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Plant Physiology and Biochemistry 103 (2016) 106e119

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Plant Physiology and Biochemistry

journal homepage: www.elsevier .com/locate/plaphy

Research article

Genome-wide characterization and comparative analysis of the MLOgene family in cotton

Xiaoyan Wang a, 1, Qifeng Ma b, 1, Lingling Dou b, Zhen Liu a, Renhai Peng a, *,Shuxun Yu b, **

a Anyang Institute of Technology, College of Biology and Food Engineering, Anyang, Henan, 455000, PR Chinab State Key Laboratory of Cotton Biology, Institute of Cotton Research of CAAS, Anyang, Henan, 455000, PR China

a r t i c l e i n f o

Article history:Received 12 December 2015Received in revised form1 February 2016Accepted 23 February 2016Available online 27 February 2016

Keywords:GossypiumMLOGene familySynteny blocksAbiotic stressPhytohormone

* Corresponding author.** Corresponding author.

E-mail addresses: [email protected] (X. Wa(Q. Ma), [email protected] (L. Dou), liuzhen378@163com (R. Peng), [email protected] (S. Yu).

1 These authors contributed equally to this work.

http://dx.doi.org/10.1016/j.plaphy.2016.02.0310981-9428/© 2016 Elsevier Masson SAS. All rights re

a b s t r a c t

In plants, MLO (Mildew Locus O) gene encodes a plant-specific seven transmembrane (TM) domainprotein involved in several cellular processes, including susceptibility to powdery mildew (PM). In thisstudy, a genome-wide characterization of the MLO gene family in G. raimondii L., G. arboreum L. and G.hirsutum L. was performed. In total, 22, 17 and 38 homologous sequences were identified for eachspecies, respectively. Gene organization, including chromosomal location, gene clustering and geneduplication, was investigated. Homologues related to PM susceptibility in upland cotton were inferred byphylogenetic relationships with functionally characterized MLO proteins. To conduct a comparativeanalysis between MLO candidate genes from G. raimondii L., G. arboreum L. and G. hirsutum L.,orthologous relationships and conserved synteny blocks were constructed. The transcriptional variationof 38 GhMLO genes in response to exogenous application of salt, mannitol (Man), abscisic acid (ABA),ethylene (ETH), jasmonic acid (JA) and salicylic acid (SA) was monitored. Further studies should beconducted to elucidate the functions of MLO genes in PM susceptibility and phytohormone signallingpathways.

© 2016 Elsevier Masson SAS. All rights reserved.

1. Introduction

Powdery mildew (PM) is an obligate fungal pathogen that cau-ses PM disease in a broad range of plants, including important cropssuch as pepper, tomato, apple, strawberry, and cotton (Glawe,2008). It is difficult to diagnose at the early stages of the disease,and it can easily spread unnoticed. According to previous obser-vations, PM disease primarily affects the leaves of sea-island cottonand upland cotton. In general, PM presents similar symptoms incotton: white or brown spots on leaf tissues, particularly at thebottom of the plant, whereas upper leaves exert some resistance.Afterwards, tissue death of diseased spots causes infected leaves tocrinkle, curl, and prematurely drop. Although blossoms and fruitsare not the initial PM fungal targets, they can also become infected.

ng), [email protected] (Z. Liu), aydxprh@163.

served.

Mildew locus O (MLO) proteins belong to a plant-specific pro-tein family containing seven transmembrane (TM) domains(Buschges et al., 1997; Devoto et al., 1999). In addition, a C-terminalcalmodulin-binding domain (CaMBD) and an extracellular N-ter-minus (Devoto et al., 1999; Kim et al., 2002a,b) have been identifiedin this family. PM resistance was first characterized in barley plantsin 1942 and the immunity was acquired because of the absence of asusceptibility gene which was named Mildew Locus O (MLO).Recessive MLO gene mutations confer durable broad-spectrumresistance to all discovered isolates of barley powdery mildewfungus Blumeria graminis f. sp hordei (Bgh) (Buschges et al., 1997;Devoto et al., 1999). Then, the discovery and identification of PMdisease resistance in other plant species, such as Arabidopsis(Consonni et al., 2006), pea (Pavan et al., 2011) and tomato (Baiet al., 2008), has confirmed that PM resistance deriving from loss-of-function mutations in MLO functional orthologue is a commonphenomenon. Therefore, broad-spectrum PM resistance in plantscould be introduced by silencing of MLO gene (Pavan et al., 2010).

Calmodulin-binding of MLO proteins promotes PM susceptibil-ity in barley (Kim et al., 2002a,b). Moreover, pharmacologicalstudies have suggested that the influx of Ca2þ ions is important for

Delta:1_given nameDelta:1_surnameDelta:1_given nameDelta:1_surnameDelta:1_given nameDelta:1_surnamemailto:[email protected]:[email protected]:[email protected]:[email protected]:[email protected]:[email protected]:[email protected]://crossmark.crossref.org/dialog/?doi=10.1016/j.plaphy.2016.02.031&domain=pdfwww.sciencedirect.com/science/journal/09819428http://www.elsevier.com/locate/plaphyhttp://dx.doi.org/10.1016/j.plaphy.2016.02.031http://dx.doi.org/10.1016/j.plaphy.2016.02.031http://dx.doi.org/10.1016/j.plaphy.2016.02.031

X. Wang et al. / Plant Physiology and Biochemistry 103 (2016) 106e119 107

MLO protein function (Kim et al., 2002a,b). Therefore, Ca2þ may bea candidate signal because plant cells generate a transient Ca2þ

signal in response to pathogen attack (Xu and Heath, 1998). Inaddition, a complex mechanism may exist during the interactionbetween MLO genes and PM. Nevertheless, there is limited infor-mation on the precise mechanism of MLO proteins. It has beenrevealed that PM fungi target MLO proteins as an access to triggerpathogenesis because vesicle-associated and actin-dependentdefence pathways are negatively regulated by functional MLOproteins in the circumstance of attempted PM penetration. Studiesin tomato (Bai et al., 2008), barley (Piffanelli et al., 2002), pepper(Zheng et al., 2013a,b), and grape (Feechan et al., 2009) confirmedthat early stages of PM infection are associated with up-regulatedexpression of MLO susceptibility-related gene, with a peak at 6 hafter inoculation.

Since the HvMLO gene was first identified in barley (Buschgeset al., 1997), MLO genes have been discovered in Arabidopsis thali-ana (Devoto et al., 2003), Oryza sativa (Liu and Zhu, 2008), Vitisvinifera (Angela Feechan et al., 2008), Triticum aestivum (Konishiet al., 2010), Glycine max (Deshmukh et al., 2014), Cucumis sativus(Zhou et al., 2013), Malus domestica (Pessina et al., 2014) and So-lanum lycopersicum (Chen and Zhang, 2014). More detailed studieshave uncovered that medium-sized gene family of MLO is plant-specific and MLO-based PM resistance is not confined to mono-cotyledones, but is also discovered in distantly related dicoty-ledones (Acevedo-Garcia et al., 2014). For example, mutant allelesof AtMLO2, one of 15 MLO genes present in A. thaliana, causedpartial resistance to the adapted strains such as Golovinomycesorontii and G. cichoracearum. Complete PM resistancewas producedwhen two other homologous genes AtMLO6 and AtMLO12were alsomutated (Consonni et al., 2006). Subsequently, two studies showedthat loss-of-function of the SlMLO1 genewas the cause of resistanceto PM disease in tomato (Bai et al., 2008; Zheng et al., 2013a,b). Itwas demonstrated that pea PM resistance was associated with loss-of-function mutations in an MLO-homologous locus (Pavan et al.,2011). The results of virus-induced gene silencing suggested thatboth CaMLO1 and CaMLO2 are involved in the susceptibility ofpepper to the PM fungus Leveillula taurica (Zheng et al., 2013a,b).Recently, NtMLO1,which is predicted to be an orthologue of tomatoSlMLO1 and pepper CaMLO2, was shown to be involved in PMsusceptibility (Appiano et al., 2015).

Apart from susceptibility/resistance to PM disease in bothmonocotyledonous and dicotyledonous plants, increasing reportshave suggested that MLO may be involved in a variety of develop-mental processes. Leaf mesophyll cells in MLO barley mutants havebeen shown to undergo spontaneous cell death, which is an indi-cation of accelerated leaf senescence (Buschges et al., 1997;Piffanelli et al., 2002). MLO family members in Arabidopsis pre-sented tissue-specific expression patterns and silencing of AtMLO7involved in pollen tube reception by the embryo sac led todecreased fertility (Zhongying Chen et al., 2006). Two additionalArabidopsis genes, AtMLO4 and AtMLO11, control root architecture,as null mutants generate asymmetrical root growth and exagger-ated curvature (Chen et al., 2009). More results have revealed thatMLO family members are involved in diverse abiotic stresses forCapsicum annuum CaMLO2 intensely induced upon exogenoustreatment of pepper leaves with the phytohormone abscisic acid(ABA) and drought stress, is shown to act as a suppressor of ABAsignalling to prevent water loss from leaves under drought condi-tions (Lim and Lee, 2014).

MLO genes have been intensively studied inmanymonocots anddicots, but very little research has focused on cotton. Publishedgenomic data on G. raimondii (DD; 2n ¼ 26) (Paterson et al., 2012),and G. arboretum (AA; 2n¼ 26) (Li et al., 2014) as well as Gossypiumhirsutum (AADD; 2n¼ 52) (Li et al., 2015) provide an opportunity to

conduct a comprehensive overview of the MLO gene family indiploid and tetraploid cotton species. In this study, we character-ized the MLO gene family in these three species with respect totheir structural, genomic and gene-expression features. Moreover,we assessed the orthologous relationships between the G. rai-mondii L., G. arboreum L. and G. hirsutum L. genomes.

2. Materials and methods

2.1. In silico identification and annotation

Genomic databases of G. raimondii L. (D5, JGI__v2.1), G. arbor-eum L. (A2, BGI _v1.0) and G. hirsutum L. (AD1, BGI _v1.0), availableat the CottonGen website (https://www.cottongen.org/) (Yu et al.,2014), were downloaded for the identification of.

MLO homologue nucleotide and protein sequences. Then,several local BLAST searches using the Arabidopsis AtMLO1 aminoacid sequence as a query were performed. Candidates with an E-value less than 1.0e�20 were estimated to beMLO homologues, andtheir gene coding regions, genomic DNA and deduced amino acidsequences were acquired. Conserved MLO domains within the ac-quired MLO sequences were confirmed by searching NCBI'sconserved domain database (http://www.ncbi.nlm.nih.gov/Structure/bwrpsb/bwrpsb.cgi) and Pfam's protein domain families(http://pfam.xfam.org/). The presence and number of TM helices inthe proteins of interest were predicted using the online softwareTMHMM (http://www.cbs.dtu.dk/services/TMHMM-2.0/).

2.2. Gene organization

The chromosomal localization of each MLO gene in G. raimondiiL., G. arboreum L. and G. hirsutum L. was deduced based on theavailable genomic information at the CottonGen database. Map-chart 2.2 software was used to visualize the distribution of MLOgenes on the chromosomes, with the exception of a small portion ofgenes that have not been localized to a chromosome (Voorrips,2002). Introns and exons of each MLO gene were determined bycomparing the cDNAs with their corresponding genomic DNA se-quences. Intron/exon composition and position were analysed bythe Gene Structure Display Server (GSDS) tool (http://gsds.cbi.pku.edu.cn/).

The MLOs of G. hirsutum were first aligned by Clustal W2 atEMBL-EBI (http://www.ebi.ac.uk/Tools/msa/clustalw2/). Geneduplication events were identified when the following conditionswere fulfilled: (1) the alignment covered more than 80% of thelonger gene, (2) the identity of the aligned regions was greater than80% of the alienable region, and (3) only one duplication event wastaken into account for tightly linked genes (Gu et al., 2002).

2.3. Phylogenetic analysis

A total of 38 GhMLO amino acid sequences together with 36other MLO homologues from 9 dicot and monocot species wereused to construct phylogenetic trees. Amino acid sequences of 36known MLOs from A. thaliana (Devoto et al., 2003), Hordeum vul-gare (Buschges et al., 1997), O. sativa (Liu and Zhu, 2008), Zea mays(Devoto et al., 2003), S. lycopersicum (Chen and Zhang, 2014), Pisumsativum (Pavan et al., 2011), V. vinifera (Angela Feechan et al., 2008),Malus domestica (Pessina et al., 2014) and C. annuum (Zheng et al.,2013a,b) were obtained based on the published information. A totalof 74 MLO protein sequences were included to perform multiplealignments using ClustalW (Thompson et al., 1994) with the defaultparameters. A neighbour-joining phylogenetic treewas constructedby MEGA 6.0 software (Tamura et al., 2013) with the pairwisedeletion option and Poisson correction model. Bootstrapping (1000

https://www.cottongen.org/http://www.ncbi.nlm.nih.gov/Structure/bwrpsb/bwrpsb.cgihttp://www.ncbi.nlm.nih.gov/Structure/bwrpsb/bwrpsb.cgihttp://pfam.xfam.org/http://www.cbs.dtu.dk/services/TMHMM-2.0/http://gsds.cbi.pku.edu.cn/http://gsds.cbi.pku.edu.cn/http://www.ebi.ac.uk/Tools/msa/clustalw2/

X. Wang et al. / Plant Physiology and Biochemistry 103 (2016) 106e119108

replicates) was used to evaluate the degree of support for aparticular grouping pattern in the phylogenetic tree.

2.4. Detection of synteny blocks

Conserved synteny blocks between MLO candidate genes fromG. raimondii L., G. arboreum L. and G. hirsutum L. were inferred byrunning the OrthoClusterDB tool available at GDR (http://genome.sfu.ca/cgi-bin/orthoclusterdb/runortho.cgi) (Ng et al., 2009). Torun OrthoCluster, the user must provide n (n � 2) genome files andone correspondence file with their corresponding orthologous re-lationships as input. Thus, orthologous groups of the MLO familyamong the three Gossypium species were previously generatedusing the OrthoMCL database (http://orthomcl.org/orthomcl/)(Fischer et al., 2011).

2.5. Plant materials, stress and phytohormone treatments

Seeds of cotton plants from the CCRI 36 cultivar (G. hirsutum L.)were surface-sterilized in 75% (v/v) absolute alcohol for 30 s and in0.1% HgCl2 (m/v) for 3min. After washing them in sterilized double-distilled water (ddH2O), seeds were sown onto sterilizedMurashige& Skoog (MS) solid medium (pH 5.8) containing 1.5% sucrose and0.7% agar. The inoculated culture tubes were placed in a growthchamber with 150 mmol m�2 s�1 fluorescent light at 25e26 �C.Seven-day-old seedlings with expanding cotyledon were trans-planted into new culture tubes supplemented with 200 mM sodiumchloride (NaCl), 200 mM mannitol (Man), 50 mM ABA, 200 mMethylene (ETH), 100 mM jasmonic acid (JA) or 1 mM salicylic acid(SA). Treated and control plants were grown under the aboveconditions, and leaf samples were collected after three weeks.Three biological replicates were included, and each sample con-tained three young leaves collected from a single plant. Collectedleaves were frozen in liquid nitrogen, and stored at �80 �C untilRNA extraction.

2.6. Real-time quantitative RT-PCR

Total RNA was extracted using the EASY spin plus RNA reagentkit RN38 (AIDLAB, Beijing, China) according to the manufacturer'sinstructions. Poly (dT) cDNAwas synthesized using the SuperscriptIII First-Strand Synthesis System (Invitrogen, USA). Primers(Table S1) for transcript analysis were designed with Premier 6.0 ofPrimer Designing Tool. The Histone 3 (AF024716) gene was used asan internal control. Quantitative Real Time-PCR (qPCR) was per-formed on an ABI 7500 system (Applied Biosystems, USA) usingSYBR Green I (with Rox) reagents to detect target products.

The running programs were as follows: holding stage at 50 �Cfor 2 min, 94 �C for 10 min, followed by 40 cycles at 95 �C for 15 s,60 �C for 1 min. Then a melting curve was generated from 65 to95 �C to examine the specificity of target sequences.

To measure differential expression of MLO genes, QRT-PCR datawas processed by 2-△△CT method (Livak and Schmittgen, 2001)and statistically analysed by Student's t test.

2.7. Ethics statement

We did not make use of human or vertebrate animal subjectsand/or tissue in our research.

3. Results and discussion

3.1. In silico characterization of Gossypium MLO homologues

Conditional searches for Gossypium MLO homologues produced

38 significant matches in G. hirsutum L., 22 in G. raimondii L. and 17in Gossypium arboreum L (Tables 1e3). Predicted MLO genesGhMLO1eGhMLO38, GrMLO1eGrMLO22 and GaMLO1eGaMLO17were numbered depending on their chromosomal location. Weconcluded that more than 90 percent of the 77 MLOs encodedproteins ranging from 400 to 600 amino acids, whereas six (onefrom G. raimondii, one from G. arboreum and four from G. hirsutum)had markedly different lengths as compared to MLO homologuesreported in the genomes of Arabidopsis (Devoto et al., 2003), V.vinifera (Feechan et al., 2009) and Cucumis sativus (Zhou et al.,2013), i.e., they were less than 300 or more than 600 aminoacids. The TMHMM2 programme predicted different orientationsand numbers of transmembrane (TM) helices in the polypeptides.The number of TM domains varied from two in GhMLO8 andGaMLO4 to eight in ten of the 77MLOs (Tables 1e3). However, 28 ofthe 77 MLO proteins had seven TM domains, which are conservedwith respect to MLO family members in monocot and dicot plantspecies (Devoto et al., 1999). Comparison of the 77 MLO cDNAs totheir genomic DNA sequences revealed that the number of exonsvaried from 8 (GhMLO8) to 18 (GhMLO22). Nearly half of the MLOgenes (32 out of 77) contained 15 exons, which seems to be acommon feature of the MLO gene family (Devoto et al., 2003). De-tails including the length of the 77 sequences and the location ofMLO domains are provided in Tables 1e3.

3.2. Genomic organization of Gossypium MLO homologues

According to the report by Kohel (Kohel, 1973), chromosomes 1to 13 of tetraploid cotton were derived from the A subgenome, andchromosomes 14 to 26 originated from the D subgenome. We wereable to map 68 out of 77 MLOs onto chromosomes in G. raimondii,G. arboreum, or G. hirsutum (Fig. 1). Generally, one or two memberswere located on most chromosomes, with the exception ofG. raimondii and G. arboretum, in which chromosomes 9 and 10contained three genes each. Furthermore, chromosome 22, origi-nating from chromosome 9 of the D subgenome in G. hirsutum,contained four MLOs. The majority of the 77 Gossypium MLO familymembers occurred as singletons, with the exception of five groups,GhMLO10eGhMLO11 (Accession NO. CotAD_12423eCotAD_12424),GhMLO27eGhMLO28 (CotAD_54310eCotAD_54311),GrMLO12eGrMLO13 (Gorai.009G078600.1eGorai.009G078700.1),GrMLO16eGrMLO17 (Gorai.010G205000.1eGorai.010G205100.1)and GaMLO16eGaMLO17 (Cotton_A_11046eCotton_A_11047),which were organized as adjacent homologues with the distanceranging from 9.4 kb to 30.8 kb (Table S4). We identified two groupsin G. hirsutum and G. raimondii and one in G. arboretum. Interest-ingly, all five gene clusters were located on chromosome 9 or 10(chr22 originating from subD_chr9), suggesting that there was aslight bias for the MLO homologue location.

Previous studies have indicated that genomic changes, includingchromosomal rearrangement, gene duplication and gene expres-sion changes, occurred during the formation of polyploid species(Cronn et al., 1999). To elucidate the expanded mechanism of theMLO gene family in G. hirsutum, we investigated genomic organi-zation of GhMLO homologues. First, we performed multiple andpairwise alignments of 38 GhMLO sequences. After comprehensiveanalysis of pairwise alignments and the physical location of eachGhMLO gene, we detected 12 pairs of homologous genes and 2tandem duplications (GhMLO1eGhMLO2/GhMLO24eGhMLO23;GhMLO10eGhMLO11/GhMLO27eGhMLO28). Related information ofhomologous genes and duplication events were presented inTable 4.

http://genome.sfu.ca/cgi-bin/orthoclusterdb/runortho.cgihttp://genome.sfu.ca/cgi-bin/orthoclusterdb/runortho.cgihttp://orthomcl.org/orthomcl/

Table 1Members of GrMLO gene family as predicted in G. raimondii cv. Shixiya1 genome.

Name Accession NO.a Chr. CDS length AA length Exons TMb MLO domain locationc MLO domain length

GrMLO1 Gorai.001G130800.1 1 1755 584 15 7 6e499 494GrMLO2 Gorai.001G200000.1 1 1857 618 14 8 29e553 525GrMLO3 Gorai.002G072500.1 2 1602 533 15 4 66e460 395GrMLO4 Gorai.002G113800.1 2 1644 547 14 6 5e485 481GrMLO5 Gorai.004G029300.1 4 1737 578 15 7 9e497 489GrMLO6 Gorai.004G106900.1 4 1314 437 14 8 4e420 417GrMLO7 Gorai.005G074900.1 5 1494 497 14 6 9e470 462GrMLO8 Gorai.005G241700.1 5 1710 569 15 6 11e507 497GrMLO9 Gorai.006G088500.1 6 1314 437 11 4 7e404 398GrMLO10 Gorai.007G193200.1 7 1683 560 15 6 9e456 448GrMLO11 Gorai.007G250700.1 7 1392 463 13 6 6e419 414GrMLO12 Gorai.009G078600.1 9 1764 587 15 7 11e505 495GrMLO13 Gorai.009G078700.1 9 1596 531 15 7 11e485 475GrMLO14 Gorai.009G118700.1 9 1455 484 14 7 6e450 445GrMLO15 Gorai.010G000800.1 10 1530 509 15 7 8e454 447GrMLO16 Gorai.010G205000.1 10 1707 568 15 8 18e475 458GrMLO17 Gorai.010G205100.1 10 1239 412 12 5 6e337 332GrMLO18 Gorai.011G030800.1 11 1545 514 14 7 20e460 441GrMLO19 Gorai.011G089600.1 11 1437 478 14 6 6e425 420GrMLO20 Gorai.011G240700.1 11 1731 576 15 8 6e495 490GrMLO21 Gorai.012G004100.1 12 1737 578 15 7 9e494 486GrMLO22 Gorai.013G197800.1 13 1515 504 13 7 7e455 449

a Available at https://www.cottongen.org/data/download/genome_JGI.b Presence and number of transmembrane (TM) helices in the proteins was predicted using the online software of TMHMM (http://www.cbs.dtu.dk/services/TMHMM-2.0/).c Presence of conserved MLO domains within the acquired MLO sequences was confirmed by searching in NCBI's conserved domain database (http://www.ncbi.nlm.nih.

gov/Structure/bwrpsb/bwrpsb.cgi).

Table 2Members of GaMLO gene family as predicted in G. arboretum genome.


GaMLO1 Cotton_A_06751 3 1671 556 15 7 9e475 467GaMLO2 Cotton_A_19376 4 1596 532 15 6 5e470 466GaMLO3 Cotton_A_00762 5 1710 569 15 6 11e507 497GaMLO4 Cotton_A_08798 5 1224 407 11 2 4e389 386GaMLO5 Cotton_A_36285 6 1302 433 13 5 4e423 420GaMLO6 Cotton_A_12678 7 1791 596 14 6 5e536 532GaMLO7 Cotton_A_06313 7 1692 563 15 7 50e490 441GaMLO8 Cotton_A_26172 8 1488 495 13 7 18e402 385GaMLO9 Cotton_A_07533 9 1467 488 14 6 6e464 459GaMLO10 Cotton_A_03932 9 1545 514 14 7 20e460 441GaMLO11 Cotton_A_23415 9 1704 567 15 7 6e484 479GaMLO12 Cotton_A_11046 10 1902 633 15 7 1e551 551GaMLO13 Cotton_A_11047 10 1602 533 15 8 11e486 476GaMLO14 Cotton_A_15078 10 1395 464 12 8 1e462 462GaMLO15 Cotton_A_20369 12 1680 559 15 5 7e475 469GaMLO16 Cotton_A_22367 13 1518 505 13 7 7e455 449GaMLO17 Cotton_A_39162 Ca1 1674 557 15 8 14e494 481

a Available at https://www.cottongen.org/data/download/genome_BGI_A2.b Presence and number of transmembrane (TM) helices in the proteins was predicted using the online software of TMHMM (http://www.cbs.dtu.dk/services/TMHMM-2.0/).c Presence of conserved MLO domains within the acquired MLO sequences was confirmed by searching in NCBI's conserved domain database (http://www.ncbi.nlm.nih.



3.3. Phylogenetic analysis

We performed a phylogenetic study on the newly identifiedGhMLO proteins. The dataset covered 38 GhMLO proteins, thecomplete A. thalianaMLO protein family AtMLO1e15 (Devoto et al.,2003), and a series of MLO homologues that have been functionallyassociated with PM susceptibility from grapevine (V. vinifera)(Angela Feechan et al., 2008), apple (Malus domestica) (Pessinaet al., 2014), barley (H. vulgare) (Buschges et al., 1997), rice (O.sativa) (Liu and Zhu, 2008), pepper (C. annuum) (Zheng et al.,2013a,b), pea (Pisum sativum) (Pavan et al., 2011), maize (Z. mays)(Devoto et al., 2003) and tomato (S. lycopersicum) (Chen and Zhang,2014). Phylogenetic analysis of total 74 MLO proteins resulted inseven distinct clades (Fig. 2). Clades I to VI were assigned accordingto a previous study of AtMLO homologues and grapevine VvMLOs

(Devoto et al., 2003; Angela Feechan et al., 2008). Two additionalclades (VII and VIII) included Rosaceae (Pisum persica, F. vesca andM. domestica) MLO homologues only, as reported by Pessina et al.(Pessina et al., 2014). Four additional G. hirsutum MLO homologuesGhMLO8, GhMLO13, GhMLO22 and GhMLO31 were grouped inclade VII with apple MdMLO18, suggesting the existence of morethan six clades in the plantMLO gene family. Eight G. hirsutumMLOhomologues (GhMLO11, GhMLO16, GhMLO21, GhMLO25,GhMLO28, GhMLO34, GhMLO36 and GhMLO38) clustered togetherin clade V with other MLO proteins, AtMLO2, AtMLO6, AtMLO12,tomato SlMLO1, pea PsMLO1, pepper CaMLO1 and CaMLO2, whichhave been experimentally shown to be required for PM suscepti-bility (e.g (Consonni et al., 2006; Pavan et al., 2011; Zheng et al.,2013a,b; Kim and Hwang, 2012).,).

Two homologues, GhMLO5 and GhMLO17 were found to

https://www.cottongen.org/data/download/genome_JGIhttp://www.cbs.dtu.dk/services/TMHMM-2.0/http://www.ncbi.nlm.nih.gov/Structure/bwrpsb/bwrpsb.cgihttp://www.ncbi.nlm.nih.gov/Structure/bwrpsb/bwrpsb.cgihttps://www.cottongen.org/data/download/genome_BGI_A2http://www.cbs.dtu.dk/services/TMHMM-2.0/http://www.ncbi.nlm.nih.gov/Structure/bwrpsb/bwrpsb.cgihttp://www.ncbi.nlm.nih.gov/Structure/bwrpsb/bwrpsb.cgi

Table 3Members of the GhMLO gene family as predicted in G. hirsutum cv. TM-1 genome.


GhMLO1 CotAD_67162 1 1332 443 13 5 6e399 394GhMLO2 CotAD_67021 1 1377 458 11 5 9e352 344GhMLO3 CotAD_28782 2 1569 522 15 7 9e449 441GhMLO4 CotAD_16944 2 1545 514 15 6 9e432 424GhMLO5 CotAD_56441 2 1842 613 15 7 5e552 548GhMLO6 CotAD_36554 3 1416 471 14 5 4e453 450GhMLO7 CotAD_62838 4 1302 433 13 5 4e423 420GhMLO8 CotAD_31993 6 768 255 8 2 7e253 247GhMLO9 CotAD_04379 8 1281 426 13 7 7e481 475GhMLO10 CotAD_12423 9 1347 448 11 6 11e401 391GhMLO11 CotAD_12424 9 1695 564 15 7 11e482 472GhMLO12 CotAD_63577 11 1470 489 14 6 6e465 460GhMLO13 CotAD_64129 12 1089 362 12 4 60e358 299GhMLO14 CotAD_30773 13 1491 496 12 6 7e446 440GhMLO15 CotAD_75839 14 1674 557 15 8 14e494 481GhMLO16 CotAD_36153 14 1767 588 15 7 6e503 498GhMLO17 CotAD_29126 15 1845 614 15 7 5e552 548GhMLO18 CotAD_45096 17 1266 421 14 7 4e404 401GhMLO19 CotAD_31735 18 1407 468 11 6 11e406 396GhMLO20 CotAD_05932 18 1416 471 14 5 4e444 441GhMLO21 CotAD_31530 19 1524 507 14 7 14e414 401GhMLO22 CotAD_20256 19 2079 692 18 8 7e463 457GhMLO23 CotAD_09253 20 1740 579 16 6 9e492 484GhMLO24 CotAD_72948 20 1236 411 11 4 44e367 324GhMLO25 CotAD_08062 22 1662 553 14 7 7e469 463GhMLO26 CotAD_01552 22 1212 403 12 5 2e369 368GhMLO27 CotAD_54310 22 1596 531 15 7 11e485 475GhMLO28 CotAD_54311 22 1725 574 15 6 11e492 482GhMLO29 CotAD_15574 24 1470 489 14 6 6e436 431GhMLO30 CotAD_06176 26 1692 563 15 5 9e482 474GhMLO31 CotAD_00651 scaffold26.1 1305 434 14 7 20e433 414GhMLO32 CotAD_07261 scaffold72.1 1437 478 13 4 9e397 389GhMLO33 CotAD_08394 scaffold190.1 1407 468 11 6 11e406 396GhMLO34 CotAD_49330 scaffold1917.1 1227 408 9 4 7e338 332GhMLO35 CotAD_39473 scaffold2046.1 1407 468 13 7 8e438 431GhMLO36 CotAD_71931 scaffold3483.1 1272 423 11 5 14e330 317GhMLO37 CotAD_75625 scaffold3566.1 1725 574 15 8 29e509 481GhMLO38 CotAD_74071 scaffold4982.1 1659 552 15 7 6e469 464

a Available at https://www.cottongen.org/data/download/genome_BGI_AD1.b Presence and number of transmembrane (TM) helices in the proteins was predicted using the online software of TMHMM (http://www.cbs.dtu.dk/services/TMHMM-2.0/).c Presence of conserved MLO domains within the acquired MLO sequences was confirmed by searching in NCBI's conserved domain database (http://www.ncbi.nlm.nih.



grouped in clade IV, which contained all monocot MLO proteins,such as barley HvMLOs, maize ZmMLO1 and rice OsMLOs func-tionally acting as PM susceptibility factors (Devoto et al., 2003).Consistent with this finding, one MLO protein from the dicot spe-cies V. vinifera (VvMLO14) (Angela Feechan et al., 2008), one ho-mologue from F. vesca (FvMLO17) (Pessina et al., 2014) and onefrom P. persica (PpMLO12) (Pessina et al., 2014) also clustered inclade IV. Such clustering results raise the question of whetherexclusively monocot MLO proteins cluster in clade IV. Analysis ofphylogenetic relationships revealed that ten G. hirsutum MLO ho-mologues were clustered in clade V and IV, which harboured alldicot and monocot MLO proteins functionally related to PM sus-ceptibility, thus making them susceptibility factor candidates. Thephylogenetic analysis performed here confirmed the presence ofclade VII, first reported in Rosaceae by Pessina et al. (Pessina et al.,2014). Additional studies should focus on the functional charac-terization of cotton MLO homologues grouped in clades IV, V andVII.

We conducted furthermultiple alignments amongMLO proteinsin clade V to identify conserved domains (Fig. 3). Twelve proteinsfrom 5 species presented a high degree of conservation in theirseven predicted TM domains, which define this protein family(Devoto et al., 2003). We also identified a calmodulin-bindingdomain consisting of a stretch of approximately 10e15 amino

acids proximal to TM domain 7 (Kim et al., 2002a,b). Moreover, twoother conserved regions within the C-terminus of several MLOproteins have been suggested to modulate PM infection (Panstruga,2005). Peptide domain I is characterized by the presence of serine(S), threonine (T) and proline (P) residues, whereas peptide domainII contains the consensus motif D/E-F-S/T-F (Fig. 3). All of theGhMLO proteins within clade V contain the two conserved domainsmentioned above, except GhMLO25 contains a modified motif II ofI-F-S-L.

3.4. Synteny block detection

Previous studies have indicated that allotetraploid cotton spe-cies were derived from an interspecific hybridization event be-tween A and D-genome diploid species (Cronn et al., 1999). Therecent availability of genome sequences for G. raimondii,G. arboreum and G. hirsutum offers great potential for comparativegenomics studies, which aim to provide insights into structures andfunctions of genomic features. First, we identified a total of 517orthologous relationships between G. raimondii, G. arboreum andG. hirsutum MLO homologues (Table S2). Because of homologousgenes and duplication events in the G. hirsutum genome, numerousmany-to-one relationships were identified. Orthologues are genesin different species that evolve from one single gene in their last

https://www.cottongen.org/data/download/genome_BGI_AD1http://www.cbs.dtu.dk/services/TMHMM-2.0/http://www.ncbi.nlm.nih.gov/Structure/bwrpsb/bwrpsb.cgihttp://www.ncbi.nlm.nih.gov/Structure/bwrpsb/bwrpsb.cgi

Fig. 1. Chromosomal localization of GrMLOs (A), GaMLOs (B), and GhMLOs (C). The relative sizes (unit, Mb) of G. raimondii (chr.D01eD13), G. arboreum (chr.A01eA13) and G.hirsutum (chr.AD01eAD26) chromosomes were consistent with published genomic data.

Table 4Homologous genes and duplication events detected in GhMLOs

SeqA AA Length SeqB AA Length Identity

Pair 1 GhMLO3 522 GhMLO4 514 95.142 GhMLO5 613 GhMLO17 614 97.723 GhMLO6 471 GhMLO20 471 97.034 GhMLO7 433 GhMLO18 421 93.825 GhMLO8 255 GhMLO22 692 95.296 GhMLO9 504 GhMLO14 496 96.177 GhMLO12 489 GhMLO29 489 97.148 GhMLO15 557 GhMLO37 574 96.419 GhMLO19 468 GhMLO33 468 98.2910 GhMLO21 507 GhMLO36 423 93.8511 GhMLO25 553 GhMLO34 408 90.6912 GhMLO30 563 GhMLO32 478 98.12Event 1 GhMLO1/GhMLO2 443/458 GhMLO24/GhMLO23 411/579 80.78/96.072 GhMLO10/GhMLO11 448/564 GhMLO27/GhMLO28 531/574 95.76/95.21


common ancestor. Such genes often retain identical biological roles

in the present-day organisms. A perfect synteny block is a

Fig. 2. Phylogenetic analysis of MLO proteins. The phylogenetic tree represents a consensus tree with branch lengths proportional to sequence distance. Numbers indicate bootstrapvalues (from 1000 replicates) that support the respective branch. The dataset includes 38 GhMLOs (GhMLO1e38) and 36 other MLO proteins from Arabidopsis thaliana, grapevine(Vitis vinifera), apple (Malus domestica), barley (Hordeum vulgare), rice (Oryza sativa), pepper (Capsicum annuum), pea (Pisum sativum), maize (Zea mays) and tomato (Solanumlycopersicum). Genbank accession numbers of translated MLO proteins used in phylogenetic analysis: AtMLO1 (Z95352); AtMLO2e15 (AF369563eAF369576); VvMLO3 (CAO18135);VvMLO4 (CAO21819); VvMLO6 (CAO66388); VvMLO9 (CAN84002); VvMLO13(CAO68971); VvMLO14(CAO66265); VvMLO17 (CAO68972); MdMLO5 (MDP0000163089); MdMLO7(MDP0000123907); MdMLO11 (MDP0000239643); MdMLO18 (MDP0000928368); MdMLO19 (MDP0000168714); HvMLO (CAB06083); HvMLO-h1 (CAB08860); OsMLO1(CAB08606); OsMLO3 (BAG93853); CaMLO1 (AAX31277); CaMLO2 (AFH68055); PsMLO1 (ACO07297); ZmMLO1 (AAK38337); SiMLO1 (AAX77013).


conserved block of genes that share exactly the same order andstrandedness and contain no mismatches compared with thechromosomes of related species.

Thenwe predicted 25, 28 and 18 conserved non-nested syntenyblocks between G. hirsutum and G. raimondii, G. hirsutum andG. arboretum, and G. raimondii and G. arboretum, respectively(Fig. 4). Notably, 12 conserved synteny blocks were discoveredamong the three Gossypium genomes (Fig. 5). Thirteen blocks werenot included in Figs. 4 and 5 because they involved genes that couldnot be localized to a specific chromosome. These conserved seg-ments contain different numbers of genes, ranging from 1 to 3. Thesize distribution of conserved non-nested blocks is shown in Fig. 6,and detailed information about each block is shown in Table S3.Desirable blocks were detected because of a close evolutionaryrelationship among these three species. In addition, because of theexistence of homologous genes, some many-to-one relationships

Fig. 3. Multiple sequence alignment of GhMLO proteins and selected MLO proteins in cladlycopersicum SiMLO1 (AAX77013) and Capsicum annuum CaMLO2 (AFH68055)) have been(Angela Feechan et al., 2008) clustered in clade V. The multiple sequence alignment was g(TM1e7) inferred from the experimentally determined topology of HvMLO (Buschges et al.,previously defined (Kim et al., 2002a,b). Two additional conserved domains I and II were preby lines above the sequences.

were generated in some blocks.A total of 83 conserved non-nested synteny blocks were pre-

dicted after pairwise comparative analysis of MLO homologues. Inparticular, genes situated on G. raimondii chromosomes 2, 5, 7, 9and 11 are predicted to have corresponding orthologues onG. hirsutum chromosomes 2, 20, 18, 22 and 22, respectively,whereas genes on G. arboreum chromosomes 5, 7, 9 and 10 aresuggested to originate from conserved blocks on G. hirsutumchromosomes 20, 2, 19 and 22, respectively (Fig. 4). The corre-sponding chromosomes that contain the largest number andhighest density of perfectly conserved synteny blocks inG. hirsutum, G. raimondii and G. arboreum are chr2-chr2D-chr7A,chr20-chr5D-chr5A and chr22-chr9D-chr10A. These data suggestthat genes within these conserved blocks may be co-regulated byspecific locus control regions (LCRs), which can control theexpression of a group of genes. This finding indicates conservation

e V based on Fig. 2. Arabidopsis thaliana (AtMLO2, AtMLO6 and AtMLO12), Solanumfunctionally characterized as susceptibility genes. Vitis vinifera VvMLO3 and VvMLO13enerated by CLUSTALX2 using default parameters. The positions of seven TM regions1997) and the approximate position of the calmodulin-binding domain (CaMDB) wereviously identified (Panstruga, 2005), and the above-mentioned domains were indicated

Fig. 4. Circos diagram of synteny blocks identified between G. raimondii, G. arboreum, and G. hirsutum MLOs. The chromosomes of G. raimondii (D01eD13), G. arboreum(A01eA13), A subgenome (AD01eAD13) of G. hirsutum, and D subgenome (AD14eAD26) of G. hirsutumwere filled with light red, light green, dark green and dark red, respectively.A total of 61 coloured lines connecting two chromosomal regions denote syntenic regions between G. raimondii, G. arboreum, and G. hirsutum. Ten blocks were not includedbecause involved genes were not localized to definitive chromosomes. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version ofthis article.)


and rearrangements of certain chromosome segments, which playan important role in the evolution or adaptive changes of thesethree close species.

3.5. Gene expression analysis during leaf development

To estimate the temporal expression patterns of GhMLO genesduring leaf development of upland cotton, we analysed publiclyavailable RNA-Seq data containing 3624 differentially expressedgenes during leaf development of 15-, 25-, 35-, 45-, 55-, and 65-day-old plants (Lin et al., 2015). Four genes, GhMLO15, GhMLO21,GhMLO25 and GhMLO38, were differentially expressed during thesix leaf developmental stages from young and mature to senescentphases (Fig. 7). The relative expression of GhMLO15 and GhMLO21 inleaves was higher than that of GhMLO25 and GhMLO38. GhMLO15was significantly up-regulated at almost all phases, although itsharply decreased at the 35-day-old stage. During leaf growthstages, GhMLO21 transcripts continuously increased, especially af-ter 35 days. GhMLO25 and GhMLO38 expression slightly increasedoverall during leaf development. In summary, four differentially

expressed genes were up-regulated during leaf development,indicating that they could be involved in the regulation of leafsenescence.

3.6. Responses of GhMLOs to stress and phytohormonal stimuli

To determine whether GhMLOs are involved in environmentalstress or phytohormone signalling pathways, we examined geneexpression of GhMLOs in response to exogenous application of salt,Mannitol, ABA, ETH, JA or SA. Experimental data demonstrated thatonly five genes were not differentially expressed and twenty geneswere sharply up-regulated or down-regulated under treatments(Fig. 8). All data of gene expression were presented in Table S5.Transcripts of eleven genes (Fig. 8A, B and C) were increased bymore than ten-fold compared to non-treated plants, whereas sixgenes (Fig. 8D and E) were found to be significantly suppressed inleaves under some of these conditions.

Previous analysis of phylogenetic relationships revealed that tenG. hirsutum MLO homologues were clustered in clade V and IV,which harboured all dicot and monocot MLO proteins functionally

Fig. 5. Circos diagrams of synteny blocks detected among GhMLOs, GrMLOs and GaMLOs. The chromosomes of G. raimondii (D01eD13), G. arboreum (A01eA13), A subgenome(AD01eAD13) of G. hirsutum, and D subgenome (AD14eAD26) of G. hirsutum were filled with light red, light green, dark green and dark red, respectively. One coloured circle withthree lines indicates a synteny block among the three genomes. Three blocks were not depicted because they did not demonstrate positioning of related genes. (For interpretation ofthe references to colour in this figure legend, the reader is referred to the web version of this article.)


related to PM susceptibility (Consonni et al., 2006; Pavan et al.,2011; Devoto et al., 2003; Zheng et al., 2013a,b; Kim and Hwang,2012). Experimental results showed that all of them were differ-entially expressed under stress or phytohormone application. Three(GhMLO6, GhMLO18 and GhMLO25) out of 38 genes were intenselyresponsive to ABA treatment. ABA is an important phytohormonethat can convert the initial stress signal, such as drought or highsalinity, into a cellular response (Gonzalez-Guzman et al., 2012; Liet al., 2011). Intense induction of these three genes by ABA sug-gests that they are specifically involved in the response of the ABAsignalling pathway. GhMLO6, GhMLO11, GhMLO17 and GhMLO28were dramatically up-regulated in upland cotton leaves cultured inthe presence of 200 mM ETH. ETH is a gas phytohormone withsignificant functions throughout the whole dicotyledon andmonocotyledon life cycle, ranging from growth and development toa variety of stress responses (Van Der Straeten and Van Montagu,1991; Qin et al., 2007). In addition, three genes (GhMLO9,GhMLO17 and GhMLO23) showed opposite expression pattern inresponse to exogenous JA and SA.

The functions of MLO family members in modulating plant

defence responses against PM and regulating cell death has beenconfirmed (Buschges et al., 1997; Consonni et al., 2006; Pavan et al.,2011; Bai et al., 2008; Piffanelli et al., 2002; Appiano et al., 2015;Kim and Hwang, 2012). However, accumulating evidence has sug-gested that MLO may be involved in a variety of abiotic stresses(Piffanelli et al., 2002; Lim and Lee, 2014; Shen et al., 2012; Qinet al., 2015). Four GmMLOs from soybean were responsive tovarious abiotic stresses and phytohormone treatments (Shen et al.,2012). The results of virus-induced silencing of CaMLO2 in chilipepper and over-expression in Arabidopsis support that CaMLO2participate in drought stress regulation, acting as a suppressor ofABA signalling (Lim and Lee, 2014). The expression of HbMLO1 fromthe rubber tree was intensely induced by diverse phytohormones(including ethephon, JA, SA, ABA, indole-3-acetic acid, and gibber-ellic acid), H2O2, and wounding treatments, but no intenseresponse to PM infection was found (Li et al., 2011). In the currentstudy, various abiotic stresses and phytohormone treatmentsinduced or suppressed the expression of 33 GhMLOs. The clearresponse of GhMLOs to stress conditions or phytohormone sup-plement suggests that they may participate in the salt, Man, ABA,

Fig. 6. Size distribution of conserved non-nested synteny blocks, obtained by OrthoCluster, preserving gene order. We did not permit mismatches and did not consider strand-edness. In total, 25, 28 and 18 conserved non-nested synteny blocks were predicted between G. hirsutum and G. raimondii (AADD- DD), G. hirsutum and G. arboretum (AADD- AA),G. raimondii and G. arboretum (DD- AA), respectively. Moreover, 12 conserved blocks of MLO candidate genes were found among the three genomes (AADD- DD- AA). Theseconserved segments contain different numbers of genes, ranging from 1 to 3 genes.

Fig. 7. Transcriptional variation of four G. hirsutum MLO genes during leaf development of 15-, 25-, 35-, 45-, 55-, and 65-day-old plants. Published RNA-Seq data (Lin et al., 2015)containing 3624 differentially expressed genes during leaf development were analysed. Four genes, GhMLO15, GhMLO21, GhMLO25 and GhMLO38 were differentially expressedduring six leaf developmental stages from young and mature to senescent phases.


Fig. 8. Relative expression analysis of twenty G. hirsutum MLO genes in response to abiotic treatments and phytohormone application. Seven-day-old seedlings were culturedwithin MS solid medium as a control (CK) or supplemented with 50 mM ABA, 200 mM sodium chloride (NaCl), 200 mM ethylene (ETH), 100 mM JA, 1 mM SA or 200 mM mannitol(Man). Treated and control plants were grown under the same conditions, and leaf samples were collected after three weeks of treatments. Three biological repeats were performed,and each sample contained three young leaves collected from one single plant. Data in the graph were mean values with standard deviation (error bar) from three replicates.Statistical analysis was conducted by Student's t-test (**P < 0.01, *P < 0.05).


ETH, JA and SA responsive signalling pathways. We propose thatfuture studies should focus on elucidating the roles of theMLO genefamily in response to environmental stimuli.

4. Conclusion

Our work led to the identification of 22 MLO homologues in G.raimondii L., 17 in G. arboreum L. and 38 in G. hirsutum L. Themajority of the 77 Gossypium MLO members were organized assingletons, with the exception of five gene clusters. After compre-hensive analysis of pairwise alignments and the physical location ofeach GhMLO gene, we detected 12 pairs of homologous genes and 2tandem duplications. Clearly, the phylogenetic analysis performedin this study confirmed the presence of clade VII, which was pre-viously reported in the Rosaceae MLO family. A total of 83 conservednon-nested synteny blocks were predicted after pairwise compar-ative analysis of MLO homologues among the three Gossypium

species. Four genes (GhMLO15, GhMLO21, GhMLO25 and GhMLO38)were differentially expressed during six leaf developmental stagesfrom young and mature to senescent phases. The general andintense response of GhMLOs to stress conditions or phytohormonesupplement suggests that MLO gene family may participate in thesalt, Man, ABA, ETH, JA and SA responsive signalling pathways inupland cotton.

Contributions

Conceived and designed the experiments: Renhai Peng, XiaoyanWang and Shuxun Yu. Performed the experiments: Xiaoyan Wang,Qifeng Ma and Lingling Dou. Analyzed the data: XiaoyanWang andQifeng Ma. Contributed reagents/materials/analysis tools: RenhaiPeng and Shuxun Yu. Wrote the paper: Xiaoyan Wang. Edited themanuscript: Xiaoyan Wang and Zhen Liu.


Acknowledgements

We would like to thank doctoral candidate Xihua Li for herassistance of Circos software. The work described in this paper wassupported by the National High-tech Research and DevelopmentProjects of China (2013AA102601), Program for Science & Tech-nology Innovation (Talents in Uinversities of Henan Province(13HASTIT026) and the Major Projects of Anyang City Science andTechnology Plan (ANKE20140208).

Appendix A. Supplementary data

Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.plaphy.2016.02.031.

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Genome-wide characterization and comparative analysis of the MLO gene family in cotton1. Introduction2. Materials and methods2.1. In silico identification and annotation2.2. Gene organization2.3. Phylogenetic analysis2.4. Detection of synteny blocks2.5. Plant materials, stress and phytohormone treatments2.6. Real-time quantitative RT-PCR2.7. Ethics statement

3. Results and discussion3.1. In silico characterization of Gossypium MLO homologues3.2. Genomic organization of Gossypium MLO homologues3.3. Phylogenetic analysis3.4. Synteny block detection3.5. Gene expression analysis during leaf development3.6. Responses of GhMLOs to stress and phytohormonal stimuli

4. ConclusionContributionsAcknowledgementsAppendix A. Supplementary dataReferences

Plant Physiology and Biochemistrydownload.xuebalib.com/xuebalib.com.36066.pdf · both CaMLO1 and...

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