Cloning and expression of a sorghum gene with homology to maize vp1. Its potential involvement in pre-harvest sprouting resistance

Pre-harvest sprouting (PHS) in sorghum is related to the lack of a normal dormancy level during seed development and maturation. Based on previous evidence that seed dormancy in maize is controlled by the vp1 gene, we used a PCR-based approach to isolate two Sorghum bicolor genomic and cDNA clones from two genotypes exhibiting different PHS behaviour and sensitivity to abscisic acid (ABA). The two 699 amino acid predicted protein sequences differ in two residues at positions 341 (Gly or Cys within the repression domain) and 448 (Pro or Ser) and show over 80, 70 and 60% homology to maize, rice and oat VP1 proteins respectively.Expression analysis of the sorghum vp1 gene in the two lines shows a slightly higher level of vp1 mRNA in the embryos susceptible to PHS than in those resistant to PHS during embryogenesis. However, timing of expression was different between these genotypes during this developmental process. Whereas for the former the main peak of expression was observed at 20 days after pollination (DAP), the peak in the latter was found at later developmental stages when seed maturation was almost complete.Under favourable germination conditions and in the presence of fluridone (an inhibitor of ABA biosynthesis), sorghum vp1 mRNA showed to be consistently correlated with sensitivity to ABA but not with ABA content and dormancy.     

The maize viviparous15 locus encodes the molybdopterin synthase small subunit

A new Zea mays viviparous seed mutant, viviparous15 (vp15), was isolated from the UniformMu transposon-tagging population. In addition to precocious germination, vp15 has an early seedling lethal phenotype. Biochemical analysis showed reduced activities of several enzymes that require molybdenum cofactor (MoCo) in vp15 mutant seedlings. Because MoCo is required for abscisic acid (ABA) biosynthesis, the viviparous phenotype is probably caused by ABA deficiency. We cloned the vp15 mutant using a novel high-throughput strategy for analysis of high-copy Mu lines: We used MuTAIL PCR to extract genomic sequences flanking the Mu transposons in the vp15 line. The Mu insertions specific to the vp15 line were identified by in silico subtraction using a database of MuTAIL sequences from 90 UniformMu lines. Annotation of the vp15-specific sequences revealed a Mu insertion in a gene homologous to human MOCS2A, the small subunit of molybdopterin (MPT) synthase. Molecular analysis of two allelic mutations confirmed that Vp15 encodes a plant MPT synthase small subunit (ZmCNX7). Our results, and a related paper reporting the cloning of maize viviparous10, demonstrate robust cloning strategies based on MuTAIL-PCR. The Vp15/CNX7, together with other CNX genes, is expressed in both embryo and endosperm during seed maturation. Expression of Vp15 appears to be regulated independently of MoCo biosynthesis. Comparisons of Vp15 loci in genomes of three cereals and Arabidopsis thaliana identified a conserved sequence element in the 5' untranslated region as well as a micro-synteny among the cereals.     

QTL analysis of falling number and seed longevity in wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.)

Pre-harvest sprouting (PHS) and seed longevity (SL) are complex biological processes of major importance for agricultural production. In the present study, a recombinant inbred line (RIL) population derived from a cross between the German winter wheat (Triticum aestivum L.) cultivars History and Rubens was used to identify genetic factors controlling these two physiological seed traits. A falling number (FN) test was employed to evaluate PHS, while SL was measured using a germination test (and the speed of germination) after controlled deterioration. FN of the population was assessed in four environments; SL traits were measured in one environment. Four major quantitative trait loci (QTL) for FN were detected on chromosomes 4D, 5A, 5D, and 7B, whereas for SL traits, a major QTL was found on chromosome 1A. The FN QTL on chromosome 4D that coincided with the position of the dwarfing gene Rht-D1b only had effects in environments that were free of PHS. The remaining three QTL for FN were mostly pronounced under conditions conducive to PHS. The QTL on the long arm of chromosome 7B corresponded to the major gene locus controlling late maturity α-amylase (LMA) in wheat. The severity of the LMA phenotype became truly apparent under sprouting conditions. The position on the long arm of chromosome 1A of the QTL for SL points to a new QTL for this important regenerative seed trait.     

Vivipary as a tool to analyze late embryogenic events in maize

In maize vivipary, the precocious germination of the seed while still attached to the ear, is the diagnostic phenotype of mutants, which are impaired in the biosynthesis or response to abscisic acid (ABA). Of the 15 genes so far described, 12 control specific steps in ABA biosynthesis, two mediate hormone response and one still has an undefined role. We have analyzed a collection of 25 independent vp isolates with the aim of determining the degree of mutational saturation that has so far been reached. Of the 25 viviparous mutants complementation tested, 22 correspond to known loci: six are allelic to vp1, another six to vp5, one to vp7, two to vp9, six to vp10 and one to w3. The remaining three represent genes not previously identified. All mutants so far tested except rea show a decrease in ABA content. As to the only two mutants (vp1 and rea) whose endogenous ABA content is not impaired, the reduction in sensitivity of the double mutant compared to the single ones suggests that the two genes control separate pathways in the ABA signal transduction. Some of the mutants in this collection have a characteristic incomplete germination that allows the embryo of the mature dry seed to resume germination. By exploiting this feature it is possible to infer, through a germination test, whether the mutant has been impaired in the acquisition of desiccation tolerance. This information provides the starting point for the dissection of the genetic basis of desiccation tolerance.     

Control of rice pre‐harvest sprouting by glutaredoxin‐mediated abscisic acid signaling

Pre‐harvest sprouting (PHS) is one of the major problems in cereal production worldwide, which causes significant losses of both yield and quality; however, the molecular mechanism underlying PHS remains largely unknown. Here, we identified a dominant PHS mutant phs9‐D. The corresponding gene PHS9 encodes a higher plant unique CC‐type glutaredoxin and is specifically expressed in the embryo at the late embryogenesis stage, implying that PHS9 plays some roles in the late stage of seed development. Yeast two‐hybrid screening showed that PHS9 could interact with OsGAP, which is an interaction partner of the abscicic acid (ABA) receptor OsRCAR1. PHS9‐ or OsGAP overexpression plants showed reduced ABA sensitivity in seed germination, whereas PHS9 or OsGAP knock‐out mutant plants showed increased ABA sensitivity in seed germination, suggesting that PHS9 and OsGAP acted as negative regulators in ABA signaling during seed germination. Interestingly, the germination of PHS9 and OsGAP overexpression or knock‐out plant seeds was weakly promoted by H₂O₂, implying that PHS9 and OsGAP could affect reactive oxygen species (ROS) signaling during seed germination. These results indicate that PHS9 plays an important role in the regulation of rice PHS through the integration of ROS signaling and ABA signaling.     

Mapping quantitative trait loci for preharvest sprouting resistance in white wheat

The premature germination of seeds before harvest, known as preharvest sprouting (PHS), is a serious problem in all wheat growing regions of the world. In order to determine genetic control of PHS resistance in white wheat from the relatively uncharacterized North American germplasm, a doubled haploid population consisting of 209 lines from a cross between the PHS resistant variety Cayuga and the PHS susceptible variety Caledonia was used for QTL mapping. A total of 16 environments were used to detect 15 different PHS QTL including a major QTL, QPhs.cnl-2B.1, that was significant in all environments tested and explained from 5 to 31% of the trait variation in a given environment. Three other QTL QPhs.cnl-2D.1, QPhs.cnl-3D.1, and QPhs.cnl-6D.1 were detected in six, four, and ten environments, respectively. The potentially related traits of heading date (HD), plant height (HT), seed dormancy (DOR), and rate of germination (ROG) were also recorded in a limited number of environments. HD was found to be significantly negatively correlated with PHS score in most environments, likely due to a major HD QTL, QHd.cnl-2B.1, found to be tightly linked to the PHS QTL QPhs.cnl-2B.1. Using greenhouse grown material no overlap was found between seed dormancy and the four most consistent PHS QTL, suggesting that greenhouse environments are not representative of field environments. This study provides valuable information for marker-assisted breeding for PHS resistance, future haplotyping studies, and research into seed dormancy.     

Polymorphic Homoeolog of Key Gene of RdDM Pathway,ARGONAUTE4_9 class Is Associated with Pre-Harvest Sprouting in Wheat (Triticum aestivum L.)

Resistance to pre-harvest sprouting (PHS) is an important objective for the genetic improvement of many cereal crops, including wheat. Resistance, or susceptibility, to PHS is mainly influenced by seed dormancy, a complex trait. Reduced seed dormancy is the most important aspect of seed germination on a spike prior to harvesting, but it is influenced by various environmental factors including light, temperature and abiotic stresses. The basic genetic framework of seed dormancy depends on the antagonistic action of abscisic acid (ABA) and gibberellic acid (GA) to promote dormancy and germination. Recent studies have revealed a role for epigenetic changes, predominantly histone modifications, in controlling seed dormancy. To investigate the role of DNA methylation in seed dormancy, we explored the role of ARGONAUTE4_9 class genes in seed development and dormancy in wheat. Our results indicate that the two wheat AGO4_9 class genes i.e. AGO802 and AGO804 map to chromosomes 3S and 1S are preferentially expressed in the embryos of developing seeds. Differential expressions of AGO802-B in the embryos of PHS resistant and susceptible varieties also relates with DNA polymorphism in various wheat varieties due to an insertion of a SINE-like element into this gene. DNA methylation patterns of the embryonic tissue from six PHS resistant and susceptible varieties demonstrate a correlation with this polymorphism. These results suggest a possible role for AGO802-B in seed dormancy and PHS resistance through the modulation of DNA methylation.     

Genetic and QTL analyses of seed dormancy and preharvest sprouting resistance in the wheat germplasm CN10955

The inheritance and genetic linkage analysis for seed dormancy and preharvest sprouting (PHS) resistance were carried out in an F₈ recombinant inbred lines (RILs) derived from the cross between “CN19055” (white-grained, PHS-resistant) with locally adapted Australian cultivar “Annuello” (white-grained, PHS-susceptible). Seed dormancy was assessed as germination index (GI7) while assessment for preharvest sprouting resistance was based on whole head assay (sprouting index, SI) and visibly sprouted seeds (VI). Segregation analysis of the F₂, F₃ data from the glasshouse and the RIL population in 2004 and 2005 field data sets indicated that seed dormancy and PHS resistance in CN19055 is controlled by at least two genes. Heritabilities for GI7 and VI were high and moderate for SI. The most accurate method for assessing PHS resistance was achieved using VI and GI7 while SI exhibited large genotype by environment interaction. Two quantitative trait loci (QTLs) QPhs.dpivic.4A.1 and QPhs.dpivic.4A.2 were identified. On pooled data across four environments, the major QTL, QPhs.dpivic.4A.2, explained 45% of phenotypic variation for GI7, 43% for VI and 20% for SI, respectively. On the other hand, QPhs.dpivic.4A.1 which accounted for 31% of the phenotypic variation in GI7 in 2004 Horsham field trial, was not stable across environments. Physical mapping of two SSR markers, Xgwm937 and Xgwm894 linked to the major QTL for PHS resistance, using Chinese Spring deletions lines for chromosome 4AS and 4AL revealed that the markers were located in the deletion bins 4AL-12 and 4AL-13. The newly identified SSR markers (Xgwm937/Xgwm894) showed strong association with seed dormancy and PHS resistance in a range of wheat lines reputed to possess PHS resistance. The results suggest that Xgwm937/Xgwm894 could be used in marker-assisted selection (MAS) for incorporating preharvest sprouting resistance into elite wheat cultivars susceptible to PHS.     

MicroRNA transcriptomic analysis of heterosis during maize seed germination

Heterosis has been utilized widely in the breeding of maize and other crops, and plays an important role in increasing yield, improving quality and enhancing stresses resistance, but the molecular mechanism responsible for heterosis is far from clear. To illustrate whether miRNA-dependent gene regulation is responsible for heterosis during maize germination, a deep-sequencing technique was applied to germinating embryos of a maize hybrid, Yuyu22, which is cultivated widely in China and its parental inbred lines, Yu87-1 and Zong3. The target genes of several miRNAs showing significant expression in the hybrid and parental lines were predicted and tested using real-time PCR. A total of 107 conserved maize miRNAs were co-detected in the hybrid and parental lines. Most of these miRNAs were expressed non-additively in the hybrid compared to its parental lines. These results indicated that miRNAs might participate in heterosis during maize germination and exert an influence via the decay of their target genes. Novel miRNAs were predicted follow a rigorous criterion and only the miRNAs detected in all three samples were treated as a novel maize miRNA. In total, 34 miRNAs belonged to 20 miRNA families were predicted in germinating maize seeds. Global repression of miRNAs in the hybrid, which might result in enhanced gene expression, might be one reason why the hybrid showed higher embryo germination vigor compared to its parental lines.     

A major QTL controlling seed dormancy and pre-harvest sprouting resistance on chromosome 4A in a Chinese wheat landrace

Wheat pre-harvest sprouting (PHS) can cause significant reduction in yield and end-use quality of wheat grains in many wheat-growing areas worldwide. To identify a quantitative trait locus (QTL) for PHS resistance in wheat, seed dormancy and sprouting of matured spikes were investigated in a population of 162 recombinant inbred lines (RILs) derived from a cross between the white PHS-resistant Chinese landrace Totoumai A and the white PHS-susceptible cultivar Siyang 936. Following screening of 1,125 SSR primers, 236 were found to be polymorphic between parents, and were used to screen the mapping population. Both seed dormancy and PHS of matured spikes were evaluated by the percentage of germinated kernels under controlled moist conditions. Twelve SSR markers associated with both PHS and seed dormancy were located on the long arm of chromosome 4A. One QTL for both seed dormancy and PHS resistance was detected on chromosome 4AL. Two SSR markers, Xbarc 170 and Xgwm 397, are 9.14 cM apart, and flanked the QTL that explained 28.3% of the phenotypic variation for seed dormancy and 30.6% for PHS resistance. This QTL most likely contributed to both long seed dormancy period and enhanced PHS resistance. Therefore, this QTL is most likely responsible for both seed dormancy and PHS resistance. The SSR markers linked to the QTL can be used for marker-assisted selection of PHS-resistant white wheat cultivars. Shi-Bin Cai and Cui-Xia Chen contributed equally to this work.     

Genetic dissection of herbicide tolerance in maize by molecular markers

In order to detect the genetic architecture of maize tolerance to Alachlor, a widely used chloroacetanilide, linkage analysis between the expression of the trait and allelic composition of molecular markers was performed. The experiment was carried out on a population of 142 recombinant inbred lines, developed starting from the F₁ between two lines with different reactivity to the herbicide, and self-fertilized for 10 generations; the lines were typed by 48 RFLP markers and 66 microsatellites (SSR). Besides seedling tolerance, evaluated as proportion of normal (non-injured) plants after herbicide treatment, other minor components of tolerance were studied: seed germination ability, pollen germination and tube growth in the presence of the herbicide. The analysis, performed by three statistical methods, revealed the presence of factors controlling seedling tolerance on seven chromosomal regions. Five QTLs appeared to be involved in seed germination ability in the presence of Alachlor, four QTLs in pollen tolerance in terms of germination and four in tube growth under stress were detected. Three loci, on chromosomes 1, 7 and 10, explained most of the variation of seedling tolerance, thus being interesting candidate for marker-assisted selection.     

利用花青素可视化跟踪表达系统快速筛选表达Cry1Ab/c基因的转基因玉米

以草甘膦抗性基因Epsps为标记基因, 在原核Kan^r基因两侧引入Cre(环化重组酶)基因识别的Lox-P位点, 同时以编码花青素合成转录因子的Bi和Cl基因为可视化选择报告基因, 构建了Bt杀虫蛋白基因Cry1Ab/c的可视化跟踪表达载体pBAC9017。用PDS1000/He基因枪转化玉米(Zea mays)自交系501的幼胚和胚性愈伤组织, 获得147个草甘膦抗性的玉米再生植株。其中106棵植株获得了结实种子, 16棵植株的结实种子有紫红色花青素基因的表达。经PCR检测表明, 外源Cry1Ab/c基因已经整合到玉米的基因组中。转基因植株种子蛋白粗提物用BT-Cry1Ab/1Ac金标免疫检测试纸条和ELISA检测, 结果表明, Cry1Ab/c在部分转基因植株后代中表达。     

Steady-state transposon mutagenesis in inbred maize

We implement a novel strategy for harnessing the power of high-copy transposons for functional analysis of the maize genome, and report behavioral features of the Mutator system in a uniform inbred background. The unique UniformMu population and database facilitate high-throughput molecular analysis of Mu-tagged mutants and gene knockouts. Key features of the population include: (i) high mutation frequencies (7% independent seed mutations) and moderation of copy number (approximately 57 total Mu elements; 1-2 MuDR copies per plant) were maintained by continuous back-crossing into a phenotypically uniform inbred background; (ii) a bz1-mum9 marker enabled selection of stable lines (loss of MuDR), inhibiting further transpositions in lines selected for molecular analysis; (iii) build-up of mutation load was prevented by screening Mu-active parents to exclude plants carrying pre-existing seed mutations. To create a database of genomic sequences flanking Mu insertions, selected mutant lines were analyzed by sequencing of MuTAIL PCR clone libraries. These sequences were annotated and clustered to facilitate bioinformatic subtraction of ancestral elements and identification of insertions unique to mutant lines. New insertions targeted low-copy, gene-rich sequences, and in silico mapping revealed a random distribution of insertions over the genome. Our results indicate that Mu populations differ markedly in the occurrence of Mu insertion hotspots and the frequency of suppressible mutations. We suggest that controlled MuDR copy number in UniformMu lines is a key determinant of these differences. The public database (http://uniformmu.org; http://endosperm.info) includes pedigree and phenotypic data for over 2000 independent seed mutants selected from a population of 31 548 F2 lines and integrated with analyses of 34 255 MuTAIL sequences.