四倍体栽培棉种高密度遗传图谱的加密及棉花红株R_1和茸毛T_1基因的精细定位
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摘要
棉花是世界性重要的经济作物之一,棉纤维的品质和色彩可以大大提升其经济价值。高密度的遗传图谱是植物基因组研究的基础。可有效阐明基因组的结构特征,为重要性状基因的精细定位和图位克隆奠定基础。棉花叶片密生茸毛和红株性状均由单显性基因控制。叶片中茸毛的发育不仅与棉纤维发育有相似之处,而且多毛具有抗虫特性。红叶中决定色素形成的基因可有效用于培育天然彩棉的育种实践中。精细定位这2个关键基因,获得其候选相关基因,进而进行结构、表达及系统进化分析,不仅可阐明其作用机理,而且可为棉花纤维品质改良、抗虫育种及天然彩棉的培育提供重要基因资源。本研究在前人构建海陆棉花种间遗传图谱的基础上,进一步开发和利用棉花新标记,进行图谱加密和染色体结构特征分析；利用加密的图谱信息,完成棉花茸毛及红株两个质量性状基因T1和R1的染色体精细定位和其候选基因的初步分析；结合已释放的不同棉种EST及基因组序列信息,进行棉花WRKY类家族基因的全基因发掘、鉴定、分类和表达初步分析。主要研究结果如下：
1、高密度海陆种间遗传图谱的构建及基因组结构分析
本研究利用新开发的gSSR引物及网上公开释放的部分CER、CGR、COT、DC、 DPL、SHIN及HAU编号的引物,与已开发的SSR标记位点进行冗余筛选,获得1431对非冗余引物,在作图亲本TM-1和Hai7124之间筛选多态性,进而完成图谱的加密工作。新获得的海陆栽培棉种种间遗传图谱包含3414个位点,总长为3677.6cM,标记间的平均距离为1.08cM,分布在26条连锁群上。
该图谱上包含941个重复位点,分别由425对引物在亲本TM-1和Hai7124间扩增产生。这些重复位点主要来源于SSR、REMAP、SRAP、AFLP、RT和In-Del等5种标记类型。其中326对SSR引物产生693个重复位点,包括287对SSR引物产生2个位点；37对产生3个位点；2对产生4个位点。分析这些重复位点的染色体分布,除位于部分同源染色体上的重复位点外,一些重复位点位于同一染色体上,另一些重复位点位于非同源的不同染色体上,表明在四倍体棉花进化进程中基因组染色体内部及染色体间发生了复制和重排等结构变异。
新构建的遗传图谱上含86个位点簇(≥5loci/cM),涉及617个位点。除Chr.1上未发现位点簇外,这86个位点簇分布在其余25个连锁群上,分布密度不均匀。其中,31个位点簇分布在At亚组上,涉及到229个位点；55个位点簇分布于Dt亚组上,涉及到388个位点。同时也检测到19个基因岛(≥5EST-SSR loci/cM)和1个反转录转座子区(≥5REMAP loci/cM).
300个位点在作图群体中不符合孟德尔遗传规律(P<0.05)。在连锁群上构成12个偏分离富集区,At亚组和Dt亚组各分布6个。这12个偏分离富集区分布在11条染色体上,其中在Chr.20上有两个偏分离富集区。在偏分离富集区上位点的偏向保持着一致性,其中8个偏向于亲本TM-1,4个偏向于杂合子。
2、两个质量性状基因的精细定位和候选基因筛选
以构建的高密度海陆种间遗传图谱为基础,分别利用(Sub16×T586)F2群体和[(Hai7124×T586)xHai7124]BC1群体,完成棉花两个重要的质量性状基因一红株R1和茸毛乃基因的精细定位。将红株R1基因定位在第16染色体标记NAU4956和NAU6752之间,遗传距离分别为2.91cM和0.49cM;将茸毛乃基因定位在第6染色体NAU5434与NAU1277之间,距最近标记NAU5434的遗传距离为0.6cM.
基于精细定位的结果和网上公开释放的棉花D基因组雷蒙德氏棉种的基因组序列信息,根据目标基因附近的标记序列映射雷蒙德氏棉基因组序列,获得目标基因所在的候选物理区域。利用Fgenesh程序对候选的物理区域进行ORF预测及BLAST2Go的功能注释及代谢特征分析,结合所克隆基因作用特征完成候选基因的确定及功能初步分析。针对色素合成代谢途径特点,筛选了6个候选基因,分别命名为RG1-RG6;参考前人拟南芥表皮毛发育研究结果,初步确定标记映射区域与棉花密生茸毛性状相关的6个候选基因,分别命名为TGI-TG60
形态及扫描电镜分析显示,随着叶龄发育,T586的叶片色素沉积越来越多叶片茸毛的发育随着叶龄发育逐渐变稀,但每一发育阶段,T586的叶片茸毛均显著高于Hai7124,在成熟叶片上,Hai7124仅叶脉及叶边缘可见茸毛分布,而T586叶片密被茸毛。选择T586和Hai7124不同叶龄的棉花叶片(叶芽、幼叶及成熟叶),进行候选基因的表达分析。Q-PCR分析表明,在控制棉花红株性状的6个候选基因中,基因RG4在T586色素沉积最多的成熟叶中,其表达量显著高于同时期Hai7124叶片中的表达量,而其他候选基因在两个亲本材料色素差异最明显时期叶片中的表达量都没有达到显著水平。在控制茸毛性状的6个候选基因中,基因TG5在T586叶片每一发育阶段的表达量与Hai7124之间均存在显著或极显著差异。同时,随着叶片的发育,在T586和Hai7124中表达水平也随之降低,与叶片表皮茸毛密度分布特征一致。其余候选基因的表达未发现规律性。综合基因表达、表型变化及扫描电镜分析结果,推测RG4和TG5是控制两个质量性状红株R1和茸毛T1的候选基因。根据BLAST2Go(?)勺功能注释表明,RG4是一个编码八氢番茄红素合酶的基因；TG5为一个bHLH类型的转录因子。
3、棉花WRKY转录因子家族全基因组分析
已有研究报道,WRKY转录因子家族参与植物的生长发育、衰老等进程,同时对各种生物胁迫和非生物胁迫都有一定的响应。在拟南芥中,AtWRKY44参与了拟南芥表皮毛形成。为了阐明WRKY转录因子家族在棉花重要性状形成中的可能角色,我们开展了WRKY转录因子家族棉花全基因组的发掘、鉴定、分类和表达初步研究。
利用在NCBI上公开释放的棉花EST序列,结合Pfam数据库提供的WRKY转录因子种子文件的全部2450条种子序列,比对棉花总EST数据库,最终得到535条WRKY类EST序列。以这535条EST序列作为探针,通过电子克隆技术,获得95个含有WRKY结构域的Contig,其中74个Contig中至少含有一个完整的WRKY结构域,暂命名为Contig1-Contig74。与拟南芥WRKY转录因子家族的进化分析显示,74个Contig属于3个组及5个亚组,其中3个组-Group Ⅰ、Group Ⅱ和Group Ⅲ,分别含有16、49和9个Contig。Group Ⅱ的五个亚组--Group Ⅱ a、Group Ⅱb、Group Ⅱc、 Group Ⅱd和Group Ⅱe分别含有5、2、25、12和5个Contig。
利用公布的雷蒙德氏棉全基因组序列草图,进一步对雷蒙德氏棉全基因组进行了WRKY转录因子家族成员的预测,共得到109个WRKY转录因子家族成员。分子进化分析将该转录因子家族成员分为3大类,其中Group Ⅰ有19个成员,Group Ⅱ有78个成员,Group Ⅲ有12个成员。进一步与来源于拟南芥的WRKY家族基因聚类结果分析,将Group Ⅱ分成Group Ⅱa、Group Ⅱb、Group Ⅱc、Group Ⅱd和Group Ⅱe五个亚组,每个组成员分别有7个、15个、29个、15个和12个。通过序列比对,发现有5个成员在WRKYGQK的保守结构域上存在有WRKYGKK的变异,分别是GrWRKY31、 GrWRKY39、GrWRKY46、GrWRKY35和GrWRKY78,并且这5个成员都属于Group Ⅱc亚组；在Group Ⅱc中的GrWRKY8还存在WRKYGHK结构域变异。除了在WRKYGQK的保守结构域上存在变异,在Group ⅠN、Group ⅠC和GroupⅢ中发现WRKY家族成员在锌指结构上也存有一定的变异。
通过序列相似性比对来自棉花EST数据库和雷蒙德氏棉基因组的WRKY转录因子,前期预测的74个Contig中,除属于Group Ⅱc亚组的Contig69、70、71和73等4个Contig外,70个Contig h(?)在二倍体D基因组雷蒙德氏棉中找到高度相似的同源序列。推测4个未找到同源序列的Contig可能是四倍体棉种在进化过程中获得的新基因或是At亚组专有的WRKY转录因子。
从陆地棉TM-1基因组中克隆获得GhWRKY19、 GhWRKY23、GhWRKY60、 GhWRKY87、GhWRKY95等5个成员全长ORF的基因组信息。其中,GhWRKY19ORF全长为1401bp,编码466个氨基酸,含有4个内含子；GhWRKY23ORF全长为942bp,编码313个氨基酸,含有4个内含子；GhWRKY60ORF全长为1068bp,编码355个氨基酸,含有2个内含子；GhWRKY87ORF全长为807bp,编码268个氨基酸,含有2个内含子；GhWRKY95ORF全长为966bp,编码321个氨基酸,含有2个内含子。不同组织、器官表达特征表明,WRKY类基因在根茎叶等营养器官中的表达量普遍高于在棉纤维中的表达量。GhWRKY38在根中高表达；GhWRKY19、GhWRKY87和GhWRKY43在叶中高表达；GhWRKY74在根和茎中高表达；而GhWRKY21和GhWRKY60在根和叶中高表达。在纤维发育进程中高表达的WRKY家族成员主要检测到GhWRKY19、GhWRKY21和GhWRKY23,其高表达的时期集中在-3dpa-5dpa,没有检测到在纤维发育后期高表达的基因。
以陆地棉抗逆品种晋棉19三叶期幼苗为试验材料,进行水杨酸(2mM SA)、茉莉酸甲酯(100μM MeJA)、脱落酸(200μM ABA)、赤霉素(500μM GA)、盐(200mM NaCl)、干旱(20%PEG6000)、乙烯(5mM ET)等非生物胁迫处理,分析WRKY类基因在非生物胁迫诱导0h、2h、4h、6h、8h、10h、12h、24h后的表达水平。结果表明：GhWRKY23、 GhWRKY43、GhWRKY60、GhWRKY74和GhWRKY87被上述七种胁迫处理诱导后表达显著提高。GhWRKY19在PEG和ET处理后,表达水平未发生显著变化,而GhWRKY21和GhWRKY38不被盐胁迫诱导。
Cotton is one of the important cash crops in the world, and the quality and color of the cotton fibers can greatly enhance its economic value. The high-density genetic map is the basement of genome research in plant. It can not only shed light on the structural features of the genome, but also lay the foundation for the fine mapping of genes related to qualitative and quantitative traits. Trichomes and red plant traits were controled by a single dominant gene in cotton. There were similarities between the development of leaf hairs and the development of the cotton fiber, and the leaf hairs had insect-resistant characteristics. Pigmentation gene of the red plant can be effectively used to cultivate natural colored cotton. To elucidate the mechanism and provide important genetic resouces for fiber quality improvement, insect resistance enhancement and natural colored cotton cultivation, we mapped the two key genes and tagged preliminary the candidate genes. In this paper, we updated our backbone genetic map and analyzed the chromosome structural features in allotetraploid cotton. Based on the high-density genetic map, two genes related to quality trait, T1and R1, were fine mapped and candidate genes were further mined. Using different cotton EST database and genome sequene of G. raimondii, the development, identification, classification of genome-wide WRKY transcription factors family in cotton were carried out and preliminary expressional analysis was performed. The main findings are as follows.
1The construction of high-density genetic map and analysis of genomic structure
Total1431primer pairs were used, including newly developed gSSR, publicly released CER, CGR, COT, DC, DPL, SHIN and HAU primers, to screen poplymorphisms between TM-1and Hai7124in order to enhance our backbone genetic map in allotetraploid cotton. The updated map consists of3,414loci in26linkage groups covering3,667.62cM with an average inter-locus distance of1.08cM. By the analysis of loci, we draw the following results:
Among2898primer pairs produced3414loci in TM-1and Hai7124,425primer pairs amplified two or more loci, resulting in total of941duplicate loci. Four marker types, morphological marker, CAPs, SNP and BAC-end, did not find duplicate loci.693duplicated loci were identified by326SSR primer pairs, with574duplicated,111triplicated, and8tetraplicated loci. Some loci were present on the homeologous chromosomes, some loci found to involve in the same chromosomes, and other loci spanned the different chromosomes, implying duplication of multiple round and genome rearrangement of both intrachromosome and interchromosome in the process of evolution.
The cluster of loci were also observed in26linkage group,86clusters involved in617loci (≥5loci/cM) were discovered on the25linkage group besides A1(Chr.01). Of them,31clusters contained229loci from At subgenome, and55clusters contained388loci from Dt subgenome.19candidate genes islands (≥5EST-SSR loci/cM) and one retrotransposon-rich region were discovered by clusters distribution of marker loci.
300loci showed non-mendelian segregation (P<0.05). A total of12segregation distortion regions (SDRs) were detected on11linkage groups. There were two SDRs in D10linkage group. Among12SDRs, six were on the At subgenome and six on the Dt subgenome, with8SDRs skewed toward G. hirutum TM-1and four SDRs skewed toward the heterozygote.
2The fine mapping of two genes related to qualitative traits and screening of candidate genes
Based on the high-density genetic map and two populations, Subl6×T586F2and [(Hai7124xT586)xHai7124] BC1populations, the red plant R1gene and the trichomes T1gene were fine mapped. As a result, R1was mapped between NAU4956and NAU6752, with only0.49cM to the nearest maker loci (NAU6752) by the Sub16×T586F2population contained1259individual plants;the T1was located between NAU5434and NAU1277, with only0.6cM to the nearest marker loci (NAU5434) by the [(Hai7124xT586)xHai7124] BC1population contained835individual plants.
To find their candidate genes, we used the sequence of markers as probes to anchor the G. raimondii genome scaffolds. The ORF were searched by Fgenesh program and functional annotation by BLAST2Go. Based on pigment synthesis pathway, we selected six candidate genes, named for RG1-RG6, for cloning R1gene; According to Arabidopsis trichomes development research, we selected six candidate genes for cloning T1gene, named for TG1-TG6.
Morphological and electron microscope scanning analysis show that the pigment accumulates is more and more in T586leaves as development process, and the density of leaf hairs of T586is significantly higher than Hai7124in each development stage, though the hairs of the leaves become sparser with the leaves development. The leaf hairs can only be observed around the nervation and the edges of the leaves of Hai7124on the mature leaves, while the leaves of T586are covered with leaf hairs of high density. Based on the study, cotton leaves of different development stage (leaf buds; young leaf; mature leaf) of T586and Hai7124were collected for further expression analysis of candidate genes. Q-PCR results indicated that among the6candidate genes, RG4gene had a significantly higher expression level in the mature leaves that accumulated the most pigment of T586than in the leaves of Hai7124of the same development stage, while others did not show significant differences. TG5gene showed significant expression difference between the leaves of T586and Hai7124at every development stage compared with other five candidate genes, and the expression level in T586and Hai7124decreased with the development of the leaves, and the results in consistant with the distribution of the density of leaf hairs, while other candidate genes did not show regularities in the expression mode. Taking all the above results into consideration, RG4and TG5are considered to be the two candidate genes that controls two important qualitative traits, namely Red plant R1and Leaf Hairs T1, respectively. Blast2Go results indicated that RG4encoded a phytoene synthase, while TG5acted as a bHLH transcript factor.
3The genome-wide analysis of WRKY transcription factor family in cotton
It has been reported WRKY transcription factors family involved in plant growth, development, senescence and response to various biotic and abiotic stress. AtWRKY44involved in trichomes development in Arabidopsis.To clarify the possible role of WRKY transcription factors family in cotton growth and development, the development, identification, classification of genome-wide WRKY transcription factors family in cotton were carried out and preliminary expressional analysis was performed.
In recent years, a large number of EST sequences of cotton have been releasd in NCBI. Using the Pfam database provides WRKY transcription factor seed file, total2450seed sequences compared to the EST database of cotton, further we got535EST sequences related to WRKY transcription factor. By the535EST sequences as probes and electronic cloning technology, we got the95contigs contained full or partial WRKY domain. Of them,21Contig lacked the zinc finger structure, and remaining74Contigs were able to find a complete WRKY domain. These74Contigs were named Contigl to Contig74. Based on the WRKY domain characteristics and evolutionary analysis,74Contigs were divided into three groups and five subgroups, Group I contained16Contigs, Group Ⅱ49and Group Ⅲ9; the Group Ⅱ was divided into five subgroup, Group Ⅱ a, Group Ⅱb, Group Ⅱc, Group Ⅱd and Group Ⅱe. The each subgroup member was5,2,25,12and5Contigs, respectively.
Due to cotton EST sequences could not cover the entire cotton transcriptome in NCBI. We scanned the G. raimondii genome scaffolds released publicly by the WRKY transcription domain. This family was divided into three categories, with included109members. Group Ⅰ contained19members, Group Ⅱ78, GroupⅢ12. By constructing the phylogenetic tree using G raimondii and arabidopsis WRKY domain, the Group Ⅱ was further divided into five subgroups, Group Ⅱa, Group Ⅱb, Group Ⅱ c, Group Ⅱd and Group Ⅱe. The each subgroup member was7,15,29,15and12, respectively.
By the alignment of the WRKY domain from G. Raimondii WRKY transcription factors, the variations of the WRKYGQK conserved domain was found. The domain of WRKYGQK become the WRKYGKK and WRKYGHK in the Group Ⅱc subgroup. The members from Group Ⅱc, including GrWRKY31, GrWRKY39, GrWRKY46, GrWRKY35and GrWRKY78, contained the variation of the WRKYGKK type, and the variation of the WRKYGHK type from GrWRKY8. The WRKY transcription factors not only included the variation of WRKYGQK domain, the zinc finger structure also had variation. The genes of zinc finger structure variation from Group Ⅰ N、Group Ⅰ C and GroupⅢ.
Integrated WRKY transcription factor from cotton EST database and the genome of G raimondii, we found that there were70Contigs are able to find highly homologous sequence in the D genome, Contig69,70,71and73did not find similar homologous sequences in the D genome. They might be new genes in the tetraploid cotton evolutionary process or they were proprietary genes of At subgenome, so they can not find homologous sequence in D genome.
Based on in silico cloning technology, we cloned several WRKY genes in G. hirsutum cv TM-1. Total the full-length sequence of five genes were obtained, GhWRKY19, GhWRKY23, GhWRKY60, GhWRKY87, and GhWRKY95. GhWRKY19ORF length is1401bp, encoding466amino acids, containing four introns; GhWRKY23ORF length is942bp, encoding313amino acids, containing four introns; GhWRKY60ORF is1068bp, encoding355amino acids, contain two introns; GhWRKY87ORF length is807bp, encoding268amino acids, containing two introns; GhWRKY95ORF is966bp, encoding321amino acid residues, containing two introns. The Q-PCR analysis in the TM-1showed that GhWRKY38highly expressed in the root; GhWRKY87and GhWRKY43in the leaves; GhWRKY74in roots and stems; GhWRKY60in the roots and leaves in high. Three WRKY members.(GhWRKY19, GhWRKY21and GhWRKY23) were highly expressed in the early fiber development. But GhWRKY19highly expressed in leaves and GhWRKY21in the roots and leaves.
Using the three-leaf stage seedlings of G hirsutum cv Jinmian19as test material, some abiotic stress treatment, such as salicylic acid (2mM SA), methyl jasmonate (100μM MeJA), abscisic acid (200μM ABA), gibberellin (of500μM GA), salt (200mM NaCl), drought (20%PEG6000), ethylene (5mM ET), were induced. We analyzed expression levels of WRKY genes in abiotic stress-induced Oh,2h,4h,6h,8h,10h,12h,24h. The results show that the expression of GhWRKY23, GhWRKY43, GhWRKY60, GhWRKY74; and GhWRKY87after i.nduced by the seven kinds stress significantly increased GhWRKY19was not induced by the PEG and ET, GhWRKY21and GhWRKY38was not induced by salt stress.
1、高密度海陆种间遗传图谱的构建及基因组结构分析
本研究利用新开发的gSSR引物及网上公开释放的部分CER、CGR、COT、DC、 DPL、SHIN及HAU编号的引物,与已开发的SSR标记位点进行冗余筛选,获得1431对非冗余引物,在作图亲本TM-1和Hai7124之间筛选多态性,进而完成图谱的加密工作。新获得的海陆栽培棉种种间遗传图谱包含3414个位点,总长为3677.6cM,标记间的平均距离为1.08cM,分布在26条连锁群上。
该图谱上包含941个重复位点,分别由425对引物在亲本TM-1和Hai7124间扩增产生。这些重复位点主要来源于SSR、REMAP、SRAP、AFLP、RT和In-Del等5种标记类型。其中326对SSR引物产生693个重复位点,包括287对SSR引物产生2个位点；37对产生3个位点；2对产生4个位点。分析这些重复位点的染色体分布,除位于部分同源染色体上的重复位点外,一些重复位点位于同一染色体上,另一些重复位点位于非同源的不同染色体上,表明在四倍体棉花进化进程中基因组染色体内部及染色体间发生了复制和重排等结构变异。
新构建的遗传图谱上含86个位点簇(≥5loci/cM),涉及617个位点。除Chr.1上未发现位点簇外,这86个位点簇分布在其余25个连锁群上,分布密度不均匀。其中,31个位点簇分布在At亚组上,涉及到229个位点；55个位点簇分布于Dt亚组上,涉及到388个位点。同时也检测到19个基因岛(≥5EST-SSR loci/cM)和1个反转录转座子区(≥5REMAP loci/cM).
300个位点在作图群体中不符合孟德尔遗传规律(P<0.05)。在连锁群上构成12个偏分离富集区,At亚组和Dt亚组各分布6个。这12个偏分离富集区分布在11条染色体上,其中在Chr.20上有两个偏分离富集区。在偏分离富集区上位点的偏向保持着一致性,其中8个偏向于亲本TM-1,4个偏向于杂合子。
2、两个质量性状基因的精细定位和候选基因筛选
以构建的高密度海陆种间遗传图谱为基础,分别利用(Sub16×T586)F2群体和[(Hai7124×T586)xHai7124]BC1群体,完成棉花两个重要的质量性状基因一红株R1和茸毛乃基因的精细定位。将红株R1基因定位在第16染色体标记NAU4956和NAU6752之间,遗传距离分别为2.91cM和0.49cM;将茸毛乃基因定位在第6染色体NAU5434与NAU1277之间,距最近标记NAU5434的遗传距离为0.6cM.
基于精细定位的结果和网上公开释放的棉花D基因组雷蒙德氏棉种的基因组序列信息,根据目标基因附近的标记序列映射雷蒙德氏棉基因组序列,获得目标基因所在的候选物理区域。利用Fgenesh程序对候选的物理区域进行ORF预测及BLAST2Go的功能注释及代谢特征分析,结合所克隆基因作用特征完成候选基因的确定及功能初步分析。针对色素合成代谢途径特点,筛选了6个候选基因,分别命名为RG1-RG6;参考前人拟南芥表皮毛发育研究结果,初步确定标记映射区域与棉花密生茸毛性状相关的6个候选基因,分别命名为TGI-TG60
形态及扫描电镜分析显示,随着叶龄发育,T586的叶片色素沉积越来越多叶片茸毛的发育随着叶龄发育逐渐变稀,但每一发育阶段,T586的叶片茸毛均显著高于Hai7124,在成熟叶片上,Hai7124仅叶脉及叶边缘可见茸毛分布,而T586叶片密被茸毛。选择T586和Hai7124不同叶龄的棉花叶片(叶芽、幼叶及成熟叶),进行候选基因的表达分析。Q-PCR分析表明,在控制棉花红株性状的6个候选基因中,基因RG4在T586色素沉积最多的成熟叶中,其表达量显著高于同时期Hai7124叶片中的表达量,而其他候选基因在两个亲本材料色素差异最明显时期叶片中的表达量都没有达到显著水平。在控制茸毛性状的6个候选基因中,基因TG5在T586叶片每一发育阶段的表达量与Hai7124之间均存在显著或极显著差异。同时,随着叶片的发育,在T586和Hai7124中表达水平也随之降低,与叶片表皮茸毛密度分布特征一致。其余候选基因的表达未发现规律性。综合基因表达、表型变化及扫描电镜分析结果,推测RG4和TG5是控制两个质量性状红株R1和茸毛T1的候选基因。根据BLAST2Go(?)勺功能注释表明,RG4是一个编码八氢番茄红素合酶的基因；TG5为一个bHLH类型的转录因子。
3、棉花WRKY转录因子家族全基因组分析
已有研究报道,WRKY转录因子家族参与植物的生长发育、衰老等进程,同时对各种生物胁迫和非生物胁迫都有一定的响应。在拟南芥中,AtWRKY44参与了拟南芥表皮毛形成。为了阐明WRKY转录因子家族在棉花重要性状形成中的可能角色,我们开展了WRKY转录因子家族棉花全基因组的发掘、鉴定、分类和表达初步研究。
利用在NCBI上公开释放的棉花EST序列,结合Pfam数据库提供的WRKY转录因子种子文件的全部2450条种子序列,比对棉花总EST数据库,最终得到535条WRKY类EST序列。以这535条EST序列作为探针,通过电子克隆技术,获得95个含有WRKY结构域的Contig,其中74个Contig中至少含有一个完整的WRKY结构域,暂命名为Contig1-Contig74。与拟南芥WRKY转录因子家族的进化分析显示,74个Contig属于3个组及5个亚组,其中3个组-Group Ⅰ、Group Ⅱ和Group Ⅲ,分别含有16、49和9个Contig。Group Ⅱ的五个亚组--Group Ⅱ a、Group Ⅱb、Group Ⅱc、 Group Ⅱd和Group Ⅱe分别含有5、2、25、12和5个Contig。
利用公布的雷蒙德氏棉全基因组序列草图,进一步对雷蒙德氏棉全基因组进行了WRKY转录因子家族成员的预测,共得到109个WRKY转录因子家族成员。分子进化分析将该转录因子家族成员分为3大类,其中Group Ⅰ有19个成员,Group Ⅱ有78个成员,Group Ⅲ有12个成员。进一步与来源于拟南芥的WRKY家族基因聚类结果分析,将Group Ⅱ分成Group Ⅱa、Group Ⅱb、Group Ⅱc、Group Ⅱd和Group Ⅱe五个亚组,每个组成员分别有7个、15个、29个、15个和12个。通过序列比对,发现有5个成员在WRKYGQK的保守结构域上存在有WRKYGKK的变异,分别是GrWRKY31、 GrWRKY39、GrWRKY46、GrWRKY35和GrWRKY78,并且这5个成员都属于Group Ⅱc亚组；在Group Ⅱc中的GrWRKY8还存在WRKYGHK结构域变异。除了在WRKYGQK的保守结构域上存在变异,在Group ⅠN、Group ⅠC和GroupⅢ中发现WRKY家族成员在锌指结构上也存有一定的变异。
通过序列相似性比对来自棉花EST数据库和雷蒙德氏棉基因组的WRKY转录因子,前期预测的74个Contig中,除属于Group Ⅱc亚组的Contig69、70、71和73等4个Contig外,70个Contig h(?)在二倍体D基因组雷蒙德氏棉中找到高度相似的同源序列。推测4个未找到同源序列的Contig可能是四倍体棉种在进化过程中获得的新基因或是At亚组专有的WRKY转录因子。
从陆地棉TM-1基因组中克隆获得GhWRKY19、 GhWRKY23、GhWRKY60、 GhWRKY87、GhWRKY95等5个成员全长ORF的基因组信息。其中,GhWRKY19ORF全长为1401bp,编码466个氨基酸,含有4个内含子；GhWRKY23ORF全长为942bp,编码313个氨基酸,含有4个内含子；GhWRKY60ORF全长为1068bp,编码355个氨基酸,含有2个内含子；GhWRKY87ORF全长为807bp,编码268个氨基酸,含有2个内含子；GhWRKY95ORF全长为966bp,编码321个氨基酸,含有2个内含子。不同组织、器官表达特征表明,WRKY类基因在根茎叶等营养器官中的表达量普遍高于在棉纤维中的表达量。GhWRKY38在根中高表达；GhWRKY19、GhWRKY87和GhWRKY43在叶中高表达；GhWRKY74在根和茎中高表达；而GhWRKY21和GhWRKY60在根和叶中高表达。在纤维发育进程中高表达的WRKY家族成员主要检测到GhWRKY19、GhWRKY21和GhWRKY23,其高表达的时期集中在-3dpa-5dpa,没有检测到在纤维发育后期高表达的基因。
以陆地棉抗逆品种晋棉19三叶期幼苗为试验材料,进行水杨酸(2mM SA)、茉莉酸甲酯(100μM MeJA)、脱落酸(200μM ABA)、赤霉素(500μM GA)、盐(200mM NaCl)、干旱(20%PEG6000)、乙烯(5mM ET)等非生物胁迫处理,分析WRKY类基因在非生物胁迫诱导0h、2h、4h、6h、8h、10h、12h、24h后的表达水平。结果表明：GhWRKY23、 GhWRKY43、GhWRKY60、GhWRKY74和GhWRKY87被上述七种胁迫处理诱导后表达显著提高。GhWRKY19在PEG和ET处理后,表达水平未发生显著变化,而GhWRKY21和GhWRKY38不被盐胁迫诱导。
Cotton is one of the important cash crops in the world, and the quality and color of the cotton fibers can greatly enhance its economic value. The high-density genetic map is the basement of genome research in plant. It can not only shed light on the structural features of the genome, but also lay the foundation for the fine mapping of genes related to qualitative and quantitative traits. Trichomes and red plant traits were controled by a single dominant gene in cotton. There were similarities between the development of leaf hairs and the development of the cotton fiber, and the leaf hairs had insect-resistant characteristics. Pigmentation gene of the red plant can be effectively used to cultivate natural colored cotton. To elucidate the mechanism and provide important genetic resouces for fiber quality improvement, insect resistance enhancement and natural colored cotton cultivation, we mapped the two key genes and tagged preliminary the candidate genes. In this paper, we updated our backbone genetic map and analyzed the chromosome structural features in allotetraploid cotton. Based on the high-density genetic map, two genes related to quality trait, T1and R1, were fine mapped and candidate genes were further mined. Using different cotton EST database and genome sequene of G. raimondii, the development, identification, classification of genome-wide WRKY transcription factors family in cotton were carried out and preliminary expressional analysis was performed. The main findings are as follows.
1The construction of high-density genetic map and analysis of genomic structure
Total1431primer pairs were used, including newly developed gSSR, publicly released CER, CGR, COT, DC, DPL, SHIN and HAU primers, to screen poplymorphisms between TM-1and Hai7124in order to enhance our backbone genetic map in allotetraploid cotton. The updated map consists of3,414loci in26linkage groups covering3,667.62cM with an average inter-locus distance of1.08cM. By the analysis of loci, we draw the following results:
Among2898primer pairs produced3414loci in TM-1and Hai7124,425primer pairs amplified two or more loci, resulting in total of941duplicate loci. Four marker types, morphological marker, CAPs, SNP and BAC-end, did not find duplicate loci.693duplicated loci were identified by326SSR primer pairs, with574duplicated,111triplicated, and8tetraplicated loci. Some loci were present on the homeologous chromosomes, some loci found to involve in the same chromosomes, and other loci spanned the different chromosomes, implying duplication of multiple round and genome rearrangement of both intrachromosome and interchromosome in the process of evolution.
The cluster of loci were also observed in26linkage group,86clusters involved in617loci (≥5loci/cM) were discovered on the25linkage group besides A1(Chr.01). Of them,31clusters contained229loci from At subgenome, and55clusters contained388loci from Dt subgenome.19candidate genes islands (≥5EST-SSR loci/cM) and one retrotransposon-rich region were discovered by clusters distribution of marker loci.
300loci showed non-mendelian segregation (P<0.05). A total of12segregation distortion regions (SDRs) were detected on11linkage groups. There were two SDRs in D10linkage group. Among12SDRs, six were on the At subgenome and six on the Dt subgenome, with8SDRs skewed toward G. hirutum TM-1and four SDRs skewed toward the heterozygote.
2The fine mapping of two genes related to qualitative traits and screening of candidate genes
Based on the high-density genetic map and two populations, Subl6×T586F2and [(Hai7124xT586)xHai7124] BC1populations, the red plant R1gene and the trichomes T1gene were fine mapped. As a result, R1was mapped between NAU4956and NAU6752, with only0.49cM to the nearest maker loci (NAU6752) by the Sub16×T586F2population contained1259individual plants;the T1was located between NAU5434and NAU1277, with only0.6cM to the nearest marker loci (NAU5434) by the [(Hai7124xT586)xHai7124] BC1population contained835individual plants.
To find their candidate genes, we used the sequence of markers as probes to anchor the G. raimondii genome scaffolds. The ORF were searched by Fgenesh program and functional annotation by BLAST2Go. Based on pigment synthesis pathway, we selected six candidate genes, named for RG1-RG6, for cloning R1gene; According to Arabidopsis trichomes development research, we selected six candidate genes for cloning T1gene, named for TG1-TG6.
Morphological and electron microscope scanning analysis show that the pigment accumulates is more and more in T586leaves as development process, and the density of leaf hairs of T586is significantly higher than Hai7124in each development stage, though the hairs of the leaves become sparser with the leaves development. The leaf hairs can only be observed around the nervation and the edges of the leaves of Hai7124on the mature leaves, while the leaves of T586are covered with leaf hairs of high density. Based on the study, cotton leaves of different development stage (leaf buds; young leaf; mature leaf) of T586and Hai7124were collected for further expression analysis of candidate genes. Q-PCR results indicated that among the6candidate genes, RG4gene had a significantly higher expression level in the mature leaves that accumulated the most pigment of T586than in the leaves of Hai7124of the same development stage, while others did not show significant differences. TG5gene showed significant expression difference between the leaves of T586and Hai7124at every development stage compared with other five candidate genes, and the expression level in T586and Hai7124decreased with the development of the leaves, and the results in consistant with the distribution of the density of leaf hairs, while other candidate genes did not show regularities in the expression mode. Taking all the above results into consideration, RG4and TG5are considered to be the two candidate genes that controls two important qualitative traits, namely Red plant R1and Leaf Hairs T1, respectively. Blast2Go results indicated that RG4encoded a phytoene synthase, while TG5acted as a bHLH transcript factor.
3The genome-wide analysis of WRKY transcription factor family in cotton
It has been reported WRKY transcription factors family involved in plant growth, development, senescence and response to various biotic and abiotic stress. AtWRKY44involved in trichomes development in Arabidopsis.To clarify the possible role of WRKY transcription factors family in cotton growth and development, the development, identification, classification of genome-wide WRKY transcription factors family in cotton were carried out and preliminary expressional analysis was performed.
In recent years, a large number of EST sequences of cotton have been releasd in NCBI. Using the Pfam database provides WRKY transcription factor seed file, total2450seed sequences compared to the EST database of cotton, further we got535EST sequences related to WRKY transcription factor. By the535EST sequences as probes and electronic cloning technology, we got the95contigs contained full or partial WRKY domain. Of them,21Contig lacked the zinc finger structure, and remaining74Contigs were able to find a complete WRKY domain. These74Contigs were named Contigl to Contig74. Based on the WRKY domain characteristics and evolutionary analysis,74Contigs were divided into three groups and five subgroups, Group I contained16Contigs, Group Ⅱ49and Group Ⅲ9; the Group Ⅱ was divided into five subgroup, Group Ⅱ a, Group Ⅱb, Group Ⅱc, Group Ⅱd and Group Ⅱe. The each subgroup member was5,2,25,12and5Contigs, respectively.
Due to cotton EST sequences could not cover the entire cotton transcriptome in NCBI. We scanned the G. raimondii genome scaffolds released publicly by the WRKY transcription domain. This family was divided into three categories, with included109members. Group Ⅰ contained19members, Group Ⅱ78, GroupⅢ12. By constructing the phylogenetic tree using G raimondii and arabidopsis WRKY domain, the Group Ⅱ was further divided into five subgroups, Group Ⅱa, Group Ⅱb, Group Ⅱ c, Group Ⅱd and Group Ⅱe. The each subgroup member was7,15,29,15and12, respectively.
By the alignment of the WRKY domain from G. Raimondii WRKY transcription factors, the variations of the WRKYGQK conserved domain was found. The domain of WRKYGQK become the WRKYGKK and WRKYGHK in the Group Ⅱc subgroup. The members from Group Ⅱc, including GrWRKY31, GrWRKY39, GrWRKY46, GrWRKY35and GrWRKY78, contained the variation of the WRKYGKK type, and the variation of the WRKYGHK type from GrWRKY8. The WRKY transcription factors not only included the variation of WRKYGQK domain, the zinc finger structure also had variation. The genes of zinc finger structure variation from Group Ⅰ N、Group Ⅰ C and GroupⅢ.
Integrated WRKY transcription factor from cotton EST database and the genome of G raimondii, we found that there were70Contigs are able to find highly homologous sequence in the D genome, Contig69,70,71and73did not find similar homologous sequences in the D genome. They might be new genes in the tetraploid cotton evolutionary process or they were proprietary genes of At subgenome, so they can not find homologous sequence in D genome.
Based on in silico cloning technology, we cloned several WRKY genes in G. hirsutum cv TM-1. Total the full-length sequence of five genes were obtained, GhWRKY19, GhWRKY23, GhWRKY60, GhWRKY87, and GhWRKY95. GhWRKY19ORF length is1401bp, encoding466amino acids, containing four introns; GhWRKY23ORF length is942bp, encoding313amino acids, containing four introns; GhWRKY60ORF is1068bp, encoding355amino acids, contain two introns; GhWRKY87ORF length is807bp, encoding268amino acids, containing two introns; GhWRKY95ORF is966bp, encoding321amino acid residues, containing two introns. The Q-PCR analysis in the TM-1showed that GhWRKY38highly expressed in the root; GhWRKY87and GhWRKY43in the leaves; GhWRKY74in roots and stems; GhWRKY60in the roots and leaves in high. Three WRKY members.(GhWRKY19, GhWRKY21and GhWRKY23) were highly expressed in the early fiber development. But GhWRKY19highly expressed in leaves and GhWRKY21in the roots and leaves.
Using the three-leaf stage seedlings of G hirsutum cv Jinmian19as test material, some abiotic stress treatment, such as salicylic acid (2mM SA), methyl jasmonate (100μM MeJA), abscisic acid (200μM ABA), gibberellin (of500μM GA), salt (200mM NaCl), drought (20%PEG6000), ethylene (5mM ET), were induced. We analyzed expression levels of WRKY genes in abiotic stress-induced Oh,2h,4h,6h,8h,10h,12h,24h. The results show that the expression of GhWRKY23, GhWRKY43, GhWRKY60, GhWRKY74; and GhWRKY87after i.nduced by the seven kinds stress significantly increased GhWRKY19was not induced by the PEG and ET, GhWRKY21and GhWRKY38was not induced by salt stress.
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