[Generation of selectable marker-free and vector backbone sequence-free Xa21 transgenic rice].

The dominant gene Xa21 with broad-spectrum and high resistance to Xanthomonas oryzae pv. oryzae (Xoo) was transferred into C418, an important restorer line of japonica hybrid rice in China using double right-border (DRB) T-DNA binary vector through Agrobacterium-mediated transformation. 17 transgenic lines were Xa21-positive with high resistance to the race P6 of Xoo through PCR analysis and resistance identification, among the total 27 independent primary transformants (T0) obtained. The subsequent analysis of the T1 progenies of these 17 T0 lines through PCR-assisted selection and resistance investigation showed that four Xa21 transgenic T0 lines could produce selectable marker-free (SMF) progenies. The frequency of primary transformants producing SMF progenies was 15%. In addition, PCR analysis also revealed these SMF progenies did not contain vector backbone sequence, and they were named as SMF and vector backbone sequence-free (SMF-VBSF) Xa21 transgenic plants. The further molecular and phenotypic analysis of the T2 and T3 progenies testified the homozygous SMF-VBSF Xa21 transgenic plants were obtained with high resistance to Xoo.

Testifying the rice bacterial blight resistance gene xa5 by genetic complementation and further analyzing xa5 (Xa5) in comparison with its homolog TFIIAgamma1.

The recessive gene xa5 for resistance to bacterial blight resistance of rice is located on chromosome 5, and evidence based on genetic recombination has been shown to encode a small subunit of the basal transcription factor IIA (Iyer and McCouch in MPMI 17(12):1348-1354, 2004). However, xa5 has not been demonstrated by a complementation test. In this study, we introduced the dominant allele Xa5 into a homozygous xa5-line, which was developed from a cross between IRBB5 (an indica variety with xa5) and Nipponbare (a japonica variety with Xa5). Transformation of Xa5 and subsequent segregation analysis confirmed that xa5 is a V39E substitution variant of the gene for TFIIAgamma on chromosome 5 (TFIIAgamma5 or Xa5). The rice has an addition gene for TFIIAgamma exists on chromosome 1 (TFIIAgamma1). Analysis of the expression patterns of Xa5 (TFIIAgamma5)/xa5 and TFIIAgamma1 revealed that both the genes are constitutively expressed in different rice organs. However, no expression of TFIIAgamma1 could be detected in the panicle by reverse transcriptase-polymerase chain reaction. To compare the structural difference between the Xa5/xa5 and TFIIAgamma1 proteins, 3-D structures were predicted using computer-aided modeling techniques. The modeled structures of Xa5 (xa5) and TFIIAgamma1 fit well with the structure of TFIIA small subunit from human, suggesting that they may all act as a small subunit of TFIIA. The E39V substitution in the xa5 protein occurs in the alpha-helix domain, a supposed conservative substitutable site, which should not affect the basal transcription function of TFIIAgamma. The structural analysis indicates that xa5 and Xa5 potentially retain their basic transcription factor function, which, in turn, may mediate the novel pathway for bacterial blight resistance and susceptibility, respectively.

[Cloning and analysis of a new NBS-LRR resistance gene family in rice].

Sequence-based gene isolation has been a practical approach for plant resistance gene cloning. In this study, RS13, a cloned rice sequence with the NBS (nucleotide-binding site) domain of resistance genes, was used as a probe to screen a bacterial artificial chromosome (BAC) library of rice variety IR64,and four positive clones were obtained. Of them the clone 14E19 covered the other three clones and was sequenced through a shotgun approach. The whole sequence of the insert fragment of 14E19 was assembled into approximately 73 kb in length. Genes on the whole assembled sequence were predicted,and four genes encoding NBS and LRR (leucine-rich repeat) domains were found, named as NL-A, B, C and D respectively. For further analysis, another longer BAC clone,106P13, covering 14E19 on the same chromosome position was identified from a BAC library of IRBB56 which had the same genome background with IR64. Ten NL-homologous copies were discovered on the sequence of the BAC clone 106P13, and four copies were identical with those on 14E19. The similar homologous sequences were also found in the genomic sequences of Nipponbare,93-11 and Guangluai4. However, NL sequences were less homologous with the known NBS-LRR resistance genes. This result indicated that NL was a new NBS-LRR gene family and was composed of ten members at least. RT-PCR and cDNA screening displayed that NL-B expressed in a bacterial blight-resistant rice variety IRBB4, indicating the gene was possibly involved in resistance reactions.

[Construction of mutant population of differential race of Xa23 resistant to rice bacterial blight and avirulence activity identification of mutants].

The mutant population of Xanthomonas oryzae pv oryzae strain differential to rice bacterial blight resistance gene Xa23 has been constructed mediated by transposon in vivo . The results of PCR amplification with specific primers and analysis of flanking sequence of mutants indicated that the foreign DNA has been integrated into X. oryzae pv oryzae genome. Four mutants with changed avirulent activity to Xa23 gene have been identified by artificial inoculation. It is possible to clone genes that are required for AvrXa23 avirulence activity using this new strategy.

Molecular evolution of the rice miR395 gene family.

MicroRNAs (miRNAs) are 20-22 nucleotide non-coding RNAs that play important roles in plant and animal development. They are usually processed from larger precursors that can form stem-loop structures. Among 20 miRNA families that are conserved between Arabidopsis and rice, the rice miR395 gene family was unique because it was organized into compact clusters that could be transcribed as one single transcript. We show here that in fact this family had four clusters of total 24 genes. Three of these clusters were segmental duplications. They contained miR395 genes of both 120 bp and 66 bp long. However, only the latter was repeatedly duplicated. The fourth cluster contained miR395 genes of two different sizes that could be the consequences of intergenic recombination of genes from the first three clusters. On each cluster, both 1-duplication and 2-duplication histories were observed based on the sequence similarity between miR395 genes, some of which were nearly identical suggesting a recent origin. This was supported by a miR395 locus survey among several species of the genus Oryza, where two clusters were only found in species with an AA genome, the genome of the cultivated rice. A comparative study of the genomic organization of Medicago truncatula miR395 gene family showed significant expansion of intergenic spaces indicating that the originally clustered genes were drifting away from each other. The diverse genomic organizations of a conserved microRNA gene family in different plant genomes indicated that this important negative gene regulation system has undergone dramatic tune-ups in plant genomes.

[Molecular mapping of a bacterial blight resistance gene Xa-25 in rice].

Xa-25 was a bacterial blight resistance gene identified in a somaclonal mutant HX-3. A doubled-haploid (DH) population including 129 stable lines was derived from anther culture of a typical japanica 02428 and a typical indica HX-3 cross. The bacterial blight strain Zhe173, a typical bacterial blight strain in Yangtze River valley, was used to test the resistance or susceptible of the DH population lines, and the results showed that the resistance lines and susceptible lines were 62 and 67, respectively. A total of 300 SSR primer pairs covering 12 rice chromosomes were used for polymorphism survey of 02428 and HX-3. Among these primers, 74 showed polymorphism between the parents. Using these polymorphic SSR markers, bulked segregant analysis was conducted on the DH population. As the result, Xa-25 was located at the terminal region of the long arm of chromosome 4 between the two SSR markers RM6748 and RM1153, the map distance between Xa-25 and the two SSR markers was 9.3 cM and 3.0 cM, respectively.

[Analysis of the transgenic rice plants derived from transformed anther calli].

Rice calli derived from anther culture were used as recipient to transfer a rice blight resistance gene, Xa21, into a japonica rice variety, Taipei 309, via Agrobacterium-mediated transformation. Seven green transgenic plants, including one mixoploid, two haploid, and four diploid plants, were regenerated. PCR, Southern blot, FISH and blight resistance analysis all indicated that Xa21 gene has been integrated into the T0 plant genomes. T1 generations of the four diploid T0 plants were further investigated for resistance segregation. Chi2 test showed that two T1 populations segregated with a ratio of 3:1, indicating that a single copy of Xa21 gene was integrated into the genome, whereas the segregation ratios of the other two T1 populations were non-Mendelian. Therefore, the four diploid transgenic plants should be heterozygous diploids.

[Breeding transgenic plants with safe or no selective markers].

The bio-safety of selective markers in transgenic plants has been a hot spot in the field of plant genetic engineering. To solve the problem of selective markers in the transgenic plants, two means of producing transgenic plants have been developed. One is the utilization of bio-safe positive selective markers which are genes mainly related to metabolism of auxins and carbohydrates. The other is the establishment of transformation systems allowing marker genes to be eliminated from the transgenic plants, which include co-ransformation, double T-DNA border vectors, site-specific recombination and transposition. All these approaches of plant genetic engineering will benefit breeding transgenic plants with bio-safety.

[Analysis and mapping of a rice repeated sequence].

A repeated sequence with a length of 560 bp, termed as DH17, was obtained during PCR amplification of rice NBS-LRR homologues. A repeated unit of 352 bp in the DH17 fragment was revealed through sequence analysis and comparison, which has a high homology with the known sequences of OS48 and TrsA, and belongs to the same repeat family. Southern hybridization displayed that there are higher DH17 copies in the genome of an indica variety, ZYQ8,than that in the genome of japonica variety, JX17. The tandom repeated DH17 sequence was mapped on the long arm end of chromosome 12 through RFLP analysis of a double haploid population derived from ZYQ8 and JX17 using DH17 as a probe.

[Genome analysis of transgenic homozygous line "Minghui 63-Xa21"].

By using rice SSRP, RAPD and AFLP molecular markers, the genome of rice transgenic line "Minghui 63-Xa21" was analyzed. 32 SSRP primers, 42 RAPD primers and 8 AFLP primers could produce obvious PCR bands in the analysis of at least 12 individual plants selected randomly from "Minghui 63-Xa21" T3 generation. Totally 550 PCR bands, equivalent to 550 genomic sites, were detected. Different individual plants of the transgenic homozygous line displayed almost the same PCR pattern. Compared with the control "Minghui 63", no difference was found in their PCR patterns. This indicated that the introduction of Xa21 into the genome of "Minghui 63" did not change these 550 genome sites and their heredity. Very few variant PCR bands were observed in some individual plants from both "Minghui 63-Xa21" and "Minghui 63". However, the variant percentage was equivalent between the transgenic line and the non-transgenic control line.

[Genetic mapping of T-DNA integration sites in Xa21 transgenic rice].

The transformation mediated by Agrobacterium has been successfully applied to rice in recent years. In the previous research we have transferred the Xa21 gene into five rice varieties of China, using Agrobacterium-mediated trasformation. In this study, T-DNA flanking sequences of Xa21 transgenic rice lines were obtained by using thermal asymmetric interlaced PCR (TAIL-PCR). The flanking sequences which are actual rice DNA were identified and located on molecular linkage map developed from a ZYQ8/JX17 double haploid (DH) population. A total of 22 T-DNA flanking rice sequences were isolated. Nineteen of them displayed RFLPs between the two parents, ZYQ8 and JX17, and were mapped on the rice chromosomes, 3, 4, 7, 9, 10, 11 and 12, respectively. The genetic mapping of T-DNA integration sites in Xa21 transgenic rice will benefit the study of position effect and stable inheritance of the transgene Xa21.

[Specific molecular markers of the rust resistance gene M4 in flax].

Flax (Linum usitatissimum L.) is an important fiber and oil-producing crop. Flax rust, caused by Melampsora lini Ehrenb. Lev., occurs worldwide and can cause severe losses in seed yield and fiber quality. In order to identify molecular markers linked to the flax rust resistant gene M4, RAPD analysis of NM4, a near-isogenic line containing the M4 gene, and the recurrent parent Bison was carried out with 540 decamer primers. The primer OPA18 could stably amplify a specific fragment, OPA18(432), in the NM4 line. The OPA18(432) marker was testified to be closely linked to the M4 gene with a genetic distance of 2.1 cM through the analysis of the F2 mapping population derived from a cross of Bison x NM4. Based on the sequence of OPA18(432), the specific PCR primers were designed, and a SCAR marker for the M4 gene was produced. Amplification of different resistant materials proved that the maker is specific for the M4 gene. This marker has been used successfully in marker-assisted selection in the flax breeding program.