TARGETED SATURATION MAPPING OF A HIGH RECOMBINATION REGION IN BARLEY
USING ESTs IDENTIFIED VIA SYNTENY TO RICE

David A. Kudrna¹, Kara Johnson¹ , Robert Brueggeman¹ , Arnis Druka¹, and Andris Kleinhofs²

 

¹Department of Crop and Soil Sciences, Washington State University,

Pullman, WA 99164-6420 USA

²Department of Crop and Soil Sciences and School of Molecular Biosciences,

Washington State University, Pullman, WA 99164-6420 USA

 

Hard-copy edition pages 13 - 21.

 

Introduction

Physical mapping identified high, medium and low recombination regions in the barley genome (Kunzel et al, 2000).  On the hypothesis that recombination occurs primarily in genes, the high and medium recombination regions are presumed to be gene rich, whereas the low recombination regions are presumed to be deficient or very low in gene sequences (Barakat, 1997).  In order to validate Kunzel's map, we are developing a  Bacterial Artificial Chromosome (BAC) based physical map for the barley chromosome 1(7H)S region defined by markers cMWG703 and MWG836.  This chromosome region encompasses approximately 40 cM (Igri x Franka map) and was projected to have a physical/genetic distance ratio of 100 kb per cM (Kunzel et al., 2000).

           

A physical BAC clone contig could be, in principle, developed by chromosome walking.  However, this is a very difficult undertaking and perhaps not possible for a large chromosome region in a large genome organism such as barley.  The alternative is saturation mapping leading to "chromosome landing", a term coined by Tanksley et al. (1995) to define an approach based on finding markers close enough to the gene of interest to reside on the same large insert clone as the gene of interest.  Saturation mapping can be practiced with random markers mapped in the whole genome.  An example of this approach is whole genome mapping with AFLP markers (Vos et al., 1995).  This technique, although very powerful, requires mapping very large numbers of markers in order to have a statistical chance of finding a marker near the target, particularly in a large genome.  A refinement of this approach is to target random markers at a specific chromosome region using bulked segregant analysis (Michelmore et al., 1991) or deletion lines to define the target.  Examples of such approaches can be found in Xu and Korban, (2000) and Gill et al. (1996).  The ideal would be to target specific markers to a specific chromosome region.  This was accomplished by Monna et al. (1997) using yeast artificial chromosome (YAC) clones to saturate the region containing the rice blast disease resistance gene Pi-b.  However, barley, like most species, does not have the luxury of a YAC clone physical map.  Previously we targeted specific rice markers to a specific barley genome region exploiting barley-rice synteny (Kilian et al., 1995).  This approach, while successful was still not good enough to saturate the specific barley genome region with markers close enough to allow "chromosome landing" with the ca. 100 kb insert BAC library available for barley (Yu et al., 2000).  Another approach is to exploit comparative mapping among many species to target specific markers to a specific chromosome region.  An example is the work of Faris et al. (2000).  Similar to the rice synteny, this approach works, but is not sufficient for complete saturation.  Here we describe an improvement of the rice synteny method where we use the rice genomic sequence to identify EST clones from barley and wheat for mapping to the region of interest.  Using this method, we have saturated the chromosome 1(7H)S target region between markers cMWG703 and MWG836 with markers and identified multiple BAC contigs.  The work is in progress, but we expect that eventually we will have a complete or nearly complete BAC contig of the region of interest.  This optimism is based on our work showing that the average barley BAC clone from the high or medium recombination regions contains 4-5 genes (Rostoks et al., 2002).  Assuming that the Triticeae (barley and wheat) EST database contains approximately 50% of all the genes, we should be able to find 2 markers per BAC clone, on the average.  There are, however, some BAC clones that contain only one gene.  Thus, some gaps may occur.  The limitations of this technique are only the available rice genomic sequence and the number of deposited Triticeae EST sequences.   

 

Methods

            The targeted gene rich region of barley chromosome 1(7H)S is flanked by the markers cMWG703 and MWG836.  Barley EST probes were selected from the syntenic rice chromosome 6 region (molecular markers C425A to S1434) by BLASTN analysis of the rice genomic sequences (http://rgp.dna.affrc.go.jp/) against the barely EST database (http://www.ncbi.nlm.nih.gov/BLAST/).  EST clones were selected as homologous to rice sequence based upon S-values greater than 70 and E-values less than 1e-10.  EST probes are available from Clemson University Genome Institute (CUGI; http://www.genome.clemson.edu/).  Probes were genetically mapped by RFLP using a set of recombinant lines selected from the North American Barley Genome Project Steptoe x Morex doubled haploid mapping population.  Protein function predictions were determined by BLASTX analysis of translated EST sequences with the protein sequence database (http://www.ncbi.nlm.nih.gov/BLAST/).

 

Physical BAC contigs were assembled manually following individual BAC confirmation with each EST.  BAC clones and screening filters are available from CUGI.

 

Results

Rice chromosome 6 from 6cM to 15 cM was identified as the barley chromosome 1(7H)S 12cM to 38 cM (cMWG703-MWG836) syntenic region by mapping rice probes in barley and barley probes in rice (data not shown).  Fortunately this rice region has been nearly completely sequenced with just one gap.

           

A total of 106 loci have been mapped to the chromosome 1(7H)S cMWG703 to MWG836 target region (Fig. 1).  Of these, 59 are ESTs (Table 1) that were identified by searching the barley EST database with rice PAC or BAC clone genomic sequences.  One is a disease resistance locus (Rcs5) and another is a morphological locus (Lga).  The remaining 47 loci were mapped previously using probes identified by synteny to wheat, rice, and oat maps or by random mapping.

           

The sequence from 19 rice PAC or BAC clones (there is considerable overlap in the sequence) from this region was used to identify a total of 159 EST clones from the barley EST data base.  Of these, 107 were genetically and/or physically mapped to 118 loci; 59 loci in the HRR.  Nine were non-polymorphic and 21 are still in process.  Many of the EST clones were single copy, 17 are duplicated and 3 hybridized to multiple (more than 10) bands on genomic Southern blots.  Additionally, 2 retroelement-like ESTs were identified by homology to the rice sequence.

           

Fifty-seven EST loci mapped outside the HRR (Table 2).  There was no obvious genetic clustering of these loci that would suggest a duplicated region in barley, however, the number of probes mapping outside the target region is higher than anticipated.  There may be several trivial explanations for this, other than chromosome rearrangements, and we are still investigating this. 

           

A total of 1149 BAC clones were identified by hybridizing the 159 identified EST clones to the barley cv. Morex 6.3X BAC library.  This identified 40 BAC contigs, but 32 singletons (no overlap with BAC clones from other probes) still remain (Fig.1).  A crude estimate of the physical size of the region covered with BAC clones to date yielded 5.5 Mb.  This estimate was derived from the 100 kb average size of the BAC clone inserts and by assuming 50% overlap for the clones in contigs.  Thus, the region is already somewhat larger than the approximately 4 Mb (40 cM x 0.1 Mb per cM) estimated by Kunzel et al. (2000).  However, given the translocation breakpoint method used and the lack of infinite breakpoints, the Kunzel et al. estimate and our data are probably within reasonable agreement.  BAC addresses identified by EST hybridization are available from our BAC database:  http://barleygenomics.wsu.edu/db3/db3.html.

 

Discussion

Using the rice-barley EST homology method, we have rapidly and efficiently more than doubled the saturation of the target region from 50 to 110 markers.  The work described here was completed at a time when only about 65,000 barley EST sequences were available in the database.  As of the end of Jan. 2002, a total of 144,000 barley and 73,000 wheat EST sequences have been deposited.  Thus, a new BLAST search of the Triticeae (barley and wheat) EST database should result in an even higher level of marker saturation.  To date we have not used the wheat EST clones, but they should work as well as the barley EST clones.  In addition, completion of the rice genomic sequencing of the syntenous region will facilitate this work.

           

Our data, showing that approximately 50% of the EST clones identified by the described procedure map to the target region, suggests that it is a valid and rapid technique for saturation mapping of specific chromosome regions in barley.  However, the number of probes mapping outside of the target region is larger than anticipated and bothersome.  Some possible trivial explanations include selection of EST with too low homology score, mapping of a related but not orthologous band, picking of the wrong EST, and library contamination.  We are investigating these ideas to determine if the ratio of those mapping to the right region vs. those mapping elsewhere can be improved.

           

The physical-genetic distance ratios are highly variable in the barley genome.   Kunzel et al. (2000) has identified the major features of the genome, but the variation within those features has remained hidden to the global look.  Thus, even in a high recombination region we can expect to find substantial variation.  We have observed that in this region. However, to date we have observed very few BAC clones that span recombination breakpoints.

 

The BAC contig of the target region, once completed, will provide access to numerous interesting genes, including seedling spot blotch resistance Rcs5, leaf wax (Cer-ze, Gsh3), long glume awn (Lga), nitrate assimilation (Nar3), winding dwarf (wnd), the chlorophyll genes Fch5, Yvs, possibly albino seedling 7 (abo7) and anthocyanin-less 1 (ant1).  Several QTL have also been mapped to this region including Fusarium head blight, heading date, yield, net blotch resistance (adult), spot blotch resistance (adult), tiller number, short and long day length, plant height, plant grain weight, kernel length and shape, and crossability with wheat. 

 

 

References

 

Barakat A., N. Carels and G. Bernardi. 1997. The distribution of genes in the genomes of Gramineae. Proc Natl Acad Sci USA 94: 6857-6861.

Faris, J.D., K.M. Haen and B.S. Gill.  2000.  Saturation Mapping of a gene-rich recombination hot spot region in wheat.  Genetics 154:823-835.

Gill, K. S., B. S. Gill, T. R. Endo and E. V. Boyko. 1996. Identification and high-density mapping of gene-rich regions in chromosome group 1 of wheat. Genetics 144: 1883-1891

Kilian, A., D. A. Kudrna, A. Kleinhofs, M. Yano, N. Kurata, B. Steffenson and T. Sasaki.  1995.  Rice-barley synteny and its application to saturation mapping of the barley Rpg1 region.  Nucleic Acids Research 23:2729-2733.

Kunzel, G., L. Korzum and A. Meister. 2000. Cytologically integrated physical restriction fragment length polymorphism maps for the barley genome based on translocation breakpoints. Genetics 154: 397-412.

Michelmore R. W., I. Paran and R. V. Kesseli.  1991.  Identification of markers linked to disease-resistance genes by bulked segregant analysis: A rapid method to detect markers in specific genomic regions by using segregating populations.  Proc. Natl. Acad. Sci. USA 88:9828-9832.

Monna, L., A. Miyao, H.S. Zhong, M. Yano, M. Iwamoto, Y. Umehara, N. Kurata, H. Hayaska and T. Sasaki.  1997.  Saturation mapping with subclones of YACs:  DNA marker production targeting the rice blast disease resistance gene, Pi-b.  Theor. Appl. Genet. 94:170-176.

Rostoks, N., Y-J Park, W. Ramakrishna, J. Ma, A. Druka, B.A. Shiloff, PJ. SanMiguel, Z. Jiang, R. Brueggeman, D. Sandhu, K. Gill, J.L. Bennetzen, and A. Kleinhofs.  2002. Genomic sequencing reveals gene content, genomic organization and recombination relationships in barely.  Functional and Integrative Genomics, in press.

Tanksley, S.D., M.W. Ganal, and G.B. Martin, G.B. 1995.  Chromosome landing: a paradigm for map-based gene cloning in plants with large genomes.  Trends in Genetics 11:63-68.

Vos, P., R., Hogers, M., Bleeker, M. Reijans, T. van de Lee,  M. Hornes, A. Frijters, J. Pot, J. Peleman, M. Kuiper and M. Zabeau.   1995.  AFLP: a new technique for DNA fingerprinting.  Nucleic Acids Research 23:4407-4414.

Xu, M.L. and S.S. Korban.  2000. Saturation mapping of the apple scab resistance gene Vf using AFLP markers.  Theor. Appl. Genet. 101:844-851.

Yu, Y., J. P. Tomkins, R. Waugh, D. A. Frisch, D. Kudrna, A. Kleinhofs, R. S. Brueggeman, G. J. Muehlbauer, R. P. Wise, and R. A. Wing.  2000.  A bacterial artificial chromosome library for barley (Hordeum vulgare L.) and the identification of clones containing putative resistance genes.  Theor Appl Genet 101: 1093-1099.

 

 

Figure 1

Click here for a .pdf file of Figure 1.

Genetic and Physical maps of the high-recombination region of barley chromosome 1 (7H)S between markers cMWG703 (bin 2) and MWG836 (bin 5).  Genetic distances are shown in centimorgans (cM).

 

Table 1.  Barley EST loci physically and/or genetically mapped in the high-recombination region of barley chromosome 1(7H)S between markers cMWG703 (bin 2) and MWG836 (bin 5).

 

EST (Acc.#)	PAC (Acc. #)	EST-PAC S-value	EST-PAC  E-value	Predicted Gene Function	Function              S -value	Function          E -value
BE230982	AB026295	SR	SR	proline-rich cell wall protein, sequence removed	SR	SR
BE060893	AP002542	206	2e-50	40S ribosomal protein S20, rice	218	2e-56
AW926257	AB026295	SR	SR	soluble starch synthase I, sequence removed	SR	SR
BF253755	AP000391	109	3e-21	Beta-5 subunit of 20S proteasome, rice	266	7e-71
BF256735	AP000399	98	2e-17	wak 1 gene, rice	160	2e-38
BF257072	AB023482	442	1e-121	splicing factor Prp8, Arabidopsis	338	2e-92
BF256448	AB023482	SR	SR	multicatalytic endopeptidase complex, sequence removed	SR	SR
BF256971	AP002071	434	1e-119	RNA-binding protein, Arabidopsis	213	7e-55
BE603328	AB026295	SR	SR	unknown protein, sequence removed	SR	SR
BE193581	AB026295	228	6e-57	MADS box protein, rice	303	1e-81
BE194830	AB023482	163	2e-37	homocitrate synthase,                               Thermus thermophilus	166	2e-40
BE216383	AP000391	117	2e-23	hypothetical protein, rice	248	8e-65
BF064968	AP000399;  AP003487	321;          321	5e-85             5e-85	unknown protein, Arabidopsis	125	3e-28
BF253546	AP002536	280	1e-72	serine / threonine phosphatase,                Nicotiana tabacum	229	1e-69
BE194136	AP002536	147	1e-32	prolyl aminopeptidase-like protein, Arabidopsis	198	2e-50
BF621935	AP002071; AP002854	248         248	5e-63                5e-63	unknown protein,  Arabidopsis	100	9e-21
BF623965	AP002542; AP000559	281             664	3e-73           0.0	arginine decarboxylase, rice	305	1e-82
BF625333	AP000399	404	1e-110	hexose carrier protein HEX6, rice	258	2e-68
BF629359	AP002854	214	8e-53	seed imbibition, Sip1, barley	243	1e-82
BF628496	AP002542; AP000559	353            353	1e-94               1e-94	Mg-protoporphyrin IX methyltransferase, rice	266	1e-70
BF631507	AP000399	133	3e-28	similar serine/threonine protein kinase, rice	233	2e-60
BF630362	AB023482	88	9e-15	kinase-like protein, Arabidopsis	113	6e-25
BF257002	AP000399	90	2e-15	similar cytochrome c oxidase subunit Vb precursor, rice	161	1e-50
BF267518	AP002854	305	2e-80	40S ribosomal protein S30, Arabidopsis	124	4e-28
BF263245	AB023482	167	2e-38	gamma adaptin 1, rice	386	1e-108
BF263968	AP000399	90	1e-15	no significant similarities
BE215412	AP002542	276	2e-71	EPSP synthase, rice	208	2e-53
BG414713	AP003632	162	6e-37	no good hits
BG415252	AP000399	305	3e-80	hypothetical protein, rice	376	1e-103

Table 1 continued

 

EST (Acc.#)	PAC (Acc. #)	EST-PAC S-value	EST-PAC  E-value	Predicted Gene Function	Function              S -value	Function E -value
BF622807	AP003632	160	2e-36	nucleosome assembly protein, Arabidopsis	218	4e-56
BF627428	AP003767	237	1e-59	proline-rich protein APG-isolog, Arabidopsis	333	1e-90
BF629541	AP003526; AP003708	188	6e-45	no goods hits
BG343171	AP003708	172	3e-40	homeodomain leucine zipper protein, rice	164	9e-41
AW982580	AP003526	194	9e-47	kinesin-like protein, Arabidopsis	309	2e-83
BG343499	AP003708	117	2e-23	serine/threonine protein kinase, Arabidopsis	172	4e-42
BE601610	AP003526	162	3e-37	putative protein, Arabidopsis	209	9e-54
BF257258	AP003708	190	9e-46	peptide methionine sulfoxide reductase, Arabidopsis	271	3e-72
BF260645	AP003487	135	7e-29	carboxypeptidase, Arabidopsis	258	4e-68
BF620281	AP003526	187	1e-44	hypothetical protein, Arabidopsis	265	3e-70
BF261716	AP003767	233	2e-58	MAP kinase kinase, Arabidopsis	171	3e-47
BF263248	AP000399	252	6e-64	arginine N-methyl transferase 1, Arabidopsis	226	5e-72
BF263807	AP003767	210	1e-51	unknown protein, Arabidopsis	169	2e-41
BF265658	AP003708	446	1e-122	root cap-specific protein, Maize	328	5e-89
BE213851	AP003708	131	1e-27	hypothetical protein, Arabidopsis	285	6e-76
BE214156	AP003487	82	1e-12	DNA-binding protein p24, Arabidopsis	90	3e-17
BF065761	AP003708	218	7e-54	hypothetical protein, Arabidopsis	273	1e-72
BF254888	AB026567c	337	1e-89	beta 5 subunit of 20S proteasome, rice	202	2e-51
BF623457	AP003454	146	3e-32	unknown protein, Arabidopsis	109	3e-23
BF254110	AP003454	277	1e-71	putative protein, Arabidopsis	135	8e-31
BE194388	AP002071; AP002854	149             149	4e-33          4e-33	beta-transducin-like protein, Arabidopsis	348	3e-95
BE602073	AB026295	168	4e-39	unknown protein, rice	199	1e-50
BG418108	AP002069	137	1e-31	hypothetical protein, rice	243	8e-64
BF261612	AP002542	735	0.0	transketolase, rice	402	1e-114
BF261621	AP002536	220	2e-54	serine/threonine protein phosphatase, alfalfa	142	3e-33
BF266534	AP004239	485	1e-134	putative lipase, Arabidopsis	226	1e-58
BE195608	AP000399	208	6e-51	early nodulin protein, rice	134	2e-30
AW926067	AP000559; AP000391	SR	SR	receptor-like protein kinase, sequence removed	SR	SR
AW926171	AP000559	SR	SR	Histone H3.2,minor, sequence removed	SR	SR
SR = sequence has been removed from the database.
c  = accession # corresponds to a mRNA rice sequence homologous to the barley EST.

 

 

 

Table 2.  Chromosome location(s) of barley EST loci that map away from the high-recombination region.

 

Chr-bin; locus	EST (Acc.#)	PAC (Acc. #)	EST-PAC S-value	EST-PAC  E-value	Predicted gene Function	Function              S-value	Function          E-value
1 (7H) - 002	BE455209	AP000391	246	2e-62	similar to lipase	303	1e-81
1 (7H) - 002	AW926881	AP000559	SR	SR	arginine decarboxylase	SR	SR
1 (7H) - 002	AW925350	AP000559; AP002542	SR	SR	hypothetical protein	SR	SR
1 (7H) - 002	BE195261	AP002542	?		granule bound starch synthase	196	8e-50
1 (7H) - 002	BF066009	AP000391	254	1e-64	hypothetical protein	72	4e-26
1 (7H) - 002	BE060779	AP000559; AP002542	76            76	5e-11                 5e-11	beta-I,3-glucanase	227	8e-57
1 (7H) - 005	BF625282	AP003019	143	2e-31	ribosomal protein 60S- L39	109	1e-23
1 (7H) - 006	BF257721	AP000559	511	1e-142	Histone H3.2,minor	266	1e-70
1 (7H) - 006	BE455049	AB023482	86	4e-14	no good hits
1 (7H) - 007	BE060921	AP000559; AP002542	292;       292	2e-76;    2e-76	NAM-like protein	258	3e-68
1 (7H) - 007	BF065540	AB023482	342	1e-91	hypothetical protein	167	4e-41
1 (7H) - 007	AW983378	AP003487; AP003767	187;    187	2e-44; 2e-44	pectate lyase	173	2e-42
1 (7H) - 007; A	AW926171	AP000559	SR	SR	Histone H3.2,minor, sequence removed	SR	SR
1 (7H) - 007; B	BF622807	AP003632	160	2e-36	nucleosome assembly protein, Arabadopsis	218	4e-56
1 (7H) - 008	BE215945	AP000399	121	5e-25	acyl-ACP thioesterase	154	3e-37
1 (7H) - 010	BG344471	AP003708	164	1e-37	beta-1 subunit of 20S proteasome	321	8e-89
1 (7H) - 012	BF259572	AP003019	473	1e-131	inorganic pyrophosphatase	303	1e-81
1 (7H) - 012	BF626991	AP002071	SR	SR	hypothetical protein	SR	SR
1 (7H) - 013	BG417554	AP003767	SR	SR		SR	SR
2 (2H) - 003	BG299346	AP003487	139	3e-30	sulfate transporter	254	3e-67
2 (2H) - 005	BF616634	AP002542	278	7e-72	unknown protein	311	5e-84
2 (2H) - 006	BE060723	AP002864	100	1e-18	Tubulin beta-3 chain	320	1e-106
2 (2H) - 008	BG417957	AP003526	85	9e-14	no good hits
2 (2H) - 008; B	BE060893	AP002542	206	2e-50	40S ribosomal protein S20, rice	218	2e-56
2 (2H) - 009	BF254704	AB026295	145	7e-32	HSPC133 protein	155	6e-37
2 (2H) - 010	BF256092	AP000391	SR	SR	no significant similarities	SR	SR
2 (2H) - 011	BF625997	AP002854	230	1e-57	alternative oxidase	253	9e-74
2 (2H) - 013	BF624194	AB023482	178	3e-42	AP2 domain-containing protein	133	8e-31
2 (2H) - 013	BF265753	AP003019	252	6e-64	H+ transporting ATPase	338	2e-92
2 (2H) - 015	BG366491	AP003526	291	5e-76	putative protein	173	5e-43
2 (2H) - 015	BF256699	AP003767	223	2e-55	no good hits
3 (3H) - 006	BF617975	AP000399	119	3e-24	arginine N-methyl transferase	137	4e-32
3 (3H) - 008; A 4 (4H) - 001; B 5 (1H) - 010; C	BE214588	AP002864	117	1e-23	tubulin beta-3 chain	407	1e-113

 

Table 2 continued

Chr-bin; locus	EST (Acc.#)	PAC (Acc. #)	EST-PAC S-value	EST-PAC  E-value	Predicted gene Function	Function              S-value	Function          E-value
3 (3H) - 011	BF257999	AP000399	SR	SR		SR	SR
4 (4H) - 002	BF258878	AP000559;         AP002542;            AP002071	SR	SR	unknown protein	SR	SR
4 (4H) - 002; A 4 (4H) - 010; B	BF258346	AP002854	490	1e-135	hypothetical protein	101	9e-21
5 (1H) - 006; A 6 (6H) - 006; B	BF065140	AP000399	78	1e-11	unknown protein	224	5e-58
5 (1H) - 007	BG369940	AP003526; AP003487	SR	SR	no good hits	SR	SR
5 (1H) - 008	BG367156	AP002069	294	4e-77	ubiquinol cytochrome c reductase	144	5e-34
5 (1H) - 008	BG414283	AP002542	162	4e-37	beta-tonoplast intrinsic protein	201	3e-51
5 (1H) - 011; A 7 (5H) - 006; B	AW983097	AB023482	82	1e-12	no good hits
5 (1H) - 013	BE060078	AP002542	86	7e-22	unknown protein	74	1e-12
6 (6H) - 005; B	BF261716	AP003767	233	2e-58	MAP kinase kinase, Arabadopsis	171	3e-47
6 (6H) - 006	BF253463	AP002864	117	1e-23	Tubulin beta-2 chain	244	8e-64
6 (6H) - 006	BG299297	AP002542	241	8e-61	granule-bound starch synthase precursor	350	8e-96
6 (6H) - 014	BG343190	AP002854	188	6e-45	TAT-binding protein	359	3e-98
6 (6H) - 014; A 3 (3H) - 015; B	BG344873	AP002854	180	1e-42	TAT-binding protein	324	4e-88
7 (5H) - 002; A 7 (5H) - 004; B	BE602168	AP003526	112	5e-22	enolase	441	1e-123
7 (5H) - 006	BF258338	AP000399	593	1e-167	hypothetical protein	249	2e-72
7 (5H) - 009	BE195592	AP002069	SR	SR	ethylene-forming-enzyme-like dioxygenase	SR	SR

SR = sequence has been removed from the database.