DNA fingerprinting of coffee germplasm and construction of molecular linkage map


OBJECTIVES

Molecular fingerprinting of elite Coffea germplasm using DNA markers

  • Establishing the taxonomic relationships of the coffee spp.. available in India based on variability across the nuclear,chloroplast and mitochondrial genomes
  • To Fingerprint the elite coffee genotypes including all the CCRI bred selections & rust differentials using RAPD,ISSR,AFLP and 'in-house' SSR markers
  • To define the parameters for marker based diversity analysis
  • Development of Reference DNA Polymorphism Panels for coffee genotypes

Development of coffee specific microsatellite markers

  • Generating coffee specific microsatellite markers from small insert genomic libraries of C.arabica & C.Canephora

Towards linkage mapping

  • To develop Mapping populations for arabica and robusta linkage mapping
  • To develop the framework linkage map in robusta background using drought specific mapping population [Kagnalla] X [CXR]

Development of Expressed Sequence Tags (ESTs)

  • Construction of EST libraries from water stressed leaf tissues of arabica and robusta variety

Isolation of Resistance gene analogues (RGAs)

  • PCR based appraoch utilising degenerate primer pairs specific to highly conserved NBS (Nucleotide Binding site) and LRR (Leucine Rich Repeat) region of plant disease resistance genes (R-genes) will be used

Development of Web-enabled database

  • A web enabled database of coffee genomics which includes the data of DNA analysis that was carried out will be available in CCMB website (trial version already available)
  • This can be a ready reference resource for other coffee researchers

Project Overview

Coffee is a perennial plantation crop. To meet the ever-changing demands of the market and the environment, a number of breeding and selection programs were initiated in India at Central Coffee Research Institute and abroad. Despite long years of effort, the progress in coffee improvemment has been slow mainly due to constraints such as

(a) Narrow genetic base (almost no variation in the cultivable arabica genepool)
(b) Lack of efficient genetic tools (genetic markers,suitable and easy selection strategies, etc.)
(c) Long breeding cycles/generation advancement

To overcome these problems, in recent years conscious efforts are underway globally to integrate the great poten-tial of molecular markers based technology(ies), which can provide impetus, dependability and directionality to theefforts on genetic improvement of coffee. The DBT supported "National Network Program on Coffee Biotechnology, 1999-2004 was the first such effort initiated in India. The main aims of the initiative were:

Molecular characterization of the selected elite coffee germplasm maintained at CCRI, to help its proper management and resourceful utilization

Generating basic materials (molecular markers/mapping populations) as a prelude to molecular (DNA)
markers based coffee breeding program and development of a framework linkage map of coffee in robusta background

Feasibility studies to develop EST database as a step towards functional genomics


Under the above program, significant leads were made. Most satisfying aspect was that the effort resulted in a platform capable of undertaking advanced molecular studies in coffee genomics in India for the first time. One additional significant outcome of this had been the Indian initiative to discuss the need/feasibility/possible mechanisms of extending the national initiative to a wider International platform for accelerating the coffee improvement efforts. Efforts to this end, originally mooted at Bangalore (27-29 January 2004), led to the formal creation of an 'International Coffee Genomics Network' at Paris, France in mid-2005; which is expected to take full shape in the coming years. In the interim, it is realized that it would be prudent to extend the leads to logical ends to meet the immediate national priorities, in the form of National Network program for next 3 or 5 years. It is in this context, that the present program is formalized.

Ongoing Project Objectives

  • Development of additional microsatellite markers : In order to expand the repertoire of useful genetic markers for linkage-analysis, efforts would be made to generate 100 additional coffee specific microsatellite markers
  • Molecular linkage mapping: The framework linkage map of Coffea canephora (using trait specific population), initiated in the first stage would be completed
  • Development of EST sequence databank: About 2000 ESTs will be developed from the normal leaf identified genotypes

Milestones Acheived so far...

Fingerprinting of Coffee germplasm

  • Genetic characterization of intra-population variability in founder germplasm of four related species using DNA markers
  • Defined the parameters for marker based diversity analysis
  • Established the taxonomic relationships of the coffee species available at CCRI in India
  • Defined the utility of nuclear ITS domain in sequence based studies to derive the realistic affinities between different Coffea species; need to analyse the whole rather than only one of the variable domain i.e., ITS 1 or ITS 2
  • The analysis on three different cellular components, nuclear, chloroplast, and mitochondrial regions suggest that organelle DNA may not be ideal candidates for phylogenetic analysis of Coffea species
  • Completed the fingerprinting of elite arabica germplasm comprising, station-bred selections and rust-differentials using RAPD, ISSR, AFLP and the ‘in-house' SSR markers; data are being analyzed
  • Cross-species amplifications using SSR markers suggest Psilanthus species to be distant relatives of Coffea species

Development of Coffee specific markers

  • Towards construction of linkage map
  • Controlled crossing (selfing of reciprocal F1 hybrids-S-2790 (HDT X Tafarikela) and S2792(Tafarikela X HDT) and 2-way backcross hybridization between diploid F1 hybrid 'S-885' (C.Congensis X C.Canephora)used as a female parent, with each of the original parent i.e, C.congensis and C.canephora was done. But the progeny seedlings from the above crosses were devastated with nematode attack. In the next attempt segregating progeny plants of drought-specific mapping population [Kagnalla] X [C X R] was made. The same were used to isolate genomic DNA for use in linkage analysis
  • The 9 SSR markers were developed from information available in public domain database
    >250 promising clones with desirable repeat motifs have been identified from 35,000 clones of HDT library for marker development
  • More than 150 new microsatellite markers were developed and validated for their use as Coffea specific SSR markers
  • Till to-date ~ 0.54 MB of genomic sequences equivalent to ~0.023% of arabica genome has been sequenced
  • It is seen that AT repeat is the most prevalent in coffee genome followed by GA and CA repeats
  • The newly developed SSR markers showed relatively better PIC values than the only 10 other such markers reported in the literature
  • A small insert partial genomic library of C. canephora has also been made
  • Studies on assessment of relative abundance of STR motifs, using southern hybridisation based slot-blot approach, revealed relatively low abundance of repeat motifs in coffee genomes

 

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