调控拟南芥花粉发育突变体zy1511的基因定位及特征分析

为了进一步研究花药花粉发育过程,我们通过EMS诱变,筛选到拟南芥雄性不育突变体zy1511。遗传分析表明,zy1511为隐性单位点突变。细胞学观察表明．突变体花药中小孢子从四分体释放出后绒毡层并没有开始退化,花药发育后期绒毡层依然部分存在。说明突变体花药绒毡层退化比野生型的要迟,因此,小孢子不能发育成正常花粉粒。利用图位克隆的方法将zv1511定位于第一条染色体上分子标记F25P12和T8L23之间134．kb的区间内。本项工作为zy1511基因的克隆及对花粉发育功能分析奠定了基础。目前尚未见到该区间内雄性不育基因的报道。因此,zy1511是控制花粉发育的尚未发现的关键基因。     

How to map ses, a mutant of Arabidopsis thaliana affecting pollen development

In Arabidopsis, map-based cloning has been developed to an effective method in mutant genetic analysis because high-density markers are available, candidate genes or genomic sequences can be amplified by PCR and transgenic techniques are simplified. Mutant ses named from shortened early-stage siliques was used as an example to show how to map a mutant in this day. By the process of bulked segregants analysis, linkage testing, large-scale and fine scale mapping, mutant ses was narrowed into a 67 kb interval from CER448792 (2000541 bp) to CER464544 (2067844 bp) crossing over the right of BAC F12K11 to the left of the BAC F4H5 including at most 22 putative genes on the top of chromosome l. In sequence-based map of Arabidopsis genes with Mutant phenotype (SMAGMP) mutant ses was between ATlg06150 (EMB1444) and ATlg08060 (MOM). The SES mapping also showed that developed markers on polymorphism site of CAPC not only were simplified and but worked well. 24 markers from CAPC used in the mapping maybe help Arabidopsis researches with others and the methods related to SES mapping also gave an example of positional cloning.     

拟南芥雄性不育突变体ms1502的遗传及定位分析

通过EMS诱变、背景纯化与遗传分析,从拟南芥（Arabidopsis thaliana）中筛选到了一棵隐性单基因控制的雄性不育突变体ms1502。细胞学观察发现,突变体在小孢子从四分体释放出后花药绒毡层过早衰亡,小孢子的内容物不正常地凝聚,最终无法形成正常的花粉粒。利用图位克隆的方法对该基因MSl502进行了定位,结果表明MS1502位于第4条染色体上分子标记F25124和T12H20之间105kb区间内。目前该区间内尚未见到花药发育必需基因（不育基因）的报道,因此MS1502是一个控制花粉发育的新基因。     

Fine mapping of an Arabidopsis thaliana male sterile mutant EC2-157

An Arabidopsis thaliana male sterile mutant EC2-157 has been isolated using an EMS mutagenesis strategy.Genetic analysis indicated that it was controlled by a single recessive gene called ms157.No pollen grains have been observed in mutant anthers.ms157 Has been mapped to a region of 74 kb located in BAC clone T6K22 on chromosome Ⅳ using a map-based cloning strategy.As no male sterile genes have been reported in this region.ms157 could be a novel gene related to fertility.The further molecular cloning and functional analysis on this gene should facilitate our understanding of A.thaliana anther development.     

Identification of Arabidopsis histone deacetylase HDA6 mutants that affect transgene expression

A mutant screen was conducted in Arabidopsis that was based on deregulated expression of auxin-responsive transgenes. Two different tightly regulated (i.e., very low expression in the absence of auxin treatment and very high expression after exogenous auxin treatment) auxin-responsive promoters were used to drive the expression of both a beta-glucuronidase (GUS) reporter gene and a hygromycin phosphotransferase (HPH)-selectable marker gene. This screen yielded several mutants, and five of the mutations (axe1-1 to axe1-5) mapped to the same locus on chromosome 5. A map-based cloning approach was used to locate the axe1 mutations in an Arabidopsis RPD3-like histone deacetylase gene, referred to as HDA6. The axe1 mutant plants displayed increased expression of the GUS and HPH transgenes in the absence of auxin treatment and increased auxin-inducible expression of the transgenes compared with nonmutant control plants. None of a variety of endogenous, natural auxin-inducible genes in the mutant plants were upregulated like the transgenes, however. Results of treatment with the DNA methylation inhibitor 5-aza-2'-deoxycytidine suggest that the axe1 mutations affect transgene silencing; however, histone deacetylase inhibitors had no affect on transgene silencing in mutant or control plants. The specific effect of AtHDA6 mutations on the auxin-responsive transgenes implicates this RPD3-like histone deacetylase as playing a role in transgene silencing. Furthermore, the effect of AtHDA6 on transgene silencing may be independent of its histone deacetylase activity.     

A collection of INDEL markers for map-based cloning in seven Arabidopsis accessions

The availability of a comprehensive set of resources including an entire annotated reference genome, sequenced alternative accessions, and a multitude of marker systems makes Arabidopsis thaliana an ideal platform for genetic mapping. PCR markers based on INsertions/DELetions (INDELs) are currently the most frequently used polymorphisms. For the most commonly used mapping combination, Columbia×Landsberg erecta (Col-0×Ler-0), the Cereon polymorphism database is a valuable resource for the generation of polymorphic markers. However, because the number of markers available in public databases for accessions other than Col-0 and Ler-0 is extremely low, mapping using other accessions is far from straightforward. This issue arose while cloning mutations in the Wassilewskija (Ws-4) background. In this work, approaches are described for marker generation in Ws-4 x Col-0. Complementary strategies were employed to generate 229 INDEL markers. Firstly, existing Col-0/Ler-0 Cereon predicted polymorphisms were mined for transferability to Ws-4. Secondly, Ws-0 ecotype Illumina sequence data were analyzed to identify INDELs that could be used for the development of PCR-based markers for Col-0 and Ws-4. Finally, shotgun sequencing allowed the identification of INDELs directly between Col-0 and Ws-4. The polymorphism of the 229 markers was assessed in seven widely used Arabidopsis accessions, and PCR markers that allow a clear distinction between the diverged Ws-0 and Ws-4 accessions are detailed. The utility of the markers was demonstrated by mapping more than 35 mutations in a Col-0×Ws-4 combination, an example of which is presented here. The potential contribution of next generation sequencing technologies to more traditional map-based cloning is discussed.     

Identification and genetic mapping of four novel genes that regulate leaf development in Arabidopsis

Molecular and genetic characterizations of mutants have led to a better understanding of many developmental processes in the model system Arabidopsis thaliana.However,the leaf development that is specific to plants has been little studies.With the aim of contributing to the genetic dissection of leaf development,we have performed a large-scare screening for mutants with abnormal leaves.Among a great number of leaf mutants we have generated by T-DNA and transposon tagging and ethylmethae sulfonate (EMS) mutagenesis,four independent mutant lines have been identified and studied genetically.Phenotypes of these mutant lines represent the defects of four novel muclear genes designated LL1(LOTUS LEAF 1),LL2(LOTUS LEAF2),URO(UPRIGHT ROSETTE),and EIL(ENVIRONMENT CONDITION INDUCED LESION).The phenotypic analysis indicates that these genes play important roles during leaf development.For the further genetic analysis of these genes and the map-based cloning of LL1 and LL2,we have mapped these genes to chromosome regions with an efficient and rapid mapping method.     

MS1516是拟南芥四分体形成和小孢子发育的必需基因

以前报道了雄性育性下降突变体ms1516,而且图位克隆的方法已将突变基因MS1516定位到拟南芥基因组第3条染色体上28kh的区间内。本文通过进一步的生物信息学分析,发现该定位区间内有一个与减数分裂有关的基因AtATM,而且等位实验结果表明rns1516和nfm0是等位突变体。细胞学分析结果表明,ms1516突变体在花药发育过程中产生多个不均等的小孢子,而且大多数的小孢子不能发育成成熟的花粉。DAPI染色的结果显示小孢子母细胞减数分裂过程中,染色体不能正常分离,对成熟花粉的扫描电镜观察结果发现突变体多数花粉形态异常。以上结果说明MS1516基因在小孢子形成和发育过程中具有重要作用。     

ST273是拟南芥绒毡层和小孢子发育的必需基因

绒毡层在拟南芥花药花粉发育过程中具有重要作用,包括分泌降解胼胝质的胼胝质酶、为花粉壁的形成提供原料以及为小孢子发育提供营养物质.本文通过对拟南芥雄性不育突变体st273的分析,研究了ST273基因在花药花粉发育过程中的功能.st273是通过T-DNA插入诱变野生型拟南芥得到的一株突变体,遗传分析表明st273是单隐性核基因控制的.利用图位克隆的方法对不育基因ST273进行了定位,结果表明ST273基因与拟南芥第三条染色体上分子标记CIW11连锁.生物信息学分析发现该分子标记附近有一个调控花粉发育的基因TDF1.测序分析结果表明在st273突变体中,TDF1基因第三个外显子上459位的碱基发生了由G459变成了A459的单碱基变化,导致ST273基因该位点提前终止突变.等位分析结果表明st273与tdf1是等位突变体.st273突变体营养生长期发育正常,但生殖生长发育出现异常.亚历山大染色结果显示st273突变体花药中没有花粉.组织切片观察结果表明,突变体花药绒毡层异常肥大且空泡化,四分体不能正常释放小孢子,最终无法形成花粉.这些结果揭示了ST273蛋白质参与调控了绒毡层和小孢子发育过程.     

Arabidopsis RTM2 gene is necessary for specific restriction of tobacco etch virus and encodes an unusual small heat shock-like protein

Arabidopsis plants have a system to specifically restrict the long-distance movement of tobacco etch potyvirus (TEV) without involving either hypersensitive cell death or systemic acquired resistance. At least two dominant genes, RTM1 and RTM2, are necessary for this restriction. Through a series of coinfection experiments with heterologous viruses, the RTM1/RTM2-mediated restriction was shown to be highly specific for TEV. The RTM2 gene was isolated by a map-based cloning strategy. Isolation of RTM2 was confirmed by transgenic complementation and sequence analysis of wild-type and mutant alleles. The RTM2 gene product is a multidomain protein containing an N-terminal region with high similarity to plant small heat shock proteins (HSPs). Phylogenetic analysis revealed that the RTM2 small HSP-like domain is evolutionarily distinct from each of the five known classes of plant small HSPs. Unlike most other plant genes encoding small HSPs, expression of the RTM2 gene was not induced by high temperature and did not contribute to thermotolerance of seedlings. The RTM2 gene product was also shown to contain a large C-terminal region with multiple repeating sequences.     

Genetic mapping of mutations using phenotypic pools and mapped RAPD markers.

Genetic markers facilitate the study of inheritance and the cloning of genes by genetic approaches. Molecular markers detect differences in DNA sequence, and are thus less ambiguous than phenotypic markers, which require gene expression. We have demonstrated a molecular approach to the mapping of mutant genes using RAPD markers and pooling of individuals based on phenotype. To map genes by phenotypic pooling a strain carrying a mutation is crossed to a strain that is homozygous for the wild-type allele of the corresponding gene. A set of primers corresponding to mapped RAPDs distributed throughout the genome and in coupling phase with respect to the wild type parent is then used to amplify DNA from wild type and mutant pools of F2 individuals. Linkage between the mutant gene and the RAPD markers is visualized by the absence of the corresponding RAPD DNA bands in the mutant pool. We developed a mathematical model for calculating the probability of linkage between RAPDs and target genes and we successfully tested this approach with the model plant Arabidopsis thaliana.     

Genetic and physical characterization of chromosome 4DL in wheat.

The long arm of chromosome 4D in wheat (Triticum aestivum L.) has been shown in previous studies to harbor genes of agronomic importance. A major dominant gene conferring Aluminum (Al) tolerance (Alt2 in 'Chinese Spring' and AltBH in 'BH 1146'), and the Knal locus controlling the K+/Na+ discrimination in saline environments have been mapped to this chromosome arm. However, accurate information on the genetic and physical location of markers related to any of these genes is not available and would be useful for map-based cloning and marker-assisted plant breeding. In the present study, using a population of 91 recombinant inbred lines segregating for Al tolerance, we provide a more extensive genetic linkage map of the chromosome arm 4DL based on RFLP, SSR, and AFLP markers, delimiting the AltBH gene to a 5.9-cM interval between markers Xgdm125 and Xpsr914. In addition, utilizing a set of wheat deletion lines for chromosome arm 4DL, the AltBH gene was physically mapped to the distal region of the chromosome, between deletion breakpoints 0.70 and 0.86, where the kilobase/centimorgan ratio is assumed to be low, making the map-based cloning of the gene a more realistic goal. The polymorphism rates in chromosome arm 4DL for the different types of markers used were extremely low, as confirmed by the physical mapping of AFLPs. Finally, analysis of 1 Mb of contiguous sequence of Arabidopsis chromosome 5 flanking the gene homologous to the BCD1230 clone (a cosegregating marker in our population coding for a ribulose-5-phosphate-3-epimerase gene), revealed a previously identified region of stress-related and disease-resistance genes. This could explain the collinearity observed in comparative mapping studies among different species and the low level of polymorphism detected in the chromosome arm 4DL in hexaploid wheat.     

Comparative mapping in Arabidopsis and Brassica, fine scale genome collinearity and congruence of genes controlling flowering time

The model dicotyledonous plant, Arabidopsis thaliana , is closely related to Brassica crop species. It is intended that information concerning the genetic control of basic biological processes in Arabidopsis will be transferable to other species. Genome collinearity and its potential to facilitate the identification of candidate genes in Arabidopsis homologous to genes controlling important agronomic traits in Brassica was investigated. Genetic mapping in B. nigra identified two loci influencing flowering time (FT), with loci on linkage groups 2 and 8 explaining 53% and 12% of the total variation in FT, respectively. The CO gene exerts an important control over FT in A. thaliana , and B. nigra homologues of CO probably also play an important role in regulating FT. B. nigra homologues of CO were identified on linkage groups 2 and 8, the homologue on group 2 was coincident with the major locus controlling FT while the homologue on group 8 was within the 90% confidence interval of the weaker FT gene. The CO homologue on group 2 exhibits abundant allelic variation suggesting that it naturally controls a wide range of flowering times. Fine-scale A. thaliana/B. nigra comparative mapping demonstrated short-range collinearity between the genomes of Arabidopsis and Brassica . Eleven DNA fragments spaced over a 1.5 Mb contig in A. thaliana were used as RFLP probes in B. nigra . Three collinear representations of the A. thaliana contig were identified in B. nigra , with one interrupted by a large chromosomal inversion. Collinearity over this range will allow the resources generated by the Arabidopsis genome project to facilitate map-based cloning in Brassica crops.     

The radish Rfo restorer gene of Ogura cytoplasmic male sterility encodes a protein with multiple pentatricopeptide repeats

A single radish nuclear gene, Rfo, restores Ogura (ogu) cytoplasmic male sterility (CMS) in Brassica napus. A map-based cloning approach relying on synteny between radish and Arabidopsis was used to clone Rfo. A radish gene encoding a 687-amino-acid protein with a predicted mitochondrial targeting pre-sequence was found to confer male fertility upon transformation into ogu CMS B. napus. This gene, like the recently described Petunia Rf gene, codes for a pentatricopeptide repeat (PPR)-containing protein with multiple, in this case 16, PPR domains. Two similar genes that do not appear to function as Rfo flank this gene. Comparison of the Rfo region with the syntenic Arabidopsis region indicates that a PPR gene is not present at the Rfo-equivalent site in Arabidopsis, although a smaller and related PPR gene is found about 40 kb from this site. The implications of these findings for the evolution of restorer genes and other PPR encoding genes are discussed.     

A genetic interval and physical contig spanning the Peronospora parasitica (At) avirulence gene locus ATR1Nd

In Peronospora parasitica (At) (downy mildew), the genetic determinants of cultivar-specific recognition by Arabidopsis thaliana are the Arabidopsis thaliana-recognised (ATR) avirulence genes. We describe the identification of 10 amplified fragment length polymorphism (AFLP) markers that define a genetic mapping interval for the ATR1Nd avirulence allele, the presence of which is perceived by the RPP1Nd resistance gene. Furthermore, we have constructed a P. parasitica (At) bacterial artificial chromosome (BAC) library comprising over 630Mb of cloned DNA. We have isolated 16 overlapping clones from the BAC library that form a contig spanning the genetic interval. BAC sequence-derived markers and a total mapping population of 311 F(2) individuals were used to refine the ATR1Nd locus to a 1cM interval that is represented by four BAC clones and spans less than 250kb of DNA. This work demonstrates that map-based cloning techniques are feasible in this organism and provides the critical foundations for cloning ATR1Nd using such a strategy.     

A simple procedure for the analysis of single nucleotide polymorphisms facilitates map-based cloning in Arabidopsis

We developed a modified allele-specific PCR procedure for assaying single nucleotide polymorphisms (SNPs) and used the procedure (called SNAP for single-nucleotide amplified polymorphisms) to generate 62 Arabidopsis mapping markers. SNAP primers contain a single base pair mismatch within three nucleotides from the 3' end of one allele (the specific allele) and in addition have a 3' mismatch with the nonspecific allele. A computer program called SNAPER was used to facilitate the design of primers that generate at least a 1,000-fold difference in the quantity of the amplification products from the specific and nonspecific SNP alleles. Because SNAP markers can be readily assayed by electrophoresis on standard agarose gels and because a public database of over 25,000 SNPs is available between the Arabidopsis Columbia and Landsberg erecta ecotypes, the SNAP method greatly facilitates the map-based cloning of Arabidopsis genes defined by a mutant phenotype.     

Jasmonate response locus JAR1 and several related Arabidopsis genes encode enzymes of the firefly luciferase superfamily that show activity on jasmonic,salicylic, and indole-3-acetic acids in an assay for adenylation

Jasmonic acid (JA) and related cyclopentanones are critical plant signaling molecules, but their mode of action at the molecular level is unclear. A map-based approach was used to identify the defective gene in the Arabidopsis JA response mutant jar1-1. JAR1 is 1 of 19 closely related Arabidopsis genes that are similar to the auxin-induced soybean GH3 gene. Analysis of fold predictions for this protein family suggested that JAR1 might belong to the acyl adenylate-forming firefly luciferase superfamily. These enzymes activate the carboxyl groups of a variety of substrates for their subsequent biochemical modification. An ATP-PPi isotope exchange assay was used to demonstrate adenylation activity in a glutathione S-transferase-JAR1 fusion protein. Activity was specific for JA, suggesting that covalent modification of JA is important for its function. Six other Arabidopsis genes were specifically active on indole-3-acetic acid (IAA), and one was active on both IAA and salicylic acid. These findings suggest that the JAR1 gene family is involved in multiple important plant signaling pathways.     

Rice molecular genetic map using RFLPs and its applications

In the past decade, notable progress has been made in rice molecular genetic mapping using genomic or cDNA clones. A total of over 3000 DNA markers, mainly with RFLPs, have been mapped on the rice genome. In addition, many studies related to tagging of genes of interest, gene isolation by map-based cloning and comparative mapping between cereal genomes have advanced along with the development of a high-density molecular genetic map. Thus rice is considered a pivotal plant among cereal crops and, in addition to Arabidopsis, is a model plant in genome analysis. In this article, the current status of the construction of rice molecular genetic maps and their applications are reviewed.