pAbstract/p pBackground/p pExpression QTL analyses have shed light on transcriptional regulation in numerous species of plants, animals, and yeasts. These microarray-based analyses identify regulators of gene expression as either cis-acting factors that regulate proximal genes, or trans-acting factors that function through a variety of mechanisms to affect transcript abundance of unlinked genes./p pResults/p pA hydroponics-based genetical genomics study in roots of a itZea mays /itIBM2 Syn10 double haploid population identified tens of thousands of cis-acting and trans-acting eQTL. Cases of false-positive eQTL, which results from the lack of complete genomic sequences from both parental genomes, were described. A candidate gene for a trans-acting regulatory factor was identified through positional cloning. The unexpected regulatory function of a class I glutamine amidotransferase controls the expression of an ABA 8-hydroxylase pseudogene./p pConclusions/p pIdentification of a candidate gene underlying a trans-eQTL demonstrated the feasibility of eQTL cloning in maize and could help to understand the mechanism of gene expression regulation. Lack of complete genome sequences from both parents could cause the identification of false-positive cis- and trans-acting eQTL./p

Abstract Drought stress greatly affects plant growth and crop yield. To understand the transcriptome dynamics during drought stress in maize seedlings, genome-wide gene expression profiling was compared between the drought-tolerant line Han21 and drought-sensitive line Ye478 using Affymetrix Maize Genome Array containing 17,555 probe sets. The results showed that in response to drought, the Han21 line had fewer probe sets with significant expression change than the Ye478 line and both lines had a common set of ~2,600 regulated probe sets under drought stress. The potential components of the abscisic acid signaling pathway were significantly identified from the common probe sets. A total of 827 probe sets with significantly differential expression between the two lines under drought stress were identified. The differential expression levels of cell wall-related and transporter genes may contribute to the different tolerances of the two lines. Additionally, we found that, compared to the sensitive line Ye478, the transcriptional levels of drought-responsive probe sets in the tolerant line Han21 recovered more quickly after re-watering, and more probe sets in the tolerant line Han21 were exclusively up-regulated at the re-watering stage. Our study provides a global gene expression dynamics of two maize inbred lines during drought stress and re-watering and will be valuable for further study of the molecular mechanisms of drought tolerance in maize.

The Maize Gene Discovery Project (MGDP) is a 5-year NSF-funded plant genome initiative that began in 1998. The MGDP collaboration involves researchers at six universities from diverse disciplines with the common goal of discovering new maize genes and developing tools for the phenotypic characterization of maize mutants. The project utilizes several approaches: EST sequencing, cDNA microarray production, and the discovery of gene function and genomic sequence through the use of a recombinant Mu1 transposon ( RescueMu ). Current achievements of the MGDP (NSF 98鈥72657) include the sequencing of over 120,000 maize ESTs from diverse cDNA libraries, and over 70,000 RescueMu flanking sequences, as well as the cataloguing of mutant seed and cob phenotypes of 23,000 maize ears, 6,200 families of maize seedlings, and 4,000 families of adult maize plants carrying MuDR/Mu and RescueMu insertion alleles. A consolidation of over 24,000 unique sequences from 19 libraries has been made into the first two of the planned set of four "Unigene" microarray slides. In addition, slides for four EST libraries have been produced. These microarray slides, EST clones, library plates of immortalized RescueMu bacterial cultures, and seed are all available online (http://www.zmdb.iastate.edu). The ZmDB website posts periodic assemblies of all maize EST and genomic sequences available from GenBank . ZmDB is also a portal for sequence analysis software designed to aid in gene discovery: MuSeqBox, GeneSeqer, and SplicePredictor . In addition, ZmDB contains links to other plant and genetics websites.

[ Jill E. Cairns ]The ability to quickly develop germplasm having tolerance to several complex polygenic inherited abiotic and biotic stresses combined is critical to the resilience of cropping systems in the face of climate change. Molecular breeding offers the tools to accelerate cereal breeding; however, suitable phenotyping protocols are essential to ensure that the much-anticipated benefits of molecular breeding can be realized. To facilitate the full potential of molecular tools, greater emphasis needs to be given to reducing the within-experimental site variability, application of stress and characterization of the environment and appropriate phenotyping tools. Yield is a function of many processes throughout the plant cycle, and thus integrative traits that encompass crop performance over time or organization level (i.e. canopy level) will provide a better alternative to instantaneous measurements which provide only a snapshot of a given plant process. Many new phenotyping tools based on remote sensing are now available including non-destructive measurements of growth-related parameters based on spectral reflectance and infrared thermometry to estimate plant water status. Here we describe key field phenotyping protocols for maize with emphasis on tolerance to drought and low nitrogen.

Plant invertases catalyze the conversion of sucrose to glucose and fructose, which are distinct signals of widely varied stress-tolerance processes, including the biosynthesis and detection of hormones under water deficit. In the invertase gene family, the candidate gene ivr2 encoding plant acid-soluble invertase plays a vital role in drought tolerance. In this study, a putative genomic sequence of ivr2 including three exons and two introns was acquired and genetically analyzed using bioinformatics and statistics, based on a partial ivr2 gene sequence of the GenBank library. The ivr2 genomic sequence data from 106 maize inbred lines were obtained using five nested primer pairs. Further analysis showed that the detected polymorphic sites were mainly located in exon-1, intron-1 and exon-2 regions; High linkage disequilibrium level and low nucleotide diversity were identified at this ivr2 locus. Association mapping combined the genotypic and phenotypic data, and a total of 48 associations showed high contributions to the variations in grain yield and its components under well-watered and water-stressed conditions over 2 years of experiments. This suggested that functional polymorphisms in ivr2 were possibly associated with maize drought tolerance. This provides reference information for efficient marker-assisted selection of superior alleles in drought tolerance breeding programs.

Different maize inbred lines are polymorphic for the presence or absence of genic sequences at various allelic chromosomal locations. In the bz genomic region, located in 9S, sequences homologous to four different genes from rice and Arabidopsis are present in line McC but absent from line B73. It is shown here that this apparent intraspecific violation of genetic colinearity arises from the movement of genes or gene fragments by Helitrons, a recently discovered class of eukaryotic transposons. Two Helitrons, HelA and HelB, account for all of the genic differences distinguishing the two bz locus haplotypes. HelA is 5.9 kb long and contains sequences for three of the four genes found only in the McC bz genomic region. A nearly identical copy of HelA was isolated from a 5S chromosomal location in B73. Both the 9S and 5S sites appear to be polymorphic in maize, suggesting that these Helitrons have been active recently. Helitrons lack the strong predictive terminal features of other transposons, so the definition of their ends is greatly facilitated by the identification of their vacant sites in Helitron-minus lines. The ends of the 2.7-kb HelB Helitron were discerned from a comparison of the McC haplotype sequence with that of yet a third line, Mo17, because the HelB vacant site is deleted in B73. Maize Helitrons resemble rice Pack-MULEs in their ability to capture genes or gene fragments from several loci and move them around the genome, features that confer on them a potential role in gene evolution.

Domestication of cereal crops, such as maize, wheat and rice, had a profound influence on agriculture and the establishment of human civilizations. One major improvement was an increase in seed number per inflorescence, which enhanced yield and simplified harvesting and storage(1,2). The ancestor of maize, teosinte, makes 2 rows of kernels, and modern varieties make similar to 8-20 rows(3). Kernel rows are initiated by the inflorescence shoot meristem, and shoot meristem size is controlled by a feedback loop involving the CLAVATA signaling proteins and the WUSCHEL transcription factor(4,5). We present a hypothesis that variation in inflorescence meristem size affects kernel row number (KRN), with the potential to increase yield. We also show that variation in the CLAVATA receptor-like protein FASCIATED EAR2 leads to increased inflorescence meristem size and KRN. These findings indicate that modulation of fundamental stem cell proliferation control pathways has the potential to enhance crop yields.

The nematode Caenorhabditis elegans is the first animal whose genome is completely sequenced, providing a rich source of gene information relevant to metazoan biology and human disease. This abundant sequence information permits a broad-based gene inactivation approach in C. elegans, in which chemically mutagenized nematode populations are screened by PCR for deletion mutations in a specific targeted gene. By handling mutagenized worm growth, genomic DNA templates, PCR screens, and mutant recovery all in 96-well microtiter plates, we have scaled up this approach to isolate deletion mutations in >100 genes to date. Four chemical mutagens, including ethyl methane sulfonate, ethlynitrosourea, diepoxyoctane, and ultraviolet-activated trimethylpsoralen, induced detectable deletions at comparable frequencies. The deletions averaged approximately 1400 bp in size when using a approximately 3 kb screening window. The vast majority of detected deletions removed portions of one or more exons, likely resulting in loss of gene function. This approach requires only the knowledge of a target gene sequence and a suitable mutagen, and thus provides a scalable systematic approach to gene inactivation for any organism that can be handled in high density arrays.

A key step during crop domestication is the loss of seed shattering. Here, we show that seed shattering in sorghum is controlled by a single gene, Shattering1 (Sh1), which encodes a YABBY transcription factor. Domesticated sorghums harbor three different mutations at the Sh1 locus. Variants at regulatory sites in the promoter and intronic regions lead to a low level of expression, a 2.2-kb deletion causes a truncated transcript that lacks exons 2 and 3, and a GT-to-GG splice-site variant in the intron 4 results in removal of the exon 4. The distributions of these non-shattering haplotypes among sorghum landraces suggest three independent origins. The function of the rice ortholog (OsSh1) was subsequently validated with a shattering-resistant mutant, and two maize orthologs (ZmSh1-1 and ZmSh1-5.1+ZmSh1-5.2) were verified with a large mapping population. Our results indicate that Sh1 genes for seed shattering were under parallel selection during sorghum, rice and maize domestication.

选取5个对干旱胁迫响应的玉米蛋白磷酸酶基因,从Mu突变体库中定向筛选到这些基因共92个Mu转座子插入的候选突变体。在田间种植这些候选突变体的后代,每个12粒。利用巢式PCR对这些后代植株的目标插入位点进行鉴定,选择阳性植株与B73杂交或回交。在92个候选突变体中,有21个为可遗传的Mu转座子插入突变体;其中8个突变体的Mu插入位点在目的基因的5'非翻译区,10个在内含子区,3个在外显子区。通过连续回交和自交,获得了这些突变体的纯合株系。本研究为这些基因的功能分析奠定了材料基础。

本研究建立了农杆菌介导的玉米合子转化方法。用农杆菌介导合子转化方法，以含有bar基因的 标准双元载体PTF102和含有bar基因和双价抗虫基因Cry1A（a）或Cry1A（c）、PTA（半夏凝集素）的载体p3300-Bt-pta转化 玉米自交系吉8902、丹340、吉4112、吉853、铁7922及PA91，直接从受体植株得到转化种子，用除草剂PPT筛选和PCR鉴定，获得转基 因植株及后代。实验分析了2002年、2003年和2004年的3批转化操作的结实率、转化率及转基因的遗传情况：经农杆菌侵染的雌穗平均结实率为 39％，合子转化频率达1％以上，转基因可以遗传下去。农杆菌介导玉米合子转化方法可以重复获得成功，表明我们成功建立起一个新的不依赖组织培养的玉米转 基因技术体系。