# Analysis of Secretary Proteins in the Genome of the Plant Pathogenic Fungus Botrytis Cinerea

Zhang Yue, Yang Jing, Liu Lin, Su Yuan, Xu Ling, Zhu Youyong, Li

Chengyun

► To cite this version:

Zhang Yue, Yang Jing, Liu Lin, Su Yuan, Xu Ling, et al.. Analysis of Secretary Proteins in the Genome of the Plant Pathogenic Fungus Botrytis Cinerea. 4th Conference on Computer and Computing Technologies in Agriculture (CCTA), Oct 2010, Nanchang, China. pp.227-237, 10.1007/978-3-642-18333-1_27 . hal-01559557

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<figure><img src="image_1.png" /></figure>
# Analysis of Secretary Proteins in the Genome of the Plant Pathogenic fungus

## *Botrytis cinerea*

ZHANG Yue, YANG Jing, LIU Lin, SU Yuan, XU Ling, ZHU Youyong, LI Chengyun*

*Key Laboratory of Agro-biodiversity and Pest Management of the Education Ministry of China, Yunnan Agricultural University, Kunming, 650201, China.*

*Corresponding author, E-mail: li.chengyun@gmail.com*

**Abstract:** The signal peptides prediction algorithm SignalP v3.0, subcellular protein location prediction algorithm TargetP v1.1, potential GPI-anchor sites prediction algorithm big-PI predictor, trans-membrane domains prediction algorithm TMHMM v2.0 and bioinformatics algorithm MEME were used to analyze 16446 protein sequences of *Botrytis cinerea*. The results showed that there were 579 deduced secretary proteins. Among these proteins, the minimum and maximum of open read frame were 102 bp and 4848 bps respectively and mean score was 1271 bps. The signal peptides' length was concentrated to 16~39 amino acids and the average length was 21.122 of these proteins contain the highly conserved host-targeting-motif RxLx within 100 residues adjacent to the signal peptide cleavage site. According to PEDNAT and COG of GenBank database, this motif's functions include metabolism modification and cell secretion etc. We blast those putative secretary proteins with RxLx motif in GenBank and found 47.54% of them have highly conserved homologues in other species, among them 74.1% have putative protein domains. This means these proteins may be more stable or earlier origin. We suppose these proteins are candidate participating in the pathogenesis of *Botrytis cinerea* but we still need more experimental evidence to confirm their definite functions.

**Key words:** *Botrytis cinerea*; signal peptide; secretary protein; host-targeting-motif

*Botrytis cinerea* belongs to Deuteromycotina and is a widespread phytopathogenic fungus causing disease in a substantial number of economically important crops [1]. It causes Gray-mold rot or Botrytis blight and affects most vegetable and fruit crops, as well as a large number of shrubs, trees, flowers, and weeds. It also has a beneficial role in the production of rare dessert wines. The genome sequence information of *Botrytis cinerea* was released in 2005 and was helpful for us to understand this ascomycete's complex developmental life cycle, Pathogenesis mechanisms and interactions with its different host plants.

Protein is the basic function element of living organism. Many pathogenic microbes could secrete kinds of proteins into the host cell to facilitate its infection process [2]. So analysis of secreted proteins in the pathogen's genome will be
helpful to reveal its pathogenesis mechanisms. The secreted proteins used to be
synthesized by ribosome and need a transport process to secrete outside the cells.
There are two mechanisms for peptides transportation. The fist is cotranslational
transfer. In this way, synthesized partial signal peptide combined to endoplasmic
reticulum and the secreted proteins were synthesized meanwhile entered the
endoplasmic reticulum, after moderated by endoplasmic reticulum and Golgi
complex they were secreted outside. The second is posttranslational translocation.
By this mechanism the complete proteins were synthesized and then were
transported for modifying with the help of leader peptide [3]. In both mechanisms,
the signal peptide has played the fundamental role. The signal peptide was usually
composed by 10 to 60 amino acids. It contained a hydrophobic region (H-region)
which was constituted by 6 to 15 amino-acid residues in the center and hydrophilic
N terminal and C terminal at the both sides [4]. According to Gunter Blobel's
signal peptide hypothesis, secretary protein's destiny was decided by its signal
peptide and this peptide will be cut off when the protein arrive its destination. So
we can decide whether a protein is a secretary protein by analysis of its signal
peptide of N terminal [5]. Several software had been developed to indentify the
signal peptide in the protein. Lee used SignalP(v2.0) analyzed 47 secretary protein
and 47 other proteins of *Candida albicans*, it shows that the putative results of this
software is credible [6].

The interactions between pathogens and their hosts is a hot spot for scientific
research recently. How the secretary proteins entered the plant cells and play their
function is still not clear now. Bacteria's type III secretary system have been
illustrated by many researchers, but the pathway of the eukaryotic pathogen's
secretary proteins is still unclear [7, 8]. There is a report revealed that during the
process of *Plasmodium Falciparum* infected erythrocyte, most of secretary
proteins which will be injected into erythrocyte contain an RxLxE/D/Q motif at 60
amino-acid residues downstream the cutting site of the signal peptide [9]. Souvik
Bhattacharjee also indicated that RgLx motif also existed in hundreds of
pathogenic secretary proteins of *Phytophthora infestans* which play the same
function as RgLxE/D/Q motifs of *Plasmodium Falciparum*. *Plasmodium
Falciparum* and *Phytophthora infestans* are far related and infect animal and plant
respectively; they should have different pathogenic process and mechanism, so
RgLx motif may be a conserved signal recognition motif of eukaryotic pathogen
[10]. In this study, we try to make use of the genome data to indicate how many
secretary proteins contained by *Botrytis cinerea* and whether RgLx motif exist in
this saprophytic fungi's secretary proteins and play pathogenic function.
# Materials and methods

The sequence data of *Botrytis cinerea* was downloaded from the database of BROAD institute (*Botrytis cinerea* strain B05.10. It totally contained 16446 putative proteins in the database (http://www.broad.mit.edu/annotation/genome/botrytis_cinerea/Home.html), by the use of signal putative software SignalP (http://www.cbs.dtu.dk/services/SignalP/) [11], subcellular organelle located software TargetP (http://www.cbs.dtu.dk/services/TargetP1.1) [12, 13], anchoring protein analysis software big-PIPredicto (http://mendel.imp.ac.at/sat/gpi/gpi_server) [14] and transmembrane helix analysis software TMHMMServer (http://www.cbs.dtu.dk/services/TMHMM) [15], we selected those proteins with signal peptide (satisfied L=-918.235-123.455*(Mean.S.score)+1983.44*(HMM scores) and L>0) [6], secreted outside the cells not to other subcellular organelle, not anchoring protein and didn't contain transmembrane helix as secretary proteins. Then we use MEME(Multiple Expectation Maximization for Motif Elicitaion) [16] to test whether these putative secretary proteins contain RxLx motif and whether there exist conserved amino-acid residue at the two sides of this motif. Then the graph was produced by software LOGO[17] according to the result of MEME. We also compared the sequence of these putative secretary proteins with RxLx motif to PEDNAT database (http://pedant.gsf.de/index.jsp) and searched them in the COG database from GeBank (http://www.ncbi.nlm.nih.gov/COG/old/xognitor.html) to find and categorized the putative functions of these proteins. At last, we blast these sequence in the GeBank (BLASTP 2.2.17 (Jun-24-2007) (http://www.ncbi.nlm.nih.gov/) try to find the homologues of these proteins and conjectured their functions.

# Results and analysis

As figure 1 showed, we get 868 proteins contained the signal peptide in the *Botrytis cinerea*'s genome, among them 579 proteins which account for 3.52% of the *Botrytis cinerea*'s genes were predicted to be secretary proteins by the method have been mentioned.

<figure><img src="image_2.png" /></figure>
<figure><img src="image_3.png" /><figcaption>Fig 2. The ORF length distribution of 284 secretary proteins in *Botrytis cinerea*</figcaption></figure>

<figure><img src="image_4.png" /></figure>
<caption>Fig 3. The signal peptide length distribution of 284 secretory proteins in *Botrytis cinerea*</caption>

The average length of the predicted secretory proteins of *Botrytis cinerea* is 1271bp; the longest one and the shortest one are 4848bp and 102bp respectively. From figure 2, we can find that the most of them are 500-2000bp, which account for 72.19% of all the genes. The average length of these secretory proteins' peptide is 21 amino-acid residues, the longest one is 39 and the shortest one is 16. The figure 3 showed the length distribution of these proteins' peptide. 540 of them are 17-25 amino-acid residues which account for 93.3% of all and proteins with 19 amino-acid residues come up to 109 proteins (18.8%).

<figure><img src="image_5.png" /><figcaption>Fig 4. Logo shows the conservation of the RxLx motif from predicted *Botrytis cinerea* secretary proteins</figcaption></figure>

The motifs are highlighted in board letters in the table. Abbreviations for amino acid residues: A, Ala; C, Cys; D, Asp; E, Glu; F, Phe; G, Gly; H, His; I, Ile; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q, Gln; R, Arg; S, Ser; T, Thr; V, Val; W, Trp; Y, Tyr.

We analyzed all these putative secretory proteins by MEME software and found that there are 122 proteins (21% of total) contain RxLx motif within the 100 amino-acid residues downstream of the cutting site of signal peptide. The report had showed that *Plasmodium Falciparum's* pathogenic secretory proteins had conserved E, D or Q fowled the RxLx motif while *Phytophthora infestans* didn't. But from the figure 4, we can found that there more A, G, L and S appeared at the downstream and upstream of the RxLx motif in *Botrytis cinerea*. A, G and L are all nonpolar amino acid and S is polar neutral amino acid. The most conserved amino-acid residue followed the RxLx motif is D. This is just the same as *Plasmodium Falciparum*.
<figure><img src="image_6.png" /><figcaption>Fig 5. Functional categorization of putative secretary proteins containing Rxlx motif which have function descriptions in COG database</figcaption></figure>

Then we categorized the putative functions of these proteins by COG of GenBank (figure 5). There are only 26 (21.3%) proteins found in COG and by categorized into 11 different kinds of functions. Most of them are related to amino-acid metabolism (23.08%). For further predicted the functions of our putative secretary proteins, we compared them in the PEDANT database but only 6 of them have the specific function description (Table 1). The most of them are

<table><caption>Tab 1. The functional description of secretary proteins containing the Rxlx motif in *Botrytis cinerea*</caption><thead><tr><td>Gene name</td><td>start</td><td>end</td><td>Functional description</td></tr></thead><tbody><tr><td>BC1G_15580</td><td>29028</td><td>29981</td><td>may act as a sorting receptor in the delivery of vacuolar hydrolases, partial</td></tr><tr><td>BC1G_05799</td><td>27623</td><td>28429</td><td>peptidylprolyl isomerase</td></tr><tr><td>BC1G_12776</td><td>79340</td><td>77335</td><td>tripeptidyl peptidase</td></tr><tr><td>BC1G_04994</td><td>285638</td><td>283980</td><td>hypothetical protein similar to alpha-L-arabinofuranosidase</td></tr><tr><td>BC1G_06540</td><td>150762</td><td>152263</td><td>hypothetical protein similar to aspartic proteinase precursor</td></tr><tr><td>BC1G_03579</td><td>412126</td><td>410768</td><td>hypothetical protein similar to aspartyl protease</td></tr></tbody></table>

related to cell metabolism and some of them also appeared in the *Phytophthora infestans*'s pathogenic secretary proteins.

<table><caption>Table 2. The blast results of putative secretary proteins include Rxlx motif</caption><thead><tr><td>Gene name</td><td>number of homologs</td><td>No. of the species containing</td><td>No. of Pathogenic species</td><td>Conserved protein domain</td></tr></thead><tbody></tbody></table>
homolog

<table>
  <tr>
    <td>BC1G_00639</td>
    <td>5</td>
    <td>2</td>
    <td>1</td>
    <td>Tannase</td>
  </tr>
  <tr>
    <td>BC1G_00978</td>
    <td>2</td>
    <td>1</td>
    <td>1</td>
    <td>Pro-kumamolisin</td>
  </tr>
  <tr>
    <td>BC1G_01009</td>
    <td>9</td>
    <td>5</td>
    <td>4</td>
    <td>N</td>
  </tr>
  <tr>
    <td>BC1G_01027</td>
    <td>18</td>
    <td>10</td>
    <td>7</td>
    <td>Peptidase_S10</td>
  </tr>
  <tr>
    <td>BC1G_01073</td>
    <td>4</td>
    <td>2</td>
    <td>1</td>
    <td>Pro-kumamolisin</td>
  </tr>
  <tr>
    <td>BC1G_01628</td>
    <td>6</td>
    <td>6</td>
    <td>4</td>
    <td>Peroxidase</td>
  </tr>
  <tr>
    <td>BC1G_01874</td>
    <td>7</td>
    <td>6</td>
    <td>3</td>
    <td>Glycine-rich protein domain</td>
  </tr>
  <tr>
    <td>BC1G_02021</td>
    <td>19</td>
    <td>11</td>
    <td>8</td>
    <td>GMC oxidoreductase; choline dehydrogenase</td>
  </tr>
  <tr>
    <td>BC1G_02163</td>
    <td>30</td>
    <td>24</td>
    <td>14</td>
    <td>Cerato-platanin</td>
  </tr>
  <tr>
    <td>BC1G_02492</td>
    <td>2</td>
    <td>2</td>
    <td>2</td>
    <td>N</td>
  </tr>
  <tr>
    <td>BC1G_02944</td>
    <td>1</td>
    <td>1</td>
    <td>1</td>
    <td>Pro-kumamolisin</td>
  </tr>
  <tr>
    <td>BC1G_03275</td>
    <td>14</td>
    <td>13</td>
    <td>8</td>
    <td>N</td>
  </tr>
  <tr>
    <td>BC1G_03557</td>
    <td>24</td>
    <td>22</td>
    <td>9</td>
    <td>N</td>
  </tr>
  <tr>
    <td>BC1G_03560</td>
    <td>4</td>
    <td>4</td>
    <td>2</td>
    <td>N</td>
  </tr>
  <tr>
    <td>BC1G_03579</td>
    <td>28</td>
    <td>22</td>
    <td>7</td>
    <td>Asp, Eukaryotic aspartyl protease</td>
  </tr>
  <tr>
    <td>BC1G_04705</td>
    <td>7</td>
    <td>6</td>
    <td>3</td>
    <td>Peroxidase</td>
  </tr>
  <tr>
    <td>BC1G_04994</td>
    <td>26</td>
    <td>18</td>
    <td>9</td>
    <td>Alpha-L-arabinofuranosidase B</td>
  </tr>
  <tr>
    <td>BC1G_05488</td>
    <td>4</td>
    <td>4</td>
    <td>2</td>
    <td>N</td>
  </tr>
  <tr>
    <td>BC1G_05765</td>
    <td>7</td>
    <td>4</td>
    <td>3</td>
    <td>Pro-kumamolisin</td>
  </tr>
  <tr>
    <td>BC1G_05799</td>
    <td>58</td>
    <td>39</td>
    <td>8</td>
    <td>FKBP-type peptidyl-prolyl cis-trans isomerase</td>
  </tr>
  <tr>
    <td>BC1G_05885</td>
    <td>5</td>
    <td>4</td>
    <td>3</td>
    <td>N</td>
  </tr>
  <tr>
    <td>BC1G_06035</td>
    <td>100</td>
    <td>43</td>
    <td>19</td>
    <td>Glycosyl hydrolase family 7; Fungal cellulose binding domain</td>
  </tr>
  <tr>
    <td>BC1G_06328</td>
    <td>6</td>
    <td>6</td>
    <td>4</td>
    <td>Bacterial alpha-L-rhamnosidase</td>
  </tr>
  <tr>
    <td>BC1G_06540</td>
    <td>14</td>
    <td>13</td>
    <td>11</td>
    <td>Asp, Eukaryotic aspartyl protease</td>
  </tr>
  <tr>
    <td>BC1G_07149</td>
    <td>24</td>
    <td>18</td>
    <td>11</td>
    <td>Peptidase_S10</td>
  </tr>
  <tr>
    <td>BC1G_07160</td>
    <td>8</td>
    <td>8</td>
    <td>6</td>
    <td>Phospholipase C</td>
  </tr>
  <tr>
    <td>BC1G_07483</td>
    <td>3</td>
    <td>3</td>
    <td>2</td>
    <td>Esterase_lipase</td>
  </tr>
  <tr>
    <td>BC1G_07899</td>
    <td>2</td>
    <td>1</td>
    <td>0</td>
    <td>Cutinase</td>
  </tr>
  <tr>
    <td>BC1G_08048</td>
    <td>5</td>
    <td>4</td>
    <td>3</td>
    <td>Amidase</td>
  </tr>
  <tr>
    <td>BC1G_08735</td>
    <td>27</td>
    <td>21</td>
    <td>12</td>
    <td>Cerato-platanin</td>
  </tr>
  <tr>
    <td>BC1G_08755</td>
    <td>50</td>
    <td>21</td>
    <td>7</td>
    <td>Glycosyl hydrolase family 15; The family 20 carbohydrate-binding module (CBM20)</td>
  </tr>
  <tr>
    <td>BC1G_09129</td>
    <td>13</td>
    <td>13</td>
    <td>10</td>
    <td>DnaJ domain</td>
  </tr>
  <tr>
    <td>BC1G_09495</td>
    <td>2</td>
    <td>2</td>
    <td>2</td>
    <td>Tannase</td>
  </tr>
  <tr>
    <td>BC1G_09611</td>
    <td>5</td>
    <td>5</td>
    <td>4</td>
    <td>DadA, Glycine/D-amino acid oxidases</td>
  </tr>
  <tr>
    <td>BC1G_10333</td>
    <td>4</td>
    <td>4</td>
    <td>1</td>
    <td>Rossmann-fold NAD(P)(+)-binding proteins</td>
  </tr>
  <tr>
    <td>BC1G_10397</td>
    <td>10</td>
    <td>10</td>
    <td>6</td>
    <td>N</td>
  </tr>
  <tr>
    <td>BC1G_10482</td>
    <td>2</td>
    <td>2</td>
    <td>1</td>
    <td>N</td>
  </tr>
  <tr>
    <td>BC1G_10768</td>
    <td>12</td>
    <td>8</td>
    <td>5</td>
    <td>Intradiol_dioxygensense_like domain</td>
  </tr>
  <tr>
    <td>BC1G_11019</td>
    <td>8</td>
    <td>8</td>
    <td>5</td>
    <td>Salicylate hydroxylase</td>
  </tr>
</table>
<table><tr><td>BC1G_11134</td><td>9</td><td>9</td><td>6</td><td>Survival protein SurE</td></tr><tr><td>BC1G_12138</td><td>8</td><td>6</td><td>3</td><td>Alpha-L-arabinofuranosidase C-terminus</td></tr><tr><td>BC1G_12157</td><td>58</td><td>29</td><td>15</td><td>Arginase family</td></tr><tr><td>BC1G_12171</td><td>11</td><td>7</td><td>4</td><td>N</td></tr><tr><td>BC1G_12200</td><td>18</td><td>14</td><td>5</td><td>Peptidase family M20/M25/M40; Acetylornithine deacetylase</td></tr><tr><td>BC1G_12456</td><td>9</td><td>7</td><td>6</td><td>Glyco_hydrolase_16</td></tr><tr><td>BC1G_12525</td><td>4</td><td>3</td><td>3</td><td>Peroxidase</td></tr><tr><td>BC1G_12619</td><td>2</td><td>2</td><td>2</td><td>N</td></tr><tr><td>BC1G_12776</td><td>3</td><td>2</td><td>2</td><td>Pro-kumamolisin</td></tr><tr><td>BC1G_12932</td><td>25</td><td>7</td><td>2</td><td>Tannase</td></tr><tr><td>BC1G_13158</td><td>2</td><td>2</td><td>1</td><td>N</td></tr><tr><td>BC1G_13581</td><td>10</td><td>6</td><td>6</td><td>N</td></tr><tr><td>BC1G_13855</td><td>7</td><td>6</td><td>3</td><td>BglC, Endoglucanase</td></tr><tr><td>BC1G_14244</td><td>86</td><td>62</td><td>13</td><td>N</td></tr><tr><td>BC1G_14398</td><td>15</td><td>12</td><td>8</td><td>DadA, Glycine/D-amino acid oxidases</td></tr><tr><td>BC1G_14702</td><td>100</td><td>36</td><td>11</td><td>Glycosyl hydrolase family 7</td></tr><tr><td>BC1G_15580</td><td>9</td><td>9</td><td>6</td><td>N</td></tr><tr><td>BC1G_15641</td><td>5</td><td>3</td><td>1</td><td>tol-pal system beta propeller repeat protein TolB</td></tr><tr><td>BC1G_16238</td><td>8</td><td>6</td><td>3</td><td>BglC, Endoglucanase</td></tr></table>

<figure><img src="image_7.png" /><figcaption>Fig 6. The location of RgLx motif and putative protein domain of 7 proteins which contain over 50 homologs in other species</figcaption></figure>
Then we blast all these proteins in the GenBank and found that 58 proteins have high conserved homologues (E-value (1×10-20 and identities) 40%) in other species (accounted for 47.54% of all predicted proteins) and most of them have putative conserved protein domains (Table 2). Then we selected 7 the most conserved (have more than 50 homologues) proteins and marked out the location of their RxLx motif and protein domains (Figure 6). We can see that in 6 proteins, the motif appeared within the first 10 amino-acid residues of the C terminals of their protein domains.

## Discussion

By application of software of bioinformatics' analysis, we found 579 putative secretary proteins and 122 (21%) of them contained Host-Targeting-motif RxLx within the 100 amino-acid residues downstream of their cutting sites in the genome of *Botrytis cinerea*. The length of most of these proteins and their signal peptide are moderate. RxLx motif spread in the secretary proteins of this kind of saprophytic fungi indicate they maybe play functions in this fungus's pathogenic secretary pathways. But we still need experiment evidence to conform this hypothesis.

By compared these proteins contain RxLx motif in the PEDANT database and COG of GenBank, we seldom found the definite description to them. For those proteins with description in COG, most of them are related to cell metabolism and cellular process. Interestingly, among these proteins, BC1G_06540 is described as an aspartic proteinase precursor. It is had already been revealed that *Botrytis cinerea* could secreted aspartic proteinase into its host cell during its pathogenic process [18]. Researchers also found that the a pathogens infected the plant, it must break through the plant cell's physical outline such as cell wall as well as changed the inner environment of host cells to fit themselves. In this process, their secretions must play the most important roles [19]. Considered other 5 RxLx motif containing proteins with definite descriptions in PEDANT database are all enzymes involved in the cell metabolism and the categorization with most proteins by COG is related to amino-acid metabolism, we supposed that maybe *Botrytis cinerea* need secreted proteinase into the plant cells in its pathogenic process.

Blast these RxLx motif containing proteins in the GenBank, we find that 47.54% of them have high conserved homologues in other species and most have
the conserved protein domains. This indicates these proteins with RXLx motif are
conserved during the evolution process or early originated in the history. Most of
the homologues contained in fungi but still some appeared in higher eukaryotes.
Among them, BC1G_14702, BC1G_14244, BC1G_05799 and BC1G_06035
contained huge number of homologues distributed in so many different kinds of
plants and animals. This indicated that maybe these genes played the irreplaceable
role in organisms as well as Botrytis cinerea. BC1G_05799 had been depicted as
peptidylprolyl isomerase in both COG and PEDANT database. This kind of
protein interconverts the *cis* and *trans* isomers of peptide bonds with the amino
acid proline and involved in many cellular process. The 6 of 7 RXLx motifs in the
highest conserved putative secretary proteins are all located at the N terminal of
their protein domains. Interestingly, the description of protein domains contained
by these 7 proteins indicated they all maybe involved in the secretion process even
in the pathogenic process. Cerato-platanin had been reported as a kind of
phytotoxic protein which was secreted by Ceratocystis fimbriata f.sp. platani [20].
FKBP_C is related to protein synthesis and locate of plant plasmids [21].
Glyco_hydro_7 is a kind of glycosyl hydrolase which was secreted to destroy
cellulose in the plant cell [22]. SpeB have been reported as a kind of virulent
effectors secreted by Group A Streptococcal [23]. GPI8 is related to the synthesis
of phosphatidylinositol anchor protein which could anchor it to the cell surface
[24]. So these proteins may play their functions in the Botrytis cinerea's secretion
pathway and contributed to its pathogenic process.

Most of pathogenic proteins reported now are secretary proteins. In this
article we predicted 579 secretary proteins and 122 of them contain
Host-Targeting-motif RXLx which could be treated as candidate pathogenic
proteins of *Botrytis cinerea*. Although we still need experiment to prove whether
these proteins contributed to pathogeneses, find these candidate proteins will
accelerate our understandings of pathogenic mechanism of *Botrytis cinerea*. Many
software used to analyze the protein had been proved effective and it is
conveniently for us to understand the information lied in the genome by the help of
them.

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