Identifying genes controlling nutrient uptake and distribution in oilseed rape (Brassica napus) Thomas D Alcock[1][5], Rory Hayden[1], Lolita Wilson[1], Ian Bancroft[2], Philip J White[3][4], Martin R Broadley[1], Neil S Graham[1] [1] School of Biosciences, University of Nottingham, Loughborough, LE12 5RD, UK [4] Kind Saud University, Riyadh 11451, Kingdom of Saudi Arabia [2] University of York, Department of Biology, Heslington, York, YO10 5DD, UK [5]
[email protected] [3] Ecological Sciences, The James Hutton Institute, Invergowrie, Dundee, DD2 5DA, UK
Introduction: • • • •
Brassica napus is an amphidiploid species, with a full set of diploid chromosomes from B. rapa and B. oleracea (Fig. 1) Processes involved in the uptake and utilisation of nutrients are complex and controlled by numerous traits Identifying markers for these traits will enable targeted breeding for improved nutrient use efficiency and biofortification Recently developed mapping techniques can be used to identify genes controlling nutrient uptake and distribution
Methods: • • • • •
Fig. 2: ASSYST population in polytunnel
Diversity set (ASSYST population1, 387 accessions) grown in polytunnels (Fig. 2) Leaf samples taken and digested prior to broad-spectrum mineral and anion analysis Associative Transcriptomics2 performed on data to locate associated genetic loci Candidate genes selected and Arabidopsis and Brassica mutants acquired Homozygous Arabidopsis mutants identified and shoot mineral concentrations assessed
Fig. 1: Schematic of the B. napus genome
Results: Leaf Calcium (Ca) & Magnesium (Mg) concentrations are very highly correlated and their control maps to similar regions of the genome: L e a f M g c o n c e n t r a t io n ( m g /k g )
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Fig. 3: Correlation between leaf Ca and Mg Data are means for each accession
Fig. 4: SNPs associated with variation in leaf Ca (A) and Mg (B). Dotted line shows significance threshold; p < 0.05, false discovery rate (FDR) corrected
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Homozygous lines of five B. rapa alleles have been identified in a candidate previously shown to function as a Mg2+ transporter in Arabidopsis3:
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Mutants in three candidate genes have reduced shoot Ca and Mg in Arabidopsis:
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Fig. 5: Shoot concentrations of Ca & Mg in Col-0 and three mutants. AtCa15 – ABC-2 type transporter; AtCa16 – Mt ATP-Mg/Pi transporter; AtCa17 – NTF2
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Fig. 6: Model of Bra.MGT7, a putative Mg2+ transporter in Brassica spp. Each circle in the chain represents an amino acid. Figure created using data from Pfam, SMART and TMHMM2.0
Fig. 7: High Resolution Melt output for Bra.mgt7-4. Potential mutants shown. A: melt curves. B: difference curves
Candidates have been identified for leaf Phosphorus (P) and Nitrate (NO3-) concentration:
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Fig. 8: SNPs associated with variation in leaf P (A) and NO3- (B). Dotted lines show significance thresholds; p < 0.05 / 0.01, FDR corrected
Discussion: • • • • •
Next steps:
Strong positive correlation between leaf Ca & Mg consistent with previous studies4 Similar Ca & Mg QTLs indicate potential common processes affect accumulation Arabidopsis mutant experiment results reveal three genes influencing Ca/Mg levels Leaf P concentration appears to be at least partly dependent on root hair traits Gene candidates for NO3- uptake look promising from inferred functions
References: 1. Bus, A et al. 2011. Theoretical and Applied Genetics 123: 1413-1423. 2. Harper AL et al. 2012. Nature Biotechnology 30: 798-802.
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Investigate Ca / Mg candidate gene interactions and their link to nutrient uptake Examine the effects of low external Ca / Mg on the growth of mutant lines Characterise homozygous Bra.mgt7 mutants to test function in Brassica Investigate tissue specific expression and effects of overexpression of Bra.MGT7 Further investigate P and NO3- candidates and link to useful agronomic traits
Acknowledgements: 3. Gebert M et al. 2009. The Plant Cell 21: 4018-4030. Supervisors: Neil Graham & Martin Broadley 4. Broadley MR et al. 2003. Journal of Experimental Botany 55: 321-336. BBSRC RIPR Programme Grant BB/L002124/1
