Comparative genomics of banana and rice genomes: Insight into an RGA cluster

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Franc-Christophe BAURENS1_{, Robert Neil Gerard MILLER}2_{, Magali LESCOT}3_{, Mathieu ROUARD}4_{, Georgios Joannis PAPPAS Jr}2_{, Takashi MATSUMOTO}5_{, Nicolas ROUX}4_{, Takuji SASAKI}5_,

Stéphanie SIDIBE BOCS1_{and Angélique D’HONT}1_. 1 UMR DAP, CIRAD, Montpellier, France

2 Bioinformatics, UCB, Brasilia, Brazil 3 _{LGPD CNRS, Marseille FRANCE}

4 CfL, Bioversity, Montpellier, France

5 RGP, NIAS, Japan Corresponding author : [email protected]

References:

[1] Miller et al. (submitted 2007)

[2] Sidibe Bocs et al. (ISMB congress, Vienna 2007) [3] Foissac et al. BMC Bioinformatics 6, 25 ( 2005) [4] Katoh et al. Nucleic Acids Res. 33, 511 (2005) [5] Guindon & Gascuel. Syst Biol 52,696 (2003) [6] Kozik et al. Bioinformatics 18, 335 (Feb 2002) [7] Pei et al. Plant Sci. 172, 1166 (2007)

[8] Pereza-Echeverria et al. Mol. Gen. Genomics (2007)

30 31 32 34 35 29 33 36 Os06g066 (67000 bp) 190 180 160 150 130 mTERF 120 ** 100 95 90 ** 60 50 40 ** 20 10 Ma4052E23 (79629 bp) 280 260 mTERF 250 240 230 220 ** 200 ** 180 * 170 160 ** 150 ** 140 ** 130 ** 110 ** 80 60 ** 50 30 20 10 * MbP032N20 (141036 bp) 10 * 180 200** 210mTERF 175 170 ** 160 150 140 130 120 * 110 ** 100 ** 90 ** 80 75 50 40 30 MaC091O16 (92764 bp) 0.0 20000.0 40000.0 60000.0 80000.0 100000.0 120000.0 140000.0 Os11g067 (140000 bp)

Retroelement, Gag-Pol genes RGA RGA fragments Other predicted genes

1 Phylogeny of Musa and rice NBS domain of RGAs

protein MAFFT [4], PhyML [5].

2 Phylogeny of Musa MARGA08 CC-NBS-LRR

Complete CDS manual alignment, phylogeny by PhyML [5].

3 Phylogeny of Musa MARGA08

CDS and fragment

manual alignment, PhyML [5].

52E23g040 32N20g160 91O16g010 91O16g110 32N20g150 91O16g100 32N20g200 32N20g010 32N20g180 32N20g130 52E23g120 91O16g200 32N20g140 91O16g090 91O16g170 32N20g110 52E23g090 Os02g04568 Os08g03326 Os11g02434 Os11g06745 Os11g06752 Os11g06736 YR9 ORYSJ Os11g06744 Os11g06739 Os11g06768 Os11g06765 Os11g06238 Os10g04739 Os03g06894 Os12g01991 Os09g03085 Os11g04791 Os11g07041 Os05g02507 Os07g04814 Os07g04813 Os09g03135 Os05g03795 Os05g03803 Os08g05430 Os10g01306 RP1D MAIZE CRE3 WHEAT Os06g06190 Os11g04291 XA1 ORYZA 100 100 100 96 83 100 100 99 84 99 64 67 98 83 92 100 96 94 61 73 92 99 95 99 70 100 0.1 32N20g020 91O16g110 32N20g150 32N20g220 91O16g090 32N20g140 32N20g110 91O16g170 52E23g120 91O16g200 52E23g040 32N20g160 91O16g100 91O16g010 52E23g090 32N20g010 32N20g130 32N20g200 32N20g180 32N20g050 100 100 100 56 100 96 99 100 100 0.05 I II III IV 32N20g140 91O16g170 32N20g110 91O16g200 52E23g120 32N20g160 91O16g100 91O16g010 91O16g090 52E23g040 32N20g150 91O16g110 32N20g020 32N20g220 32N20g060 52E23g010 52E23g100 91O16g180 91O16g120 32N20g130 32N20g010 32N20g250 52E23g090 91O16g080 32N20g200 32N20g180 32N20g240 91O16g075 52E23g095 91O16g175 32N20g050 52E23g060 100 99 61 100 100 100 53 85 100 100 56 64 100 94 100 65 0.05 III IV II I

Results

The 3 BAC clones belong to the same RGA locus:

Sequence comparison using ACT and mLagan Vista [2] have shown that the 3 BACs share more than 30Kb of genomic sequence. Using automatic predictions (Eugene [3]) and subsequent manual annotation, presence of mitochondrial transcription termination factor gene (mTERF) in the 3 BACs have been reported together with presence of a Serine threonine-protein kinase gene (STK) in two of the BACs (see purple lines in Figure 4).

RGA sequence alignment:

Multiple alignments were performed on the 34 RGAs using Mafft, ClustalW and finally checked manually. MARGA08 cluster (90% identity) contains both complete and fragmented RGAs in two orientations with most of the genes on the reverse strand (see Figure 4). MARGA08 coding sequences (CDS) contain several microsatellite motifs which could contribute to diversity of sequence and potentially to functionnal shifts.

Phylogenetic analysis:

Based on sequence alignments, phylogenetic analyses have been performed using PhyML [5]. Phylogenetic analysis of both banana and rice RGAs shows that CC-NBS-LRR clusters evolved independently in the two species (see tree 1), moreover, distances within Musa are smaller than in rice indicating more recent duplication events in Musa. Four groups of genes have been defined (highlighted in color: green, blue, orange and red). 32N20g50 gene present on the direct strand is clearly separated from other RGAs present on the reverse strand (see tree 2). Analysis of MARGA08 family members including pseudogenes and fragments indicate that group IV contains all gene and frag-ments oriented on the direct strand (see tree 3) and group III contain all other fragfrag-ments (light green).

Introduction

Cultivated bananas are highly sterile, parthenocarpic, vegetatively propagated plants. Most of the economically important clones are polyploid and very suceptible to disease (e.g. fungi, bacteria, viruses).

MARGA08 probe [1], a resistance gene analog (RGA) of coiled coil - nucleic binding site - leucine rich repeat (CC-NBS-LRR) type was used to screen three banana BAC libraries.

Two from wild diploid species potentially source of resistance to Black sigatoka disease: Musa acuminata spp. burmannicoides Calcutta 4 (AA-MA4), Musa balbisiana “Pisang Klutuk Wulung” (BB-MbP) and

one from a very susceptible triploid cultivated clone Cavendish - Grande Naine (AAA-MAC).

Positive BAC clones were subjected to BAC Fingerprint and RFLP analysis [1]. These revealed that MARGA08 is organized in cluster in the three banana clones. One BAC containing MARGA08 cluster per banana accession was sequenced and compared.

4 Musa - Rice RGA cluster comparison using GenomePixelizer [6]

Othologous relationships identified using BBMH, ACT, Lagan/Vista [2] and phylogenetic analysis, are indicated by colored lanes. Star indicates potential expression (see Table); no star: pseudogenes and fragments.

Conclusion and propects

The MARGA08 gene ancestor seems to be mono-exonic.

RGA cluster seems to be the result of a complex pattern of amplification involving gene du-plication before and after M. acuminata / M. balbisiana speciation.

Further investigations will involve (i) the fine study of microsatellite polymorphism inside coding sequence, (ii) the determination of the type of selection affecting MARGA 08 genes by computing ω = dN/dS ratio, (iii) the complete sequencing of MARGA08 locus in Musa, including haplotypes and (iv) comparison with recent RGAs discovered in banana [7-8]

M ARG A08 L (aa) CDS structure Expressed M icrosatellites

32N20g010 1000 2 introns, 4 sm all internal ∆ M ay be (*) _{(S)14-(A)1-(E)21}

32N20g130 1070 1 exon Predicted (**) _{(S)5-(A)3-(E)11}

32N20g180 926 1intron, 1 large internal ∆ M ay be (*) _{(S)3-(A)4-(E)9}

52E23g090 1057 1 exon Predicted (**) _{(S)3-(A)0-(E)4}

52E23g010 793 1 retro, N-term C-term ∆ Ψ _{(S)5-(A)0-(E)0}

32N20g060 931 3 stops in fram e, N-term ∆ Ψ _{(S)9-(A)0-(E)0}

91O 16g120 860 N-term ∆ M ay be (*) _{(S)6-(A)4-(E)18}

32N20g020 1100 1 retro Ψ _{(S)5-(A)5-(E)4}

91O 16g110 1067 1 exon Predicted (**) _{(S)10-(A)4-(E)5}

91O 16g010 1057 1 stop in fram e M ay be (*) _{(S)6-(A)5-(E)6}

91O 16g090 1064 1 exon _{Predicted (**) (S)12-(A)0-(E)11}

Table Musa Resistance Gene Analog features: MARGA08 cluster [1]

L: protein length, ∆: deletion, Ψ: pseudogene