Molecular evidence of barley yellow dwarf virus replication/movement suppressed by the resistance gene Bdv2.

A wheat-Thinopyrum intermedium translocation line YW642 possesses the resistance to GAV serotype of barley yellow dwarf virus (BYDV), in which the resistance gene Bdv2 is derived from the chromosome 7X of Thinopyrum intermedium group 7. It is interesting to analyze BYDV accumulation content in the resistant and susceptible wheat plants for controlling BYDV disease and understanding the resistance mechanism against BYDV. In the paper, semi-quantitative reverse-transcription PCR (RT-PCR) was used to detect and quantify BYDV-GAV in the resistant and susceptible plants using specific primers for the coat protein (CP) and RNA-dependent RNA polymerase (RdRp) genes of BYDV-GAV serotype. On the inoculation site, the amount of the virus in the resistant wheat line (YW642) was much lower compared to the susceptible sib line (YW641). There was small amount of the virus could be detected in YW642 at 2-5 days post infestation (dpi), afterwards the amount of virus decreased and no virus could be detected at 14 and 16 dpi. In the uninoculated upper leaves, no BYDV was detected in YW642 from 1 to 14 dpi, while the virus could be detected at 3 dpi and then accumulated rapidly in YW641. These results showed at molecular level that the replication and/or movement of BYDV-GAV were strongly suppressed in YW642, presumably owing to the action of the BdV2 gene.

[Identification of the triploid hybrid chromosomes of Elymus canadensis L. x Hordeum brivisubulatum Link. by genomic in situ hybridization].

AThe intergeneric F1 hybrid between Elymus canadensis L. and Hordeum brivisubulatum Link. is a triploid (2n = 3x = 21 ), in which 7 chromosomes,that is equal to the base number of parent's chromosomes, were lost. In order to indentify the chromosome constitution of the triploid F1 hybrid of E. canadensis L. x H. brivisubulatum Link., the genomic in situ hybridization of F1 root tip cell chromosome DNA that H. brivisubulatum H1 H1 H2H2 genome DNA labeled with Biotin-16-dUTP was conducted using E. canadensis SSH(c)H(c) genome as blocking DNA. The result showed that the chromosomes of triploid F1 hybrid consisted of 7 chromosomes from E. canadensis S genome and 14 chromosomes from H. brivisubulatum H1 H2 genome while the 7 chromosomes of Hc genome of E. canadensis were lost.

[Construction, characterization and screening of a transformation-competent artificial chromosome (TAC) library of wheat-Thinopyrum intermedium translocation line with resistance to barley yellow dwarf virus].

A transformation-competent artificial chromosome (TAC) library was constructed from the genomic DNA of wheat-Th. intermidium translocation line HW642 that harbor the barley yellow dwarf virus (BYDV) resistance gene derived from Th. intermidium. The library consists of 2.3 x 10(6) clones with an average insert size of 22kb, representing approximately 2.5 haploid genome equivalents and is able to give a greater than 95.77% probability of isolating single-copy DNA sequences from this library. The library was stored as frozen cultures in 24 96-well formats, each well containing approximately 1000 different clones. TAC clones containing interest gene could be identified by the pooled PCR technique. A sequence characterized amplified region (SCAR) marker cosegregated with BYDV resistance gene, derived from a simple sequence repeat (SSR) or microsatellite marker wms37 of wheat, was applied to screen the TAC library. Twelve clones were successfully selected by the pooled PCR method. PCR products were identified by hybridizing with the SCAR marker band of Th. intermidium. Out of 12 clones, 10 positive clones restricted by Hind III were shown to hybridize with genomic DNA of Th. intermidium. These results showed evidences that the 10 clones could be used as candidate clones for isolation of BYDV resistance and its related genes, and the TAC library is a useful resource for isolating genes.

[Development of new PCR markers specific to a Thinopyrum intermedium chromosome 2Ai-2 and cloning of the St-specific sequences].

To meet the need of selecting translocation lines, some new RAPD markers for 2Ai-2 chromosome of Th. intermedium were identified in the paper. Out of 320 RAPD primers, 2 specific primers, OPO05 and OPM04, can amplify respectively a specific band with size of about 650 bp and 1400 bp in the BYDV resistant materials containing the chromosome 2Ai-2, including Th. intermedium, wheat-Th. intermedium partial amphipoild Zhong 4 Awnless, addition lines Z1, Z2 and Z6, F1(Z2/Wan7107) and substitute line ZD28 etc, but absent in all the materials lacking the 2Ai-2 chromosome, including susceptible wheat parents and other wheat-Th. intermedium addition lines L1 and Z4. The RAPD markers specific to chromosome 2Ai-2, OPO05(650) and OPM04(1400), may be located on the St genomic region of 2Ai-2 chromosome by PCR analysis on Th. intermedium (E1E2St), Pseuderogneria strigosa (St), Th. elongatum (E), Haynaldia villosa (V), Secale cereale (R), Hordeum vulgare (H), Aegilops squrrosa (D) and Triticum aestivum (ABD) genomic DNA. The specific bands of RAPD markers OPO05(650) and OPM04(1400) were isolated and cloned. After the clones were subjected to restrict digestion analysis, PCR and Southern hybridization analysis, some clones were sequenced. Based on the sequences, 1 pair of primers SC-O5(U + L) and 1 pair of primers SC-M4(U + L) were designed, synthesized and used to amplify the materials with and without 2Ai-2 chromosome. The results showed that the SCAR markers of chromosome 2Ai-2, SC-O5 and SC-M4, were converted successfully from the RAPD markers OPO05(650) and OPM04(1400). The Th. intermedium fragments amplified by the primers of SC-O5 (U + L) and SCM4(U + L) were cloned and analyzed. The results of Southern hybridization indicated that TiSCO5, the cloned fragment of Th. intermedium amplified by primers of SC-O5(U + L) was a low-copy sequence specific to St genome, and another sequence TiSCM4, the cloned fragment of Th. intermedium amplified by primers of SC-M4(U + L) was a repeat sequence specific to St genomic. The sequences will be new probes to detect St genomic chromatin.