Maximal sequence length of exact match between members from a gene family during early evolution

Maximal sequence length of exact match between members from a gene family during early evolution 1 first-author 2 1 2 †E-mail:fanlj@zju.edu.cn †E-mail:fanlj@zju.edu.cn fanlj@zju.edu.cn Institute of Crop Science, Zhejiang University, Hangzhou 310029, China Institute of Crop Science, Zhejiang University, Hangzhou 310029, China Institute of Bioinformatics/IBM Biocomputational Lab, Zhejiang University, Hangzhou 310029, China Institute of Bioinformatics/IBM Biocomputational Lab, Zhejiang University, Hangzhou 310029, China Mutation (substitution, deletion, insertion, etc.) in nucleotide acid causes the maximal sequence lengths of exact match (MALE) between paralogous members from a duplicate event to become shorter during evolution. In this work, MALE changes between members of 26 gene families from four representative species ( Arabidopsis thaliana , Oryza sativa , Mus musculus and Homo sapiens ) were investigated. Comparative study of paralogous’ MALE and amino acid substitution rate ( d A < 0.5) indicated that a close relationship existed between them. The results suggested that MALE could be a sound evolutionary scale for the divergent time for paralogous genes during their early evolution. A reference table between MALE and divergent time for the four species was set up, which would be useful widely, for large-scale genome alignment and comparison. As an example, detection of large-scale duplication events of rice genome based on the table was illustrated. Maximal length of exact match (MALE) Divergent time Gene family Minimal length of exact match (MILE) Genome alignment INTRODUCTION 1 Gene duplication provides a main resource of new genes in genomes (Ohno, 1970 1970 ; Brown, 1999 1999 ). Sequences of two paralogous genes from a duplication event will become different from each other along with evolutionary processes. And the difference in sequences caused by substitution, deletion, insertion of nucleotide acid will cause maximal sequence lengths of exact match (MALE) between paralogous members from a gene family to become shorter during evolution. As for example, of a newborn gene, its sequence is the same as that of its parental (paralogous) gene, i.e. the MALE equals to the length of the original sequence. During the evolutionary process, the MALE would become shorter gradually when genetic mutations occurred. As for gene families, the exact trend of the MALE changes among paralogous genes still remain obscure, and a detailed description of the relationship between MALE and evolutionary time is needed. For random sequences, the longest match and its statistical significance between two random DNA sequences has been well documented in early study on modelling a random DNA sequence alignment (Arratia et al., 1986 1986 ; Karlin and Altschul, 1990 1990 ). In large-scale genome sequence alignment, a parameter named minimal sequence length of exact match (MILE), was usually used in corresponding algorithms to limit the searching space and return their results in practical time, such as algorithms implicated by MUMmer, a very fast and widely used program for large-scale genome alignment and comparison (Delcher et al., 2002 2002 ). In such algorithms, all exact matches between two target genomic sequences will be located at the first step. All exact matches under the value of MILE which was set beforehand, will be ignored in the next alignment. Gene sequences (coding sequences or translated amino acid sequences) on the genomic sequences are usually used in genome alignment such as PROmer of MUMmer packet, which translates target genomic sequence into protein sequences through six open reading frames. In practice, the value of MILE is usually regulated (default value was set as 6 aa in PROmer) for rational hits to be returned. High MILE value chosen, leads to fewer hits returned. Apparently, to gene sequences, MILE implicates some kinds of similarity, or evolutionary scale. But the evolutionary significance of MILE in genome alignment has not been reported yet. In this study, we investigated the MALE changes of main gene families from four representative species ( Arabidopsis thaliana , Oryza sativa , Mus musculus and Homo sapiens ) during evolution. Our results indicated that MALEs were related with evolutionary time significantly during early evolution of gene families, and four curve functions between MALE and evolutionary time were created for the four species using data on their gene families, respectively. Based on the four functions, a reference table between MALE and its corresponding evolutionary time was also constructed for the four species. As an important application of our study on genome alignment, an example of study on large-scale duplication events of rice genome was illustrated. MATERIALS AND METHODS 1 Sequence data source 2 Protein sequences of 9 gene families (GTPBP, SCDehydRed, MFS, UDPGlycTnsf, GSDLLipase, Polygalns, SubtilisinSP, CytP450 and Calmod) of Arabidopsis thaliana were downloaded from http://www.tc.umn.edu/~cann0010/genefamilyevolution/index.html http://www.tc.umn.edu/~cann0010/genefamilyevolution/index.html and 17 gene families of other three species (LTP, Peroxidase, ABC, NbsLrr and CytP450 of Oryza sativa ; Mage, Hox, ABC, Potassium voltage-gated channel, Matrix metalloproteinase, kallikrein and CytP450 of Homo sapiens ; ABC, Collagen, Hox, Synaptotagmin and CytP450 of Mus musculus ) were selected from the protein database of NCBI ( http://www.ncbi.nlm.nih.gov http://www.ncbi.nlm.nih.gov ). Average number of members of a gene family was 98 (ranging from 92 to 112). Gene Family Criteria: Amino acid sequences of members from gene families must have over 40% sequence similarity and contain all key amino acid signature motifs of the corresponding gene family. Two hundred random sequences were created using PERL program. Estimation of MALE and amino acid substitution rates of paralogous genes 2 MALEs between paralogous genes from a gene family were calculated using mummer program of MUMmer package (Delcher et al., 2002 2002 ). The amino acid substitution rate ( d A ) was estimated using the aaml program of PAML package (Yang, 1999 1999 ) with the Dayhoff matrix. The divergence time was calculated based on a molecular clock rate of 9×10 −10 nonsynonymous substitutions per site per lineage per year and 2.25 nonsynonymous substitutions per amino acid change (Lynch and Conery, 2000 2000 ; Goff et al., 2002 2002 ). RESULTS 1 MALE changes during evolution 2 In order to show an enlarged picture of MALE changes of gene families during evolution, those big size gene families from four representative species were chosen. A total of 26 gene families averaging 98 members per family were used in this study. For brevity, we depicted only one big gene family, cytochrome P450 (Fig. 1 1 ). The other families, however, did behave as we had expected, and consistent with their evolutionary clade. A clear changing trend between MALE and amino acid substitution rate ( d A ) of paralogs from a gene family could be observed in the dot-plot of the Arabidopsis P450 gene family for low d A values ( < 0.5) (Fig. 1a 1a ), and therefore, a curve function could be created to fit it significantly (Fig. 1c 1c ). The results indicated that MALEs of paralogous genes decreased sharply at the beginning period of evolution ( d A < 0.1) and then became slower during early evolution ( d A < 0.5). Along with the further accumulation of mutations ( d A > 0.5), MALE decreased very slowly and ultimately stayed around a fixed value (6–10 aa). MALEs distribution of random sequences indicated that almost (95%) of their MALEs fell into a range of 3–6 aa (Fig. 1d 1d ). Besides the random sequences by computer simulation, we also investigated the MALE using real biological sequences by random by choosing 93 protein sequences of 27 species from Swiss-Prot protein database. The results were similar to those by computer simulation, and suggested that MALEs were related with divergence time significantly during early evolution of the gene families. The trend of MALE changes of P450 gene family presented almost the same pattern in four different species (Fig. 1c 1c ), representing a wide range of the biological kingdom ( Arabidopsis and Oryza sativa for dicot and monocot plants, Homo sapiens and Mus musculus for vertebrate). The results suggested that MALEs of a common gene family tended to behave similarly during evolution in different species. Then how did the different gene families in the same species behave? Based on nine gene families from Arabidopsis , the results showed that MALE changes (lines) of different gene families were basically the same (Fig. 1b 1b ), although there existed some differences among them, which actually stemmed from the different evolutionary speed among genes and gene families in a genome. MALE and divergent time 2 The above results indicated that MALE changes could be considered as a good scale of evolutionary time during early evolution of gene families, and different gene families from the same species basically present a common pattern of MALE changes during evolution. This pointed out the possibility of setting up a reference table relating MALE and evolutionary time at a species (genome) level. The table would be useful and convenient for sequence analysis at genome level, such as large-scale genome alignment. In such a table (Table 1 1 ), MALEs and corresponding d A values and divergence times were averaged from representative gene families of the four species. Therefore, duplicate events that occurred in a genome at different times during evolution can be estimated by just choosing different MALE values based on the table. A detailed example will be illustrated in the next section. 2 MALE (aa) 2 Arabidopsis 2 Oryza 2 Mus 2 Homo MY d A MY d A MY d A MY d A 7 164 (191) 0.6576 145 (177) 0.5807 168 (189) 0.6701 165 (190) 0.6592 8 158 (185) 0.6332 142 (173) 0.5671 163 (186) 0.6526 162 (187) 0.6462 9 152 (179) 0.6097 138 (169) 0.5539 159 (182) 0.6355 158 (185) 0.6335 10 147 (173) 0.5871 135 (166) 0.5410 155 (179) 0.6189 155 (182) 0.6210 11 141 (167) 0.5653 132 (162) 0.5284 151 (176) 0.6027 152 (179) 0.6087 12 136 (161) 0.5444 129 (158) 0.5161 147 (173) 0.5870 149 (177) 0.5967 13 131 (156) 0.5242 126 (155) 0.5040 143 (170) 0.5716 146 (174) 0.5850 14 126 (151) 0.5047 123 (151) 0.4923 139 (167) 0.5567 143 (172) 0.5735 15 121 (146) 0.4860 120 (148) 0.4808 136 (164) 0.5421 141 (169) 0.5622 16 117 (141) 0.4680 117 (145) 0.4696 132 (162) 0.5279 138 (167) 0.5511 17 113 (136) 0.4506 115 (141) 0.4586 129 (159) 0.5141 135 (165) 0.5402 18 108 (132) 0.4339 112 (138) 0.4479 125 (156) 0.5007 132 (162) 0.5296 19 104 (127) 0.4178 109 (135) 0.4375 122 (153) 0.4876 130 (160) 0.5191 20 101 (123) 0.4023 107 (132) 0.4273 119 (151) 0.4748 127 (158) 0.5089 21 97 (119) 0.3874 104 (129) 0.4173 116 (148) 0.4624 125 (155) 0.4989 22 93 (115) 0.3730 102 (126) 0.4076 113 (145) 0.4503 122 (153) 0.4891 23 90 (111) 0.3592 100 (123) 0.3981 110 (143) 0.4385 120 (151) 0.4794 24 86 (107) 0.3458 97 (121) 0.3888 107 (141) 0.4271 117 (149) 0.4700 25 83(104) 0.3330 95 (118) 0.3797 104 (138) 0.4159 115 (147) 0.4607 26 80(100) 0.3207 93 (115) 0.3709 101 (136) 0.4050 113 (145) 0.4516 27 77(97) 0.3088 91 (113) 0.3622 99 (133) 0.3944 111 (142) 0.4427 28 74 (94) 0.2973 88 (110) 0.3538 96 (131) 0.3841 109 (140) 0.4340 29 72 (90) 0.2863 86 (108) 0.3455 94 (129) 0.3741 106 (138) 0.4255 30 69 (87) 0.2757 84 (105) 0.3375 91 (127) 0.3643 104 (136) 0.4171 35 57 (74) 0.2282 75 (94) 0.2999 80 (116) 0.3191 94 (127) 0.3776 40 47 (62) 0.1889 67 (84) 0.2665 70 (106) 0.2795 85 (118) 0.3418 45 39 (53) 0.1564 59 (75) 0.2368 61 (98) 0.2448 77 (110) 0.3094 50 32 (44) 0.1294 53 (67) 0.2105 54 (89) 0.2144 70 (102) 0.2801 55 27 (37) 0.1071 47 (60) 0.1871 47 (82) 0.1878 63 (95) 0.2536 60 22 (32) 0.0887 42 (53) 0.1662 41 (75) 0.1645 57 (89) 0.2296 65 18 (27) 0.0734 37 (48) 0.1477 36 (69) 0.1441 52 (82) 0.2078 70 15 (22) 0.0608 33 (42) 0.1313 32 (63) 0.1262 47 (77) 0.1882 75 13 (19) 0.0503 29 (38) 0.1167 28 (58) 0.1105 43 (71) 0.1703 80 10 (16) 0.0416 26 (34) 0.1037 24 (53) 0.0968 39 (66) 0.1542 85 9 (13) 0.0345 23 (30) 0.0922 21 (49) 0.0848 35 (62) 0.1396 90 7 (11) 0.0285 20 (27) 0.0819 19 (45) 0.0743 32 (58) 0.1264 95 6 (10) 0.0236 18 (24) 0.0728 16 (41) 0.0651 29 (54) 0.1144 100 5 (8) 0.0196 16 (21) 0.0647 14 (37) 0.0570 26 (50) 0.1036 Reference tables for four species were considered in this study (Table 1 1 ). Based on datasets of their representative gene families, the logarithm equations between MALEs and their d A values of the four species were constructed: y =−41.11ln( x )−0.11 ( R 2 =0.92) ( Arabidopsis ), y =−39.79ln( x )−11.92 ( R 2 =0.94) ( Oryza sativa ), y =−34.14ln( x )−5.45 ( R 2 =0.91) ( Mus musculus ) and y =−44.54ln( x )−10.36 ( R 2 =0.90) ( Homo sapiens ). In Table 1 1 , two divergent (duplication) times, mean and maximal time (plus value of mean and two standard deviations) and their corresponding MALE are listed for every species. Maximal time of a MALE means that duplicate events of most ( > 95%) gene families with the MALE occurred before the time. Application of MALE 2 An important objective of MALE application is the analysis of large-scale genome alignment and comparison. Here we displayed an example to illustrate how to detect large-scale duplication blocks in rice genome using above reference table. In large-scale genome sequence alignment, the minimal sequence length of exact match (MILE) was usually used in corresponding algorithms to limit searching space and return their results in practical time. When a particular MILE was chosen, it means that those duplicate genes with less sequence than MALE, which equates to the MILE, would be ignored and not be returned. For example, when MILE parameter is set at 30 aa in a genome alignment using program PROmer of MUMmer packet, it means only those genes would be focused on that originated within an evolutionary time, say, 84 million years for rice (Table 1 1 ), and other genes will be absolutely screened out. Fig. 2 2 showed the selected inter-chromosome alignment results (between chromosome 1 and 5; 11 and 12) of rice using PROmer program of MUMmer packet. Two levels (30 and 100 aa) of MILE were chosen, which mean those duplicate genes with less than 30 or 100 MALE will be ignored and not be returned. Apparently, there were changes in the dot-plots between chromosome 1 and 5 using the two different MILE parameters: a syntenic line was observed when 30 MILE was chosen and it vanished as MILE increased to 100. The situation of chromosome 11 and 12 was different from that of chromosome 1 and 5: their syntenic line almost kept stable when MILE value was changed. It is obvious that large-scale duplication blocks were detected in both alignments when the 30 aa MILE value was chosen (Fig. 2a 2a and Fig. 2b 2b ). According to the reference table (Table 1 1 ), 30 aa MILE corresponds to divergent time of 84 million years ago, i.e. many (~50%) genes appeared 84 million years ago and most genes ( > 95%) that appeared 105 million years ago, were ignored or scanned off, and only the genes originated within 84 millions years were focused on. The results showed that the two large-scale duplicate events both occurred 84 million years ago. But at 100 aa MILE level, only one duplicate block between chromosome 11 and 12 was detected (Fig. 2d 2d ), which indicated that the duplicate event occurred more recently, about 16 million years ago, and another duplicate event occurred even earlier, around 21–84 million years ago. The results also implied that two large-scale duplication events should occur during early evolution of rice genome. All chromosomes of rice were further detected using PROmer with 30 and 100 MALE, respectively (See complementary materials at http://ibi.zju.edu.cn/bioinplant/data/ http://ibi.zju.edu.cn/bioinplant/data/ ). Many duplicate blocks were detected at 30 MALE levels, as in chromosome 1 and 5. The blocks did not overlap each other and covered most parts of rice chromosomes. The results suggested that beside the recent duplication (~5 Mb) of chromosome 11 and 12, a whole-genome duplication occurred in rice genome about 20–84 million years ago. Our results were consistent with those of Paterson et al.( 2004 2004 ), who suggested that a whole-genome duplication and a duplicate event between chromosome 11 and 12 occurred ~70 million years ago and recently, respectively. DISCUSSION 1 Our studies on MALE changes of main gene families from four representative species during evolution indicated that MALEs are related to divergent time significantly during early evolution of gene families, and a reference table between MALE and its corresponding divergent time was set up. An important application of the table is large-scale genome analysis, such as genome alignment. A successful example of application of the table to detect the large-scale duplication events in rice genome is given in this paper. The release of the high-quality genome sequence of various organisms makes possible the analysis of chromosomal behavior and other evolutionary processes. For large-scale genome sequence analysis, our reference table between MALE and divergent time provides an estimate of divergent times of target genes concerned. But it must be cautioned that the table is associated with a large degree of uncertainty when it deals with ancient ( d A > 0.5) divergent events of duplicated genes because the MALEs of those ancient duplicate genes were not sensitive to divergent time (Figs. 1a–1c 1a–1c ). A rational default value of MILE for genome alignment should be set to guarantee that hits with biological significance are returned. Our analysis on random protein sequences indicated that few (<1.1%) MALE between random sequences were over 6 aa. Therefore, 6 aa seems to be a good starting point for genome alignment. Of course, more strictly speaking, length (such as 7 aa, less than 0.04% of total random MALEs are over this length) could also be chosen for some particular analysis. Another traditional evolutionary scale is nucleotide acid synonymous substitutions rate ( K s), which is widely used in analysis of evolutionary divergence of genes. In this study, the scale was not used because higher K s values ( K s < 2.0, even 1.0) are associated with a large degree of uncertainty due to saturation of substitutions (Li, 1997 1997 ; Blanc and Wolfe, 2004 2004 ). References R Arratia , L Gordon , MS Waterman . An extreme value theory for sequence matching . Ann Stat , 1986 . 14 971 - 993 . . An extreme value theory for sequence matching G Blanc , KH Wolfe . Widespread paleopolyploidy in model plant species inferred from age distributions of duplicate genes . Plant Cell , 2004 . 16 1667 - 1678 . . Widespread paleopolyploidy in model plant species inferred from age distributions of duplicate genes TA Brown . Genomes , 1999 . : Oxford BIOS Scientific Publishers Limited , . Genomes AL Delcher , A Phillippy , J Carlton , SL Salzberg . 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Phylogenetic Analysis by Maximum Likelihood (PAML) We thank Prof. Mingwei Gao and Dr. Shaukat Ali for their critical reading. 10.1631/jzus.2005.B0470 Project supported by the National Natural Science Foundation of China (Grant Nos. 30270810, 90208022 and 30471067) and IBM Shared University Research (Life Science), China 2004-11-16 2005-02-19 2005-05-22 2022-01-05 Copyright © 2005, Journal of Zhejiang University Science 2005 Journal of Zhejiang University-SCIENCE B (Biomedicine and Biotechnology) 06 6B