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Structural and functional analyses of the wheat genomes based on expressed sequence tags (ESTs) related to abiotic stresses J. Ramalingam, M.S. Pathan, O. Feril, Miftahudin, K. Ross, X.-F. Ma, A.A. Mahmoud, J. Layton, M.A. Rodriguez-Milla, T. Chikmawati, B. Valliyodan, R. Skinner, D.E. Matthews, J.P. Gustafson, and H.T. Nguyen
Abstract: To gain insights into the structure and function of the wheat (Triticum aestivum L.) genomes, we identified 278 ESTs related to abiotic stress (cold, heat, drought, salinity, and aluminum) from 7671 ESTs previously mapped to wheat chromosomes. Of the 278 abiotic stress related ESTs, 259 (811 loci) were assigned to chromosome deletion bins and analyzed for their distribution pattern among the 7 homoeologous chromosome groups. Distribution of abiotic stress related EST loci were not uniform throughout the different regions of the chromosomes of the 3 wheat genomes. Both the short and long arms of group 4 chromosomes showed a higher number of loci in their distal regions compared with proximal regions. Of the 811 loci, the number of mapped loci on the A, B, and D genomes were 258, 281, and 272, respectively. The highest number of abiotic stress related loci were found in homoeologous chromosome group 2 (142 loci) and the lowest number were found in group 6 (94 loci). When considering the genome-specific ESTs, the B genome showed the highest number of unique ESTs (7 loci), while none were found in the D genome. Similarly, considering homoeologous group-specific ESTs, group 2 showed the highest number with 16 unique ESTs (58 loci), followed by group 4 with 9 unique ESTs (33 loci). Many of the classified proteins fell into the biological process categories associated with metabolism, cell growth, and cell maintenance. Most of the mapped ESTs fell into the category of enzyme activity (28%), followed by binding activity (27%). Enzymes related to abiotic stress such as b-galactosidase, peroxidase, glutathione reductase, and trehalose-6-phosphate synthase were identified. The comparison of stress-responsive ESTs with genomic sequences of rice (Oryza sativa L.) chromosomes revealed the complexities of colinearity. This bin map provides insight into the structural and functional details of wheat genomic regions in relation to abiotic stress. Key words: wheat, EST, bin mapping, abiotic stress. Re´sume´ : Afin de mieux connaıˆtre la structure et la fonction des ge´nomes du ble´ (Triticum aestivum L.), les auteurs ont identifie´ 278 EST en lien avec les stress abiotiques (froid, chaleur, se´cheresse, salinite´ et aluminium) parmi 7 671 EST de´ja` assigne´s a` des chromosomes du ble´ par cartographie. Des 278 EST de stress, 259 (811 locus) ont e´te´ assigne´s a` des re´gions chromosomiques par analyse de de´le´tions. La distribution de ces EST chez les sept groupes de chromosomes home´ologues a e´te´ analyse´e. Celle-ci n’e´tait pas uniforme sur l’ensemble des diffe´rentes re´gions chromosomiques des trois ge´nomes du ble´. Les bras courts et longs des chromosomes du groupe 4 ont montre´ plus de locus au sein des re´gions distales par rapport aux re´gions proximales. Des 811 locus, 258, 281 et 272 ont e´te´ localise´s sur les ge´nomes A, B et D, respecReceived 13 December 2005. Accepted 19 June 2006. Published on the NRC Research Press Web site at http://genome.nrc.ca on 6 December 2006. Corresponding Editor: T. Schwarzacher.‘ J. Ramalingam. Division of Plant Sciences, University of Missouri, Columbia, MO 65211, USA; Department of Plant Molecular Biology and Biotechnology, Tamil Nadu Agricultural University, Coimbatore 641 003, India. M.S. Pathan, O. Feril, B. Valliyodan, R. Skinner, and H.T. Nguyen. Division of Plant Sciences, University of Missouri, Columbia, MO 65211, USA. Miftahudin and T. Chikmawati. Department of Agronomy, University of Missouri, Columbia, MO 65211, USA; Department of Biology, Bogor Agricultural University, Bogor 16144, Indonesia. K. Ross and J.P. Gustafson.1 USDA-ARS-PGRU, Columbia, Department of Agronomy, University of Missouri, Columbia, MO 65211, USA. X.-F. Ma. Department of Agronomy, University of Missouri, Columbia, MO 65211, USA; Forage Improvement Division, The Samuel Roberts Noble Foundation Inc., 2510 Sam Noble Parkway, Ardmore, OK 73401, USA. A.A. Mahmoud. Department of Agronomy, University of Missouri, Columbia, MO 65211, USA. J. Layton. Department of Agronomy, University of Missouri, Columbia, MO 65211, USA; DuPont Agriculture and Nutrition, Pioneer Hi-Bred International, Inc, 810 Sugar Grove Avenue, Dallas Center, IA 50063, USA. M.A. Rodriguez-Milla. Department of Agronomy, University of Missouri, Columbia, MO 65211, USA; Biochemistry and Molecular Biology Department, University of Nevada, Reno, NV 89557, USA. D.E. Matthews. USDA–ARS–WRRC, Albany, CA 94710, USA. 1Corresponding
author (e-mail:
[email protected]).
Genome 49: 1324–1340 (2006)
doi:10.1139/G06-094
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tivement. Le plus grand nombre de locus de stress abiotique a e´te´ trouve´ sur les chromosomes home´ologues du groupe 2 (142 locus) et le plus faible nombre a e´te´ observe´ chez le groupe 6 (94 locus). Quant aux EST spe´cifiques d’un ge´nome, c’est le ge´nome B qui en montrait le plus (7 locus), tandis qu’aucun n’a e´te´ observe´ au sein du ge´nome D. Pareillement, en ce qui concerne les EST spe´cifiques d’un groupe d’home´ologues, le groupe 2 en montrait le plus avec 16 EST uniques (58 locus), suivi du groupe 4 avec 9 EST uniques (33 locus). Plusieurs des prote´ines de fonction connue appartenaient a` des cate´gories associe´es au me´tabolisme, a` la croissance cellulaire et a` l’home´ostasie. La majorite´ des EST cartographie´s tombaient dans la cate´gorie des prote´ines a` activite´ enzymatique (28 %) ou a` domaine de liaison (27 %). Les enzymes implique´es dans le stress abiotique, comme la b-galactosidase, la peroxydase, la glutathione re´ductase et la tre´halose-6-phosphate synthase, ont e´te´ identifie´es. La comparaison des EST de stress avec les se´quences des chromosomes du riz (Oryza sativa L.) a re´ve´le´ les complexite´s de la coline´arite´. Cette carte apporte un e´clairage sur les de´tails structuraux et fonctionnels des re´gions ge´nomiques du ble´ en rapport avec la re´sistance aux stress abiotiques. Mots cle´s : ble´, EST, cartographie par re´gions, stress abiotique. [Traduit par la Re´daction]
______________________________________________________________________________________ Introduction Plants are continuously exposed to multiple abiotic environmental factors, including water availability, temperature variation, and the mineral content of soil, all of which can affect crop growth, development, yield, and quality. To survive under abiotic stress, plants have developed various mechanisms to perceive external signals and to manifest adaptive responses to the environment with proper physiological and morphological changes (Bohnert et al. 1995). In the past, the efforts to improve stress tolerance through conventional breeding and genetic engineering have been limited by the complexity of stress responses. The development of new tools for comparative genomics has made rapid progress in the discovery of novel genes responsible for crop improvement. As of 29 April 2005 a total of 26 773 945 ESTs from different plant and animal species have been sequenced and deposited into the database of the National Center for Biotechnology Information (NCBI, http://www.ncbi.nlm.nih. gov/dbEST). Wheat, with 589 455 ESTs, is the major shareholder among the plants. In 1999, the National Science Foundation funded a project to study the structure and function of the expressed portion of wheat genomes by deletion mapping over 8000 Triticeae unigene ESTs to hexaploid wheat (http:// wheat.pw.usda.gov/NSF; Gustafson et al. 2004). Cultivated wheat, comprising 3 diploid genomes (AA, BB, and DD), is widely adapted to diverse ecological conditions. Abiotic stresses caused by drought, heat, cold, and soil salinity are known as the major factors in the reduction of wheat yield (Boyer 1982; Tolmay 2001; Pellegrineschi et al. 2004). Although the polyploid nature of wheat enables it to adapt successfully to different ecological conditions, the complex genome structure makes it difficult to locate the genes responsible for stress reactions. The complete sequencing of cultivated wheat is problematical owing to its large genome size (16 000 Mb; Arumuganathan and Earle 1991; Bennett and Leitch 2005). The identification and sequencing of gene-rich regions in wheat is hindered by genome repetition (Feuillet and Keller 2002; Yan et al. 2003). Mapping and characterizing ESTs offers a manageable approach to the complex architecture and functioning of this species and may help unravel the genetics of stress responses. Extensive gene redundancy is known to exist across all metabolic pathways in both rice and Arabidopsis thaliana
(Goff et al. 2002). Multicopy genes in wheat may facilitate the tightly regulated expression of specific isoenzymes in specialized tissue or in response to environmental challenges (Lange et al. 2000; Dixon 2001; Goff et al. 2002). Genes involved in regulatory processes should be quite diverse, whereas those involved in essential processes such as cell division and protein synthesis are expected to be largely conserved (Poethig 2001). The aim of the present study was to identify, locate, and characterize ESTs related to abiotic stress (drought, heat, cold, salinity, and aluminum) using the 7671 deletionmapped EST unigene sequences, which were developed from 37 different cDNA libraries of wheat and closely related species created from different tissues, developmental stages, and growing conditions (Zhang et al. 2004; Lazo et al. 2004). By physically mapping ESTs to chromosome deletion bins and categorizing them as to biological and molecular functions, chromosomal regions of interest to abiotic stress might be identified. Comparison of the stress-related Triticeae ESTs to rice genome sequences might further illuminate structural, functional, and evolutionary characteristics of the wheat genomes.
Materials and methods Plant materials All of the aneuploid stocks used in the present analyses were developed in the hexaploid wheat Triticum aestivum ‘Chinese Spring’. The ‘Chinese Spring’ aneuploid stocks used in the present study included 21 nullisomic–tetrasomic (NT) lines, 24 ditelocentric (DT) lines (Sears 1954, 1966; Sears and Sears 1978), and 101 deletion lines (Endo 1990; Endo and Gill 1996). Successive, overlapping, deleted regions of the deletion lines delimited a physical region on a chromosome arm called a ‘‘bin’’ (http://wheat.pw.usda.gov/ NSF/deletionuse.gif). The deletion bin assignments indicated the physical location of each deletion interval to a specific chromosomal region based on the fractional length (FL) values established by Endo and Gill (1996). The most proximal in each arm was delimited by the most proximal breakpoint and by the centromeric breakpoint in the relevant ‘Chinese Spring’ NT and DT aneuploid stocks (Qi et al. 2003). These genetic stocks allowed for the assignment of ESTs to specific bins on individual chromosome arms (Peng et al. #
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2004; Conley et al. 2004; Munkvold et al. 2004; Miftahudin et al. 2004; Linkiewicz et al. 2004; Randhawa et al. 2004; Hossain et al. 2004). The 101 wheat deletion stocks were obtained from B.S. Gill (Kansas State University, Manhattan, Kans.) and the NT and DT aneuploids were obtained from the USDA – Sears Collection of wheat genetic stocks (University of Missouri, Columbia). cDNA libraries and abiotic stress related ESTs The details of libraries, EST development, and tissues involved can be obtained at http://wheat.pw.usda.gov/NSF/ project/library.html (Zhang et al. 2004; Lazo et al. 2004). As of 29 September 2003 a total of 7671 unique ESTs from 37 different cDNA libraries were mapped. From these 7671 mapped ESTs, abiotic stress related (drought, heat, cold, salinity, and aluminum) ESTs were identified using the Gene Ontology (GO) consortium database, which was downloaded from http://www.geneontology.org and used for sequence comparison with mapped EST sequences. The EST nucleotide sequences (actually the consensus sequences of their contigs unless they were singletons) were searched directly using BLASTx against the protein sequences in Swiss Prot+TrEMBL. BLASTx performs a translation of the nucleotide sequence in all 6 reading frames and searches all of them against the protein sequences (Altschul et al. 1990). The BLASTx significant level used was E value < 10–5. The GO categories of the proteins that matched were obtained from the GOA database, http://www.ebi.ac.uk/ GOA/. These matches were at various levels in the GO hierarchy, primarily leaf nodes. To summarize them at a level relevant to abiotic stress, a Perl script using GO::AppHandle (http://www.godatabase.org/dev/go-db-perl/doc/ go-db-perl-doc.html) was used to traverse the hierarchy and assign each match to higher levels. Levels 2, 3, 4, and 5 below the primary GO nodes biological_process and molecular_function were examined. The best results were from biological process level 3 and molecular function level 2. Each matched unigene was classified based on both biological process and molecular function, according to the GO database standard (Fig. 1A and 1B). Those ESTs linked to GO database abiotic stress responsive proteins were selected and used for further analyses. The 278 abiotic stress related ESTs are available as a supplementary table at http://wheat.pw.usda.gov/pubs/2005/Ramalingam/. Abiotic stress responsive ESTs As of 29 September 2003, 7671 ESTs were deletion mapped in wheat chromosomes. The ESTs were developed from a diverse set of 37 cDNA libraries including several from tissues subjected to stress conditions, which provided representative samples of stress-related ESTs. These 7671 mapped ESTs were classified using the biological process and molecular function ontologies according to the GO database. Among the mapped ESTs, 4184 significantly matched with at least 1 protein sequence in the GO database. To investigate the detailed function of each EST, the GO database was analyzed at level 4 for both molecular activity and biological process. Of the 4184 EST hits to the protein database, 278 (6.7%) were identified as abiotic stress related proteins (E < 10–5). The library details of selected abiotic stress related ESTs are presented in Table 1. The physical
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location and biological function of those ESTs were further analyzed. Deletion mapping All procedures used for genomic DNA isolation, restriction endonuclease digestion, gel electrophoresis, and DNA gel blot hybridization were as described by Akhunov et al. (2003) and are available at http://wheat.pw.usda.gov/NSF/ project/. For Southern analysis, genomic DNA from each aneuploid and deletion stock was digested with EcoRI. l DNA, digested with HindIII and BstEII, was used as a size marker. Deletion mapping was performed by hybridizing the cDNA clone corresponding to each EST to a Southern blot of DNA from a panel of wheat aneuploid and deletion stocks (Qi et al. 2003, 2004). Absence of a particular restriction fragment in the lane for a particular stock indicated that the locus was distal to the corresponding deletion breakpoint (Fig. 2). The EST analyses were carried out in 10 mapping laboratories, and all analyzed images are available at http://wheat.pw. usda.gov/cgi-bin/westsql/map_locus.cgi. Wheat-rice comparative analysis All abiotic stress responsive EST nucleotide sequences were compared with the ordered BAC and (or) PAC clones of the rice genome (http://www.ncbi.nlm.nih.gov/BLAST/). The best hit (at more than 70% identity with E value £ 10–5 and at least 100 bp sequence length) was used for synteny analysis.
Results As of 29 September 2003, 7671 ESTs were deletionmapped in wheat chromosomes. Among the mapped ESTs, 4184 significantly matched with at least one protein sequence in the GO database. Of the 4184 EST hits to the protein database, 278 (6.7%) were identified as abiotic stress related proteins (E < 10–5). The library details of selected abiotic stress related ESTs are presented in Table 1. The 278 stress-related ESTs detected 1401 restriction fragments accounting for 882 loci. Nineteen of these ESTs (71 loci) hybridized to the same fragments in all the deletion lines tested as well as the ditelosomics and nullitetrasomics, and therefore could not be mapped and were omitted from the analysis. The lack of polymorphism in the hybridization of these 19 ESTs was probably due to a much higher homology among these orthologous genes on the 3 genomes. Qi et al. (2004) reported that they could not assign 601 EST probes to a chromosome or a chromosome bin for various reasons including missing DNA lanes, poor blots, and comigrating fragments. The 19 abiotic stress related ESTs fall under this latter category. This left 259 ESTs (811 loci), which were mapped throughout the wheat genome. Of the 811 total loci, 68 were deletion mapped to chromosomes or chromosome arms but not to deletion bins. Because the number of abiotic stress related loci detected in the A, B, and D genomes were not uniform, the data could not be pooled for ANOVA and a w2 test was used to show significant differences between and among genomes, among chromosomes, chromosome arms, and gene density. The B genome contained more EST loci (281) than either the A (258) or D (272) genomes ( 2, p > 0.05), but the differences were not #
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Fig. 1. Gene ontology classification of ESTs mapped on wheat chromosomes corresponding to various biological processes (A) and molecular activities (B).
Table 1. Library details of abiotic stress related ESTs. Library name AS040E1X SC024E1X TA001E1X TA005E1X TA006E1X TA006E2N
Genus species Aegilops speltoides Secale cereale Triticum aestivum Triticum aestivum Triticum aestivum Triticum aestivum
Genotype F2sa ‘Blanco’ ‘Cheyenne’ ‘Chinese Spring’ ‘Chinese Spring’ ‘Chinese Spring’
Tissue Anther Anther Endosperm Seedling Shoot Shoot
Stage Premeiotic Mature 5–30 DPAb 5d Seedling Seedling
TA007E1X TA008E1X TA008E3N
Triticum aestivum Triticum aestivum Triticum aestivum
‘Chinese Spring’ ‘Chinese Spring’ ‘Chinese Spring’
Seedling Root Root
5d Seedling Seedling
TA009XXX
Triticum aestivum
‘Sumai3’
Spike
Mature
TA012XXX TA015E1X TA016E1X TA017E1X TA018E1X TA019E1X
Triticum Triticum Triticum Triticum Triticum Triticum
aestivum aestivum aestivum aestivum aestivum aestivum
‘Brevor’ ‘Chinese ‘Chinese ‘Chinese ‘Chinese ‘Chinese
Spring’ Spring’ Spring’ Spring’ Spring’
Embryo Seedling Crown Spike Spike Spike
Mature dormantc 14 d Seedling Post anthesis Post anthesis Pre anthesis
TA027E1X TA031E1X TA032E1X TA037E1X
Triticum Triticum Triticum Triticum
aestivum aestivum aestivum aestivum
TAMW101 ‘Chinese Spring’ ‘Chinese Spring’ ‘Chinese Spring’
Leaf Flag leaf Spike Sheath
Full tillering Full tillering Post anthesis Seedling
TM043E1X TT039E1X Total
Triticum monococcum Triticum turgidum
DV92 ‘Landon-16’
Shoot apex All types
7-week-old plant Mature
Treatment Normal Normal Normal Dehydration Etiolated and unstressed Etiolated, unstressed, and normalized Cold stress Etiolated and unstressed Etiolated, unstressed, and normalized Fusarium graminearum challenged Imbibedd 2 h heat-shock cycles Vernalized 20, 30, 45 DAPe 5, 10, 15 DAP 2 cm spike to yellow anther 78% and 65% RWCf Heat stress Heat stress 5–20 DAP Salt stress 12h and 7d hydroponics Early reproductive apex Normal
No. of ESTs 10 13 17 1 12 11 5 30 32 5 1 8 14 5 23 58 4 8 7 4 8 2 278
a
F2s from 2-12-4-8-1-1-1-(1) PI36909-12-811-(1). Days post anthesis. c Mature, dormant seeds. d Seeds imbibed in 20 mmol/L ABA for 12 h at 30 8C. e Days after anthesis. f Relative water content. b
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Fig. 2. Physical bin map of wheat homoeologous chromosomes showing the number of stress-responsive EST loci in different deletion bins. The numbers on the left indicate the number of ESTs (EST loci) mapped to the short and long arms of each chromosome. The colorcoding corresponds to the distribution pattern of ESTs in the deletion bins. Yellow signifies bins with more than 10 abiotic stress related EST loci, whereas blue signifies empty bins.
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Fig. 2. (continued).
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Fig. 2. (continued).
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Fig. 2. (concluded).
Table 2. Distribution of abiotic stress related EST loci among the homoeologous chromosome groups. Homoeologous groupa 1 2 3 4 5 6 7 Total
Physical length (m) 31.3 35.5 36.0 32.3 34.0 32.4 33.9 235.4
Observed loci 96 142 115 122 139 94 103 811
Expected locib 107.8 122.3 124.0 111.3 117.2 111.6 116.8 .
p valuec 0.0235
.
a
Combined data for 3 genomes. The expected loci are based on chromosome physical length (Gill et al. 1991). c Chi-square test indicated the probability of departure from the expected loci. b
significant. The homoeologous chromosome groups with the greatest number of abiotic stress related loci were groups 2 (142 loci) and 5 (139 loci). The differences in EST loci distribution among the homoeologous groups was significant ( 2, p < 0.05, Table 2). The differences in EST loci distribution within homoeologous group 2, among chromosomes 2A, 2B, and 2D, was also significant ( 2, p < 0.006, Table 3). Compared with the A and D genomes, the B genome
had a significantly ( 2, p < 0.05) greater number of EST loci in all the homoeologous groups except 2 and 4. The total number of observed fragments ranged from 1 to 24, with an average of 5 restriction fragments and 2.9 mapped loci per EST. The ESTs that detected all 3 homoeologues without cross hybridizing to other chromosomes amounted to 17 that mapped to homoeologous group 1 chromosomes, 18 to group 2, 15 to group 3, 12 to group 4, 15 each to groups 5 and 6, and 10 to group 7. Gene density was defined in the present study as the ratio of mapped loci to the expected. Gene density, in this study, was higher in the D-genome chromosomes of all of the homoeologous chromosome groups, except for groups 5 and 6. Chromosome 2D showed the highest relative gene density (1.41) followed by 1D (1.32). The lowest gene densities (
