May 1998 / Accepted: 17 September 1998     

新疆地区番茄潜叶蛾遗传多样性的ISSR分析

[目的]番茄潜叶蛾已成为世界性番茄的重要害虫,对番茄产业的发展造成了严重的威胁,研究其遗传多样性有利于揭示不同地理种群的遗传变异结构。[方法]采用ISSR分子标记技术分析了20个地理种群番茄潜叶蛾的遗传多样性和遗传结构特征。[结果]15条引物扩增出137条ISSR条带,其中多态性条带占96.35%,所有个体显示了各自独特的ISSR图谱。ISSR的标记遗传多样性结果表明,20个番茄潜叶蛾地理种群遗传距离大小范围为0.0065~0.1623,遗传一致度范围为0.8502~0.9935。种群变异来源分析表明,25.36%的遗传变异来自种群间,74.64%的变异来源于种群内部。UPGMA系统发育分析结果表明,各地理种群的聚类与地理位置无较强的关联性。[结论]番茄潜叶蛾尚处在入侵早期阶段,且具备频繁入侵和多点的特征。防控上要注意加强检疫,阻绝多点入侵来源。     

Inoculation and colonization of coffee seedlings (Coffea arabica L.) with the fungal entomopathogen Beauveria bassiana (Ascomycota: Hypocreales)

The fungal entomopathogen Beauveria bassiana became established as an endophyte in coffee seedlings grown in vitro and inoculated with B. bassiana suspensions in the radicle. The fungus was recovered as an endophyte 30 and 60 days postinoculation, from stems, leaves, and roots, and at 60 days postinoculation one of the isolates was also recovered as an epiphyte. Fusarium sp., Rhodotorula sp., and four bacterial morpho-species were also detected, indicating these were present as endophytes in the seed.     

Genetic diversity of wild Cymbidium goeringii (Orchidaceae) populations from Hubei based on Inter-simple sequence repeats analysis

Cymbidium goeringii is a diploid and nonrewarding, bumblebee-pollinated species, which is distributed in China, Japan and Korea Peninsula. This species is now highly endangered due to the mass collection and forest clearance in China. In the present study, we investigated the distribution of genetic variation within and between eleven populations of Cymbidium goeringii in central China by using Inter-simple sequence repeats (ISSR) markers. Eleven primers produced a total of 127 clear and reproducible bands of which 112 were polymorphic. High genetic diversity was detected in Cymbidium goeringii for both population level (P = 63.1%; He = 0.194 5) and species level (P = 88.2%; He = 0.262 8). A higher level of genetic differentiation was detected among populations (G _ST = 0.244 0, F _ST = 0.220 7) with Nei’s G _ST analysis and analysis of molecular variance (AMOVA), and no correlation was found between geographical and genetic distance. Genetic drift rather than gene flow played an important role in forming the present population structure of Cymbidium goeringii. Limited gene flow among populations and gene drift increase the extinction risk of local populations. Some conservation concerns are therefore discussed together with possible strategies for implementing in situ and ex situ conservation. __________ Translated from Biodiversity Science, 2006, 14(3): 250–257 [译自: 生物多样性] Equally contributed authors     

Genetic variation in five Mediterranean populations of Juniperus phoenicea as revealed by inter-simple sequence repeat (ISSR) markers

BACKGROUND AND AIMS: The assessment of the genetic variability and the identification of isolated populations within a given species represent important information to plan conservation strategies on a genetic basis. In this work, the genetic variability in five natural populations of Juniperus phoenicea, three from Sardinia, one from Cyprus and the last one in the Maritime Alps was analysed by means of ISSRs, on the hypothesis that the latter could have been a refugial one during the last glaciation. METHODS: ISSRs were chosen because of their ability to detect variation without any prior sequence information. The use of three primers yielded 45 reproducible, polymorphic bands, which were utilized to estimate the basic parameters of genetic variability and diversity. KEY RESULTS: All of the populations analysed harboured an adequate amount of genetic variability, with H(S) = 0.1299. The proportion of genetic diversity between populations has been estimated by G(ST) = 0.12. The three Sardinian populations are separated, as tested by AMOVA, from the Cyprus and the continental ones. CONCLUSIONS: The results indicate that geographical isolation has represented a major barrier to gene flow in Juniperus phoenicea. This work represents a first step towards a full genetic characterization of a conifer from the Mediterranean, a world biodiversity hotspot confronted with climate change, and thus contributes towards the planning of genetics-informed conservation strategies.     

A comparison of genetic variation among wild and cultivated Morus Species (Moraceae: Morus) as revealed by ISSR and SSR markers

In this study, inter-simple sequence repeats (ISSR) ans simple sequence repeat (SSR) markers were used to investigate genetic diversity of 27 mulberry accessions including 19 cultivated accessions (six M. multicaulis, three M. alba, two M. atropurpurea, two M. bombycis, one M. australis, two M. rotundiloba, one M. alba var. pendula, one M. alba var. macrophylla, and one M. alba var. venose) and 8 wild accessions (two M. cathayana, two M. laevigata, two M. wittiorum, one M. nigra and one M. mongolica). ISSRs and SSRs were compared in terms of their informativeness and efficiency in a study of genetic diversity and relationships among 27 mulberry genotypes. SSRs presented a higher level of polymorphism and greater information content. All index values of genetic diversity both markers analyzed using Popgene 32 software indicated that within wild species had higher genetic diversity than within cultivated species. Cultivation may caused the lose of genetic diversity of mulberry compared with wild species revealed by ISSR and SSR markers. The mean genetic similarity coefficients among all mulberry genotypes ascribed by ISSR and SSR matrices were 0.7677 and 0.6131, respectively. For all markers a high similarity in dendrogram topologies was obtained although some differences were observed. Cluster analysis of ISSR and SSR using UPGMA method revealed that the wild species are genetically distant from the domesticated species studied here. The correlation coefficients of similarity were statistically significant for both marker systems used. Principal coordinates analysis (PCA) for ISSR and SSR data also supports their UPGMA clustering. These results have an important implication for mulberry germplasm characterization, improvement, molecular systematics and conservation.     

Allozyme variation within and among populations of onion (Allium cepa L.) from West Africa

Local germplasm of onion (Allium cepa L.) in West Africa is threatened by extinction. Sixteen populations of onion collected in five countries in West Africa were investigated for isozyme polymorphism using four polymorphic enzyme systems (ADH, MDH, 6-PGDH and PGI) among nine enzyme systems assayed. This is the first report on the genetic diversity of local landraces of onion. The inheritance of two dimeric enzyme systems PGI and MDH was demonstrated using F₂ progeny arrays. The PGI system revealed a single locus with three alleles, and the MDH system revealed three loci with four alleles. Four polymorphic systems revealed nine alleles (adh-a1 and a2, mdh-c1 and c2, 6-pgdh-a1 and a2, and pgi-a1, a2 and a3) in the 16 local populations observed. The mean number of alleles per polymorphic locus was 2.25, and 67% of the alleles were present in all populations. Allele 6-pgdh-a2 was present in only two landraces (from Niger and Nigeria); it is considered to be a rare allele (frequency approximately 2%). Among the 16 populations, within-population diversity was greater (90%) than between-population diversity (10%). Genetic distance analyses showed an aggregate of all populations except for two, which originated from Nigeria, an English-speaking country. Received: 24 August 1995 / Accepted: 26 February 2001     

Genetic diversity within and among the wild populations of Murraya koenigii (L.) Spreng., as revealed by ISSR analysis

Murraya koenigii (L.) Spreng., commonly known as curry leaf plant, is found in the different hilly regions of India. In the present study, fifty-nine accessions representing eight wild populations of M. koenigii were analyzed using thirteen ISSR primers. A total of 152 bands were amplified, out of which, 136 were polymorphic corresponding to 89.47% polymorphism across the accessions. The pairwise population genetic distances were calculated for all the populations that varied from 0.05 to 0.13 between the populations of M. koenigii. AMOVA and Nei’s genetic diversity analysis revealed higher genetic variations within populations than among the populations. The clustering of populations in the dendrogram was not in congruence with geographical affiliations. The results indicate that the ISSR method is sufficiently informative and powerful to estimate the genetic diversity in M. koenigii populations. As M. koenigii is an important wild plant genetic resource, therefore, information on genetic variability might be a potential source as breeding material for development of commercially valuable traits in M. koenigii plants.     

Genetic analysis and gene mapping of a rice few-tillering mutant in early backcross populations ( Oryza sativa L.)

A rice mutant,G069, characteristic of few tiller numbers, was found in anther culture progeny from theF ₁ hybrid between anindica-japonica cross, Gui630×02428. The mutant has another two major features: delayed tillering development and yellowing apex and margin on the mature leaves. As a donor parent,G069 was further backcrossed with the recurrent parent,02428, for two turns to develop aBC ₂F₂ population. Genetic analysis in theBC ₂F₂ population showed that the traits of few-tillering and yellowing apex and margin on the mature leaves were controlled by one recessive gene. A pool of equally mixed genomic DNA, from few-tillering individual plants inBC ₂F₂, was constructed to screen polymorphism with simple sequence repeat (SSR) markers in comparison with the02428 genome. One SSR marker and three restriction fragment length polymorphism (RFLP) markers were found possibly linked with the recessive gene. By using these markers, the gene of few-tillering was mapped on chromosome 2 between RFLP marker C424 and S13984 with a genetic distance of 2.4 cM and 0.6 cM, respectively. The gene is designatedft1.     

Rhizogenesis in callus from Conference pear (Pyrus communis L.) protoplasts

Large numbers (ca 6×10⁶ protoplasts/g f.wt) of viable (80%) protoplasts were isolated from embryo-callus tissues of Conference pear using an enzyme mixture which contained 2.0% (w/v) Meicelase, 2.0% (w/v) Rhozyme HP-150 and 0.03% (w/v) Macerozyme R-10. A medium based on ammonium-free MS salts and supplemented with 2.0 mg/l NAA, 0.5 mg/l BAP and 9% (w/v) mannitol supported protoplast division and the proliferation of multicellular colonies. Colonies were taken to the callus stage on a medium which contained MS salts plus 0.1 mg/l 2,4-D and 0.1 mg/l BAP. Roots were regenerated from these protoplastderived calli on MS medium with 0.1 mg/l NAA, 5.0 mg/l BAP and 50 mg/l casein hydrolysate.Abbreviations BAP 6-benzylaminopurine - CPW13M CPW salts medium [15] with 13% (w/v) mannitol - FDA fluorescein diacetate, f. wt-fresh weight - MS Murashige and Skoog [14] - NAA -naphthaleneacetic acid - PE plating efficiency (%) - 2,4-D 2,4-dichlorophenoxyacetic acid