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Exploitation of gene technology in white lupins - AR0142

Description
The main aim of the project will be to add significantly to the techniques available for facilitating the breeding of new genotypes of white lupin (Lupinus albus) particularly suited to UK farming conditions. This will be achieved by identifying QTLs for important traits and locating them on a molecular marker map, which will be generated within the project.

Work thus far with molecular markers in white lupin has shown the utility of exploiting molecular markers and the potential to identify QTLs (1), which might then be exploited by breeders. However, to generalise the potential of this technology in lupin needs the generation of a saturated map, onto which traits and QTLs can be introduced in a more facile manner. It is anticipated that the project will benefit from our existing links in Europe and worldwide, in particular by joining an international effort centred on a lupin marker/QTL/genome analysis being established in Perth, Australia, in the initiation of which we were instrumental.

The facilitation of breeding for improved genotypes of white lupin for the UK is a fundamental factor in raising the potential for home-produced protein. As has been noted in the ENTEC report (1998) on “Home-Produced Proteins for Animal Feed” and in the recent review by MAFF of “Minor Combinable Crops R&D”, the short fall in protein produced in the UK has been brought into clearer focus with: the rising cost of soya bean products; Agenda 2000 adoption; the “fallout” from the BSE crisis and the general move away from animal based protein sources. Against the background of this obvious need to fill “the gap” with affordable but high quality protein, lupins, as noted in both these reports, is a clear candidate. This is further emphasised by the nitrogen fixing attributes of lupin, along with its characteristic of allowing phosphate mobilisation. Thus lupin must also be seen as a major player in the move to more integrated farming systems and environmentally sustainable agricultural practices.

Therefore the project aims at the further identification of QTLs, important in relation to morphological, agronomic, quality and processing traits and locating these by developing a saturated molecular marker map of white lupin (which will be alongside those of other lupin species, which other researchers plan to develop in parallel) This will produce a readily transferable technology to allow the necessary breeding and selection operations to be achieved more efficiently. Thus, the project will facilitate the more rapid selection of suitable genotypes of white lupins, which will then be transferable and exploitable within the UK agricultural sector.
Objective
1. Qualitative and Quantitative trait loci: We have already generated 25 putative markers associated with important traits in L albus. Markers have been identified as being associated with differential expression of 6 characters, some of which are determined by major genes while other show a continuous distribution of phenotypic expression. We are currently carrying out initial investigations on their linkage values. However, this will be preliminary and restricted to the particular markers without a mechanism to refine the search for more closely linked markers – as will become possible with the developing map. We will therefore plan during the project to map these markers onto the emerging map as it progresses. We will look for linkage with fresh markers and hence define those that lie closer the putative QTL – hence making them more usefully applied in breeding (note that one very closely linked marker or two less tightly linked, but flanking markers, will serve to effectively tag the character of interest).

2. New Quantitative trait loci: The family structure that will characterise the inbreeding process of the population will allow the potential to screen, on a family basis, for the expression of quantitative traits and so look for new QTLs associated with molecular markers. The attempts that are being made in a range of species to screen for QTLs using data from individual F2 plants (or animals) are destined to failure (as is becoming all too apparent to those who argued otherwise). However, the application of a simple “progeny testing” approach will allow us not only to score the progenies for a range of characters but also allow the differentiation of homozygous and heterozygous marker loci (both marker technologies to be employed give mostly dominant markers). This will allow us to achieve our target given above and holds the potential to extend this to other characters (such as alkali and cold tolerance) that we are currently devising the necessary protocols and screens (MAFF Studentship).

3. Marker techniques: The intention is to continue to exploit PCR based techniques. This will be partly by further application of the Inter Simple Sequence Repeats (ISSR) which have already proved applicable and informative in our hands with lupins. The methodology will be extended in year one to exploit further possibilities to generate additional polymorphic markers segregating in the F2 population. This will include investigating a further 25 anchored primers; using combinations of two primers; adapting the protocols to allow running out the markers on sequencing gels; and carrying out restriction of DNA after PCR. This should generate in the order of 100 polymorphic markers within 24 months. The parameters needed to optimise the technique of Amplified Fragment Length Polymorphism (a technique that is now running within the research group here) will be optimised and then applied to the F2 population. This will be anticipated to generate a further 100 polymorphic markers within 36 months. After the initial development of AFLPs we will then proceed to optimise the protocol for running them via the DNA sequencer and directly recording the patterns generated using the software package. This procedure has yet to be tried with lupins but promises to generate reliable, repeatable markers in larger quantities than the “manual” methods.

4. Development of a mapping population: we will exploit 2 existing F2 populations on which we have already carried out some marker analysis. These represent populations, the parents of which were chosen to be as diverse as possible and on which we are currently examining the putative QTLs so far identified. One of these populations will be passed through successive rounds of selfing to produce a set of about 100 inbred lines (or at least inbred to F7) by Year 3. We will also take advantage of our extensive lupin germplasm collection to screen lines to look for additional markers which might not segregate in the particular population we have chosen to look at in detail and thus further to the generic applicability of our final marker set.

5. Linkage map development: The polymorphic markers generated above in the F2 population will serve to produce an initial marker map with an anticipated 300-350 markers by the end of the 3rd Year. This will then allow comparison with the maps that are being developed by other groups, particularly those working with Prof. Mike Jones and Dr Rob Potter in Perth, Australia. With supporting paperwork from us they have been granted money to set up a database, with international inputs, on the lupin genome. In addition they will be developing and extending their marker work on L angustifolius, which should also allow us access to further primers with a high possibility of them being informative.

6. Inheritance of markers: As we proceed through the inbreeding process we will use the families generated to look for segregation of our detected markers and allow us to therefore attribute recombination values to the markers and hence refine significantly the crude F2 map, noted above. This should allow the definition of the significant linkage groups which will iterate towards defining the chromosomes by the 3 – 4th year.

7. Linkage Map: By the end of the fourth year we will have developed a map with about 350 molecular markers and will have identified markers that are significantly linked with many traits of direct relevance to lupin breeders.
Time-Scale and Cost
From: 1999

To: 2003

Cost: £196,730
Contractor / Funded Organisations
University - Reading
Keywords
Arable Farming              
Crop Improvement              
Crops              
Farming