The exploitation of the genetic variation generated to produce genetic markers for marker-assisted breeding in crop plants
Examples of recent projects.
1. Use of methylation-sensitive restriction endonucleases to target coding sequences in genomes.
A CASE studentship on wheat (with Dr. Peter Jack) - (Dr) Kate Parsley (kw211@mole.bio.cam.ac.uk) - and a Cambridge Commonwealth studentship on oil palm - (Dr) Farah Hafeez (fh218@cam.ac.uk)
In plant genomes, one of the most common modifications of DNA is the addition of a methyl group to the 5' position of the Cytosine base. There is also a correlation between methylation and inactivation of genes.
We have used methylation-sensitive endonucleases, NotI and MluI to construct test numbers of Bacterial Artificial Chromosomes (BACs) for hexaploid bread wheat (Triticum aestivum) and for oil palm (Elaeis guineensis Jacq). These have been analysed by arraying clones onto membranes and screening them with a number of probe sequences and also by end-sequence comparison and database searching. A total of 180 wheat end-sequences (60 NotI-derived and 120 MluI-derived clones) and 492 oil palm (MluI-derived) end-sequences were examined and compared with T. monococcum BAC clones kindly provided by Jorge Dubcovsky and HindIII oil palm BAC end-sequences, respectively. The results suggest significant enrichment for coding sequences in both species.
2. Development of genetic markers for oil palm (E. guineensis Jacq.)
African oil palm (E. guineensis Jacq.)
Oil palm is approximately 7 times more productive in terms of oil/hectare/year than the best yielding temperate oil crops and is extensively planted in South-East Asia and a number of countries in South America and Africa.
Because it is a tree crop, breeding selection is a slow process, with a typical round of selection taking between 12 and 16 years. By developing genetic markers for this species in a project funded by New Britain Palm Oil Ltd (NBPOL - Papua New Guinea), it is hoped to improve the management of germplasm, investigate new sources of variation for introduction into breeding programmes and, eventually, to allow selection of traits using markers before the trait is itself expressed. This could significantly decrease the breeding cycle time.
Development of a new marker system for oil palm.
At the Department of Genetics, University of Cambridge, we investigated one family of the complement of mobile genetic elements in oil palm, specifically Ty1/copia-like retrotransposons, with a view to generating a non-patented, polymorphic, multi-locus marker system. This work was carried out by (Dr) Zuzana Price (z.price@gen.cam.ac.uk) as a CASE PhD studentship with NBPOL. Using degenerate primers, fragments have been amplified from a key gene (part of the reverse transcriptase gene which is essential to the element's ability to move within the genome). Fragments were amplified from an individual of African oil palm (E. guineensis Jacq), from an individual of South American oil palm (E. oleifera) and from an individual of coconut (Cocos nucifera). Using Paup 4.0, a sequence comparison has been carried out with over 90 cloned sequences from this research, and also with sequences that were already present in the database.
Representative sequences are presented in the dendrogram in Figure 1. From this, classes A, B1, B2 and C were identified, with >95% confidence, by boot-strap scores. More details of this are given in (2) and (5)
Figure 1. The cylindrical figure at the top represents a generic intact 'Ty1/copia-like' retrotranposon. The arrows indicate where conserved sequences allow the amplification of a short region of the Reverse Transciptase (RT) gene. The dendrogram is generated in Paup 4.0 and based on sequence comparison.
'?' indicate non-palm Ty1/copia-like retrotransposons from the database. 'l' represents palm Ty1/copia-like retrotransposons. Black indicates that sequences are derived from the database (NCBI/EMBL). Blue indicates sequences cloned in this research from coconut (Cocos nucifera), red sequences cloned in this research from African oil palm (E. guineensis) and green sequences cloned in this research from South American oil palm (E. oleifera). All sequences cloned in this research were cloned from a single individual of the appropriate palm.
This work has gone on to develop a retrotransposon-based marker system, Inter-Retrotransposon Amplified Repeats (IRAP) for oil palm. Markers derived from this system have been mapped in a breeding cross and added to a map consisting of AFLP markers (as a comparison) and microsatellite markers, also developed within this project. The first seven linkage groups (of 16) are shown in Figure 2.
Figure Two: First seven linkage groups of the 16 expected, for a marker map constructed in DM635. This is a cross central to the NBPOL breeding programme and consists of 67 heterozygous individual palms.
Markers in red represent microsatellites (developed here), markers in green represent AFLPs and markers in blue represent IRAP markers (developed here). The positions of IRAP markers are also emphasised by the use of blue arrows.
IRAP may offer a particular advantage for investigating genetic relationships within accessions of palms, as the insertion of a retrotransposon is an irreversible event and will be reflected in the offspring derived from that individual.
Over 100 microsatellite markers have also been developed and partially characterised and further work on all marker systems developed is currently on-going in a project being run by Dr Zuzana Price at Genetics, Cambridge University.
With the establishment of the new campus at Semenyih (University of Nottingham in Malaysia; UNIM), new collaborative oil palm programmes are being developed and are described below.
Current projects:
3. EU FP6 INCO-DEV 'BAMLINK' [link3]
Building on fifteen years of research on bambara groundnut at Nottingham University, a four year programme- 'Bamlink'- has been funded by the EU (start date Jan 1st 2006).
Led by Dr Sayed Azam-Ali (Programme Coordinator; sayed.azam-ali@nottingham.ac.uk) at Nottingham with nine other partners (2 European, 4 African and 3 Indian) the programme aims to investigate the genetics and extend the current knowledge of the physiology of bambara groundnut (Vigna subterranea). This underutilised species (which is highly drought tolerant) has great potential as an important part of the diet in developing countries. The programme will develop physical and informational resources to facilitate subsequent breeding efforts and will see a unique collaboration between Africa and India in developing an important food security crop and also value-added products from this African legume.
Nottingham will develop a dense molecular map in an intra-specific (wide) cross and will carry out a detailed multi-environment QTL analysis. Microsatellite and Conserved Orthologous Set (COS) markers will be developed for Bambara groundnut and made available to the research community through this programme. Nottingham will also use the Tropical Crops Research Unit to uncouple abiotic stress factors, such as heat and drought
(TCRU: http://www.nottingham.ac.uk/tcru/ Dr Sayed Azam-Ali: http://www.nottingham.ac.uk/biosciences/ah/academic/azam.html)
Dr Shravani (Rakhi) Basu has been appointed to this programme, after completing a PhD at Nottingham in which she developed the first controlled crosses in Bambara groundnut and produced an initial AFLP-based molecular map. Initial QTL analysis in this cross reveals several genes involved in domestication of this crop.
Mr Odireleng (Ozie) Molosiwa joined the group in October 2007 on a Commonwealth PhD Scholarship and will be examining ways to characterise the extent of genetic variation in bambara groundnut landraces and to utilise this information to help account for the phenotypic variation observed within landraces.
Dr Rakhi Basu (shravani.basu@nottingham.ac.uk) tel: 0115 9516048
Mr Ozie Molosiwa (odireleng.molosiwa@nottingham.ac.uk) tel: 0115 9516048
A Bambara groundnut plant from a cultivated landrace - seed are produced at the base of the plant. The wild (V. s. spontenea forms has a spreading habit)
4. The BBSRC wheat 'Big-Ears' project
Building on research initiated by Dr John Foulkes (john.foulkes@nottingham.ac.uk; http://www.nottingham.ac.uk/biosciences/ah/academic/foulkes.html), Prof. Matthew Reynolds, CIMMYT ( m.reynolds@cgiar.org ) and Prof. Roger Sylvester-Bradley, ADAS (roger.sylvester-bradley@adas.co.uk) and pursued through 2 PhD studentships, we have recently been awarded funding by the BBSRC to investigate the basis of 'ear fertility' in a Doubled Haploid population of a cross between UK winter wheat (Rialto) and a CIMMYT spring wheat 'large rachis' line (L14).
The project is for 4 years, is led by Dr Foulkes and is a collaboration between Nottingham and CPB-Twfords (Plant Breeders, Thriplow) with advice from CIMMYT and ADAS. The programme will investigate the physiological basis of the 'large ear' phenotype and determine whether a longer rachis would alleviate sink limitation in UK wheat, permitting an increase in yield in elite breeding materials.
A CIMMYT wheat parent ear (left) and UK Rialto (right)
The programme started in July 2006 and involves the development of a genetic map within this cross to allow QTL analysis and physiological work to begin to understand the basis of the extended rachis trait and its effect on yield traits. The genetic factors for fertility/large rachis will be introgressed into CPB-Twyford breeding material through a back-crossing programme and the value of this trait for UK agriculture evaluated. These backcross lines will also be invaluable for understanding the genetic and physiological basis for this trait.
Dr Gracia Vargas Ribas was appointed on three year BBSRC post-Doctoral position in June 2007 and heads the research programme, focusing on the molecular genetic aspects of the programme.
Mr Adam Docherty was appointed to an HGCA/Nottingham University PhD studentship in August 2005 and is primarily working on the initial development of the genetic map in the 140 DH line population. He is also examining contrasting DH lines to try to understand the physiological basis of the trait.
Prof. Jian An Huang was a visiting Fellow on a Chinese Government scholarship from Sep 2005 - Sep 2006 and worked with Adam to begin to development the genetic map.
Mr. Jayalath deSilva is a physiology research technician on the project and is involved in the collection of physiological data.
Ms Julietta (Jools) Marques is a molecular lab technician who is also working part-time on this programme.
Gracia Vargas Ribas (gracia.ribasvargas@nottingham.ac.uk) tel: 0115 951 6048
Adam Docherty (adam.docherty@nottingham.ac.uk) tel: 0115 951 6036
Jayalath deSilva (jayalath.desilva@nottingham.ac.uk) tel : 0115 951 6036
Julietta Marquez (julietta.marquez@nottingham.ac.uk) tel : 0115 951 6036
BBSRC BB/D008972/1 Project 'Raising the ceiling on UK wheat yields - introgression and assessment of novel 'large-ear' CIMMYT germplasm into UK pre-breeding lines' (May 2006 - April 2010; PI: Foulkes, Co-PIs: Mayes, Wilson (zoe.wilson@nottingham.ac.uk ; http://www.nottingham.ac.uk/biosciences/plantsci/staff/zoe_wilson.html )
5. Oil palm molecular genetics and marker-assisted breeding.
Building on the recent establishment of the new Malaysian campus at Semenyih in 2005 and of Biosciences at Semenyih in 2006, a number of areas of cross-site oil palm research are under development. These will be greatly facilitated by the completion of joint research labs in a collaboration between UoN, UNIM and AAR. These are due to open in January 2008.
Details of intial programmes will appear shortly.
6. Homologous recombination in crop plants.
In linked PhD programmes, we aim to investigate aspects of gene targeting during genetic transformation and meiotic recombination in parallel in model and crop species. This is in collaboration with Dr Philippe Vain (philippe.vain@bbsrc.ac.uk) and Prof. John Snape at the John Innes Centre for Plant Research (Norwich, UK).
We are also currently growing a wheat neutron deletion population at Nottingham which was developed in the spring cultivar "Highbury" by Prof. Snape at JIC. This material is at the M8 generation and will be examined for relevant phenotypes in the coming year. We have developed a screening method to identify the absence of a homeologue within a particular line, even when there is no obvious phenotype. This will allow detailed studies of such lines.
Beginning to understand homologous recombination in crop species is one of the key developments needed in the next decade.
Mr Chanate Malumpong began a PhD in October 2005 and is funded by a Thai Government Fellowship Grant. He is working primarily on examining the effects of over-expressing rice homologues of known genes involved in recombination on gene targeting and meiotic recombination in Arabidopsis and rice. He is also characterising the effects of Tos17 insertional inactivation of the same genes in rice plants.
Mr Upendra Devisetty began a PhD in October 2005, partly funded by a University of Nottingham award. He is working primarily on identifying wheat material with missing homeologues of known genes involved in recombination and evaluating any phenotypic effects.
Dr Katie Mayes is a post-doc appointed in Feb 2006 on a part-time contract funded by a University of Nottingham New Lecturer Fund ('laying the foundations for research into crop meiosis') and has been preparing constructs for over-expression of the selected RAD51 family genes in a number of species.
Harvesting ear rows from the neutron deletion population based on 'Highbury' from Prof. John Snape (JIC)
Chanate Malumpong (chanate.malumpong@nottingham.ac.uk)
Upendra Devisetty (upendra.devisetty@nottingham.ac.uk);
Telephone: 0115 9516048
Katie Mayes (katie.mayes@nottingham.ac.uk)
7. The comparative genetics of Onocerin production in Restharrows.
Restharrows (Ononis repens and O. spinosa in the UK) were originally agricultural weeds with significant tap-roots (hence the name, as they would arrest the harrow or plough). Restharrows also produce a substance in their roots called Onocerin. This cyclic, symmetrical, compound appears to be derived from a modification of the beta-amyrin pathway common to most plants. It is also possible that production of this compound may assist in the colonisation of marginal land. Ononis repens maritima is common on shingle beaches on the east coast and is easily found in Suffolk.
A principle components analysis of microsatellite data (10 loci) examining population structure between the main four UK 'species'.
The work builds on previous research which has isolated an almost complete clone of the beta-amyrin-like gene from Ononis repens and we aim to use comparative genetics to clone and investigate genes in the pathway involved in the production of this rare polycyclic compound. This is being pursued through a CASE PhD studentship with Prof. Jerry Roberts (Jeremy.roberts@nottingham.ac.uk; Nottingham) and Dr Peter Dean (peter@cambio.fsnet.co.uk; http://www.cambio.co.uk; Cambio) to which Steven Hayes was appointed to a 4 year BBSRC PhD CASE award in October 2005.
Ononis repens maritima (on a beach) and Ononis repens (in a meadow)
Steven Hayes (steven.hayes@nottingham.ac.uk); tel: 0115 95 16048
Recent Papers
5. Mayes, S., Stadler, S., Basu, S., Murchie, E., Massawe, F., Kilian, A., Roberts, J.A., Mohler, V., Wenzel, G., Beena, R., Sheshshayee, M.S. and Azam-Ali, S.N. BAMLINK - a cross disciplinary programme to enhance the role of bambara groundnut (Vigna subterranea L. Verdc.) for food security in Africa and India. Acta Horticulturae 806(1): 137-150.
6. Haddrill, P.R., Shuker, D.M., Amos, W., Majerus, M.E.N. and Mayes, S. (2008) Patterns of female multiple mating in wild and laboratory populations of the two-spot ladybird, Adalia bipunctata (Coleoptera: Coccinellidae) revealed by microsatellite analysis. Molecular Ecology 17: 3189-3197
7. Al-Rugaishi, I.A., Davey, M., Alderson, P. and Mayes, S. (2008) Genetic relationships and genotype-tracing in date palms (Phoenix dactylifera L.) in Oman, based on microsatellite markers. Plant Genetic Resources: Characterisation and Utilisation: 6(1) 70-72.
8. Mayes, S., Hafeez, F., Price, Z., MacDonald, D., Billotte, N. and Roberts, J.A. (2008) 'Molecular research in oil palm, the key oil crop for the future' In 'Genomics of Tropical Crop Plants.' (eds Moore, P.H. and Ming, R.) Springer, NY.
9. Kloda J.M., Dean P.D.G., MacDonald, D.M. and Mayes, S. (2007). Using principle component analysis to compare genetic diversity across polyploidy levels within plant complexes: an example from British Restharrows (Ononis spinosa and Ononis repens).' Heredity. doi:10.1038/sj.hdy.6801044
10. Haddrill, P.R., Shuker, D.M., Mayes, S. and Majerus, M.E.N. (2007). Transient benefits of multiple mating in the two-spot 1 ladybird, Adalia2 bipunctata. European Journal of Entomology 104(3): 393-398.
11. Basu S., Roberts, J.A., Azam-Ali, S. and Mayes, S (2007). 'Development of microsatellite markers for Bambara groundnut (Vigna subterranea (L) Verdc.) Molecular Ecology Notes 7(6): 1326-1328; DOI: 10.1111/j.1471-8286.2007.01870.x
12. Basu, S., Mayes, S., Davey, M., Roberts, J.A., Azam-Ali, S.N., Mithren, R. and Pasquet, R.S. (2007) Inheritance of 'domestication' traits in bambara groundnut (Vigna subterranea L. Verdc.) Euphytica 157:59-68; DOI: 10.1007/s10681-007-9396-4
13. Price, Z., Hafeez, F., Billotte, N and Mayes, S. (2007) Plant genome. Biodiversity and Evolution - oil palm. Vol.1, Part D. A.K Sharma and A. Sharma (eds). Kluwer Publishing ISBN: 978-1-57808-420-2.
14. Basu, S., Roberts, J.A., Azam-ali, S.N. and Mayes, S. (2007) Bambara Groundnut. In: Kole, C.M., ed. Genome Mapping and Molecular Breeding in Plants .: Pulses, Sugar and Tuber Crops. 3. Springer, pp. 159-173
15. Mayes, S., Holdsworth, M.J., Pellegrineschi, A. and Reynolds, M. (2005) 'Allying genetic and physiological innovations to improve productivity of wheat and other crops.' pp 89-122 in 'Yields of farmed species - constraints and opportunities in the 21st century'. Eds. R Sylvester-Bradley & J Wiseman: Nottingham University Press, Nottingham.
16. Mayes, S., Parsley, K., Sylvester-Bradley, R., May, S. and Foulkes, M.J. (2005). 'Integrating Genetic information into plant breeding programmes: how will we produce new varieties from molecular variation using bioinformatics?' Annals of Applied Biology 146: 223-237.
17. Kloda, J.M., Dean, P.D.G., MacDonald, D.M. and Mayes, S (2004). 'Isolation and characterisation of microsatellite loci in Ononis repens, Leguminosae.' Molecular Ecology Notes 4: 596-598.
18. Freeman, S., West, J., James, C., Lea, V. and Mayes, S. (2004). 'Development of microsatellite markers for tea (Camellia sineensis).' Molecular Ecology Notes. 4:324 - 326.
19. Price, Z., Schulman, A.H. and Mayes, S (2004). 'Development of new marker methods - an oil palm example.' Plant Genetics Resources: Characterisation and Utilisation 1: 2/3, 105-115
20. Kamau E.M., Mayes, S. and Barrett, J.A. (2003). 'Isolation and characterisation of microsatellite loci in Schizolobium parahyba (Leguminosae).' Molecular Ecology Notes 3, 469-470.
21. Haddrill, P.R., Majerus, M.E.M. and Mayes, S. (2002). 'Isolation and characterisation of highly polymorphic microsatellite loci in 2-Spot Ladybirds, Adalia bipunctata'. Molecular Ecology Notes 2, 316-319.
22. Price, Z., Dumortier, F., MacDonald, D. and Mayes, S. (2002). 'Characterisation of copia-like elements in oil palm.' Theoretical and Applied Genetics. 104, 5 pp.860- 867.
23. Rance, K., Mayes, S., Price, Z., Jack, P.L. and Corley, R.H.V. (2001).'Quantitative Trait Loci for yield components in oil palm (E. guineensis Jacq.)' Theoretical and Applied Genetics 103, 1302 - 1310
24. Mayes, S., Jack, P.L. and Corley, R.H.V. (2000). 'The application of molecular markers in a specific breeding programme for oil palm.' Heredity 85, pp. 288-293.
Previous publications
25. Mayes, S., Jack, P.L., Marshall, D.F. and Corley, R.H.V., (1997) Construction of a RFLP genetic linkage map for oil palm (Elaeis guineensis Jacq.). Genome, 40(1), 116-122.
26. Mayes, S., James, C.M., Horner, F., Jack P.L. and Corley, R.H.V. (1996) The application of restriction fragment length polymorphism for the genetic fingerprinting of oil palm (E. guineensis Jacq). Molecular Breeding 2, 175-180.
27. Jack, P.L., Dimitrejevic, T.A.F. and Mayes, S. (1995) Assessment of nuclear, mitochondrial and chloroplast RFLP markers in oil palm (Elaeis guineensis Jacq.). Theoretical and Applied Genetics, 90(5), 643.
28. Jack, P.L. and Mayes, S. (1993) Use of molecular markers for oil palm breeding. II - Use of DNA markers (RFLPs). Oleagineux, 48(1), 1.
1. Bode A. Okukolu, Sean Mayes, Florian Stadler, N.Quat Ng, Iyiwole Fawole, Dominique Dumet, Sayed N. Azam-Ali, Albert G. Gilbert, Chittaranjan Kole. 2011. Genetic diversity in bambara groundnut (V. subterranea L verdc.) as revealed by phenotypic descriptors and DArT marker analysis. Genetic Resources and Crop Evolution in press. 2. Upendra Kumar Devisetty, Katie Mayes and Sean Mayes. 2010. The RAD51 and DMC1 homeologous genes of bread wheat: molecular characterisation and expression analysis. BMC Research Notes 3:245. 3. Rubyogo Jean Claude, Matthew Dickinson, Sean Mayes and Teshale Assefa (2009) Assessment of Different Bean Seed Grades and Sources from Ethiopia Using Molecular Tools. Journal of New Seed 10:293-301.
4. Taah, K.J., Alderson, P.G., Power, J.B. and Mayes, S. (2009). Effects of auxin type, root length and shoot size on ex vitro survival of micropropagated avocado shoots. Ghana Journal of Horticulture 7: 18-25.