Comparative mapping of the barley Ppd-H1 photoperiod response gene region,which lies close to a junction between two rice linkage segments

Comparative mapping of cereals has shown that chromosomes of barley, wheat, and maize can be described in terms of rice "linkage segments." However, little is known about marker order in the junctions between linkage blocks or whether this will impair comparative analysis of major genes that lie in such regions. We used genetic and physical mapping to investigate the relationship between the distal part of rice chromosome 7L, which contains the Hd2 heading date gene, and the region of barley chromosome 2HS containing the Ppd-H1 photoperiod response gene, which lies near the junction between rice 7 and rice 4 linkage segments. RFLP markers were mapped in maize to identify regions that might contain Hd2 or Ppd-H1 orthologs. Rice provided useful markers for the Ppd-H1 region but comparative mapping was complicated by loss of colinearity and sequence duplications that predated the divergence of rice, maize, and barley. The sequences of cDNA markers were used to search for homologs in the Arabidopsis genome. Homologous sequences were found for 13 out of 16 markers but they were dispersed in Arabidopsis and did not identify any candidate equivalent region. The implications of the results for comparative trait mapping in junction regions are discussed.     

Fine genetic mapping fails to dissociate durable stem rust resistance gene Sr2 from pseudo-black chaff in common wheat (Triticum aestivum L.)

The broad-spectrum stem rust resistance gene Sr2 has provided protection in wheat against Puccinia graminis Pers. f. sp. tritici for over 80 years. The Sr2 gene and an associated dark pigmentation trait, pseudo-black chaff (PBC), have previously been localized to the short arm of chromosome 3B. In a first step towards the positional-based cloning of Sr2, we constructed a high-resolution map of this region. The wheat EST (wEST) deletion bin mapping project provided tightly linked cDNA markers. The rice genome sequence was used to infer the putative gene order for orthologous wheat genes and provide additional markers once the syntenic interval in rice was identified. We used this approach to map six wESTs that were collinear with the physical order of the corresponding genes on rice chromosome 1 suggesting there are no major re-arrangements between wheat and rice in this region. We were unable to separate by recombination the tightly linked morphological trait, PBC from the stem rust resistance gene suggesting that either a single gene or two tightly linked genes control both traits.     

大麦产量相关基因HvYrg1的克隆及植物RNA干扰载体的构建

大麦谷粒是受多个数量性状基因(QTL)控制的复杂性状,而RING E3泛素连接酶在决定大麦产量和蛋白质降解途径中起到极为重要的作用。本研究按照同源克隆的方法,依据水稻、拟南芥、玉米、小麦和酵母等E3泛素连接酶保守区域设计引物,采用RT-PCR方法从西藏大麦中克隆出产量相关基因HvYrg1全长cDNA序列,包括完整的开放阅读框架(ORF)1 275 bp,编码蛋白为424个氨基酸(GenBank No. EU333863)。同源性比较结果显示,它与GenBank上已报道的水稻GW2基因同源性最高为86%。以植物表达载体pCAMBIA2300-35s-OCS质粒为基础,构建由35s启动子调控的HvYrg1基因的RNA干扰载体pCAM-RNAi-HvYrg1。这一载体的成功构建为研究该基因在作物产量的功能鉴定打下了很好的基础。     

Reciprocal control of flowering time by OsSOC1 in transgenic Arabidopsis and by FLC in transgenic rice

In a screen for MADS box genes which activate and/or repress flowering in rice, we identified a gene encoding a MADS domain protein (OsSOC1) related to the Arabidopsis gene AtSOC1. AtSOC1 and OsSOC1 show a 97% amino acid similarity in their MADS domain. The rice gene contains a large first intron of 27.6 kb compared to the 1 kb intron in Arabidopsis. OsSOC1 is located on top of the short arm of chromosome 3, tightly linked to the heading date locus, Hd9. OsSOC1 is expressed in vegetative tissues, and expression is elevated at the time of floral initiation, 40-50 days after sowing, and remains uniformly high thereafter, similar to the expression pattern of AtSOC1. The constitutive expression of OsSOC1 in Arabidopsis results in early flowering, suggesting that the rice gene is a functional equivalent of AtSOC1. We were not able to identify FLC-like sequences in the rice genome; however, we show that ectopic expression of the Arabidopsis FLC delays flowering in rice, and the up-regulation of OsSOC1 at the onset of flowering initiation is delayed in the AtFLC transgenic lines. The reciprocal recognition and flowering time effects of genes introduced into either Arabidopsis or rice suggest that some components of the flowering pathways may be shared. This points to a potential application in the manipulation of flowering time in cereals using well characterized Arabidopsis genes.     

Isolation and mapping of rFUS6, a rice orthologue of Arabidopsis thaliana FUS6.

COP9 complex is one of the most important components that act in repressing photomorphogenesis in Arabidopsis thaliana. FUS6 has been identified as one of eight subunits of the COP9 complex in Arabidopsis. Using Arabidopsis Fus6 cDNA as a probe, we screened a rice root cDNA library and a rice genomic library. A 1730-bp cDNA was obtained, which has an open reading frame corresponding to 441-amino-acid. This 441 amino acids putative protein has 67% identity with Arabidopsis COP11/FUS6 (AtFUS6) and 40% identity with human GPS1, an AtFUS6 orthologue. So we designated this novel gene as rFUS6. The 6.2-kb genomic sequence of rFUS6 was also obtained. Sequence comparison showed that the rFUS6 gene had six exons and five introns. Sequence inspection of the 5'-flanking region revealed the presence of some potential light-regulated cis-elements such as a G-box, GT-1 binding sites, and a TGACG motif. Southern hybridization with rice total DNA showed that rFUS6 was perhaps a single copy gene. The rFUS6 locus was mapped by hybridization with a rice BAC library membrane and the results showed that rFUS6 had a locus at 16.3 cM of chromosome 1.     

Cloning and characterization of a gene encoding wheat starch synthase I

 A cDNA clone, and a corresponding genomic DNA clone, containing full-length sequences encoding wheat starch synthase I, were isolated from a cDNA library of hexaploid wheat (Triticum aestivum) and a genomic DNA library of Triticum tauschii, respectively. The entire sequence of the starch synthase-I cDNA (wSSI-cDNA) is 2591 bp, and it encodes a polypeptide of 647 amino-acid residues that shows 81% and 61% identity to the amino-acid sequences of SSI-type starch synthases from rice and potato, respectively. In addition, the putative N-terminal amino-acid sequence of the encoded protein is identical to that determined for the N-terminal region of the 75-kDa starch synthase present in the starch granule of hexaploid wheat. Two prominent starch synthase activities were demonstrated to be present in the soluble fraction of wheat endosperm by activity staining of the non-denaturing PAGE gels. The most anodal band (wheat SSI) shows the highest staining intensity and results from the activity of a 75-kDa protein. The wheat SSI mRNA is expressed in the endosperm during the early to mid stages of wheat grain development but was not detected by Northern blotting in other tissues from the wheat plant. The gene encoding the wheat SSI (SsI-D1) consists of 15 exons and 14 introns, similar to the structure of the rice starch synthase-I gene. While the exons of wheat and rice are virtually identical in length, the wheat SsI-D1 gene has longer sequences in introns 1, 2, 4 and 10, and shorter sequences in introns 6, 11 and 14, than the corresponding rice gene. Received: 5 June 1998 / Accepted: 29 September 1998     

Identification of perennial ryegrass (Lolium perenne (L.)) and meadow fescue (Festuca pratensis (Huds.)) candidate orthologous sequences to the rice Hd1(Se1) and barley HvCO1 CONSTANS-like genes through comparative mapping and microsynteny

Microsynteny with rice and comparative genetic mapping were used to identify candidate orthologous sequences to the rice Hd1(Se1) gene in Lolium perenne and Festuca pratensis. A F. pratensis bacterial artificial chromosome (BAC) library was screened with a marker (S2539) physically close to Hd1 in rice to identify the equivalent genomic region in F. pratensis. The BAC sequence was used to identify and map the same region in L. perenne. Predicted protein sequences for L. perenne and F. pratensis Hd1 candidates (LpHd1 and FpHd1) indicated they were CONSTANS-like zinc finger proteins with 61-62% sequence identity with rice Hd1 and 72% identity with barley HvCO1. LpHd1 and FpHd1 were physically linked in their respective genomes (< 4 kb) to marker S2539, which was mapped to L. perenne chromosome 7. The identified candidate orthologues of rice Hd1 and barley HvCO1 in L. perenne and F. pratensis map to chromosome 7, a region of the L. perenne genome which has a degree of conserved genetic synteny both with rice chromosome 6, which contains Hd1, and barley chromosome 7H, which contains HvCO1.     

Construction of a full-length cDNA library from young spikelets of hexaploid wheat and its characterization by large-scale sequencing of expressed sequence tags

The polyploid nature of wheat is a key characteristic of the plant. Full-length complementary DNAs (cDNAs) provide essential information that can be used to annotate the genes and provide a functional analysis of these genes and their products. We constructed a full-length cDNA library derived from young spikelets of common wheat, and obtained 24056 expressed sequence tags (ESTs) from both ends of the cDNA clones. These ESTs were grouped into 3605 contigs using the phrap method, representing expressed loci from each of the three genomes. Using BLAST, 3605 contigs were grouped into 1902 gene clusters, showing that loci of the three genomes are not always expressed. A homology search of these gene clusters against a wheat EST database (15964 gene clusters) and a rice full-length cDNA database (21447 gene clusters) revealed that a quarter of the wheat full-length cDNAs were novel. A protein database of Arabidopsis was used to examine the functional classification of these gene clusters. The GC-content in the 5 -UTR region of wheat cDNAs was compared to that of rice. Forty-three genes (3.5% of wheat cDNAs homologous to those of rice) possessed distinct GC-content in the 5 -UTR region, suggesting different breeding behaviors of wheat and rice.     

Epistatic interaction between vernalization genes Vrn-Am1 and Vrn-Am2 in diploid wheat

Genes Vrn-A(m)1 and Vrn-A(m)2 control the vernalization requirement in diploid wheat (Triticum monococcum). The epistatic interaction between these two genes on flowering date was studied here using a factorial analysis of variance. One hundred and two F2 plants were classified according to their genotypes for molecular markers tightly linked to Vrn-A(m)1 and Vrn-A(m)2. Mean comparisons showed that the VrnA(m)2 allele for winter growth habit was dominant to the vrn-A(m)2 allele for spring growth habit and that the Vrn-A(m)1 allele for spring growth habit was dominant to the vrn-A(m)1 allele for winter growth habit. A significant interaction was found between these two genes, suggesting that they work in the same developmental pathway. Plants homozygous for the recessive vrn-A(m)2 allele for spring growth habit flowered earlier than plants from the Vrn-A(m)2 class independently of the alleles present at Vrn-A(m)1. However, differences in heading date between plants with the Vrn-A(m)1 allele and those with the vrn-A(m)1 allele were significant only when the dominant Vrn-A(m)2 allele was present. A genetic model for the action of these two vernalization genes is proposed in which the role of Vrn-A(m)1 is to counteract the Vrn-A(m)2-mediated delay of flowering.     

Arabidopsis phosphoribosylanthranilate isomerase: molecular genetic analysis of triplicate tryptophan pathway genes.

Phosphoribosylanthranilate isomerase (PAI) catalyzes the third step of the tryptophan biosynthetic pathway. Arabidopsis PAI cDNAs were cloned from a cDNA expression library by complementation of an Escherichia coli trpC- PAI deficiency mutation. Genomic DNA blot hybridization analysis detected three nonallelic genes encoding PAI in the Arabidopsis genome. DNA sequence analysis of cDNA and genomic clones indicated that the PAI1 and PAI2. All three PAI polypeptides possess an N-terminal putative plastid target sequence, suggesting that these enzymes all function in plastids. The PAI1 gene is flanked by nearly identical direct repeats of approximately 350 nucleotides. Our results indicate that, in contrast to most microorganisms, the Arabidopsis PAI protein is not fused with indole-3-glycerolphosphate synthase, which catalyzes the next step in the pathway. Yeast artificial chromosome hybridization studies indicated that the PAI2 gene is tightly linked to the anthranilate synthase alpha subunit 1 (ASA1) gene on chromosome 5. PAI1 was mapped to the top of chromosome 1 using recombinant inbred lines, and PAI3 is loosely linked to PAI1. cDNA restriction mapping and sequencing and RNA gel blot hybridization analysis indicated that all three genes are transcribed in wild-type plants. The expression of antisense PAI1 RNA significantly reduced the immunologically observable PAI protein and enzyme activity in transgenic plants. The plants expressing antisense RNA also showed two phenotypes consistent with a block early in the pathway: blue fluorescence under UV light and resistance to the anthranilate analog 6-methylanthranilate. The extreme nucleotide conservation between the unlinked PAI1 and PAI2 loci suggests that this gene family is actively evolving.     

Genes responding to vernalization in hexaploid wheat

Genotype-specific gene expression in response to vernalization in common wheat was examined by the differential display method. Two near-isogenic lines of Vrn-A1 ( Vrn-A1 for the spring type and vrn-A1 for the winter type) were treated by vernalization of developing embryos in detached-ear cultures. This treatment was effective to promote vrn-A1 genotypes to head at a time equivalent to that of Vrn-A1. Differential cDNA fragments were isolated by the RT-PCR method from embryos subjected to vernalization treatments for 2- and 4-weeks at DAP10 and DAP20 stages, respectively. Among 110 differential cDNA fragments isolated, 48 were examined for their chromosomal locations and designated as wec ( wheat- embryo cold treatment) genes. Seven wec genes showed genotype-specific expression in response to vernalization. The statistical analysis utilizing two recombinant inbred lines showed that four wec genes were significantly associated with heading factors.