Effects of Aegilops longissima chromosome 1Sl on wheat bread-making quality in two types of translocation lines.

KEY MESSAGE: Two wheat-Ae. longissima translocation chromosomes (1BS·1SlL and 1SlS·1BL) were transferred into three commercial wheat varieties, and the new advanced lines showed improved bread-making quality compared to their recurrent parents. Aegilops longissima chromosome 1Sl encodes specific types of gluten subunits that may positively affect wheat bread-making quality. The most effective method of introducing 1Sl chromosomal fragments containing the target genes into wheat is chromosome translocation. Here, a wheat-Ae. longissima 1BS·1SlL translocation line was developed using molecular marker-assisted chromosome engineering. Two types of translocation chromosomes developed in a previous study, 1BS·1SlL and 1SlS·1BL, were introduced into three commercial wheat varieties (Ningchun4, Ningchun50, and Westonia) via backcrossing with marker-assisted selection. Advanced translocation lines were confirmed through chromosome in situ hybridization and genotyping by target sequencing using the wheat 40 K system. Bread-making quality was found to be improved in the two types of advanced translocation lines compared to the corresponding recurrent parents. Furthermore, 1SlS·1BL translocation lines displayed better bread-making quality than 1BS·1SlL translocation lines in each genetic background. Further analysis revealed that high molecular weight glutenin subunit (HMW-GS) contents and expression levels of genes encoding low molecular weight glutenin subunits (LMW-GSs) were increased in 1SlS·1BL translocation lines. Gliadin and gluten-related transcription factors were also upregulated in the grains of the two types of advanced translocation lines compared to the recurrent parents. This study clarifies the impacts of specific glutenin subunits on bread-making quality and provides novel germplasm resources for further improvement of wheat quality through molecular breeding.

Development of wheat-Dasypyrum villosum T6V#4S·6AL translocation lines with enhanced inheritance for powdery mildew resistance.

KEY MESSAGE: New translocation lines with T6V#4S·6AL in the Ph1 and ph1b backgrounds were developed with improved inheritance of powdery mildew resistance. The wheat-Dasypyrum villosum T6V#4S·6DL translocation line Pm97033, which exhibits strong powdery mildew (PM) resistance, was developed many years ago, but has limited application in wheat breeding. One of the major reasons for this is that the translocation chromosome has low transmission rate, which makes it difficult to obtain ideal genotype through recombination with other elite agronomic traits in a limited segregating population. Further modifications are thus needed to make better use of this genetic resource. In this study, Pm97033 and the T6V#2S·6AL translocation line NY-W were hybridized with the CS ph1b mutant, and two F1 hybrids were hybridized with each other. Then, plants homozygous for the ph1b deletion carrying the alien chromosome arm(s) 6V#2S and 6V#4S were identified from the segregating populations using molecular markers. New T6V#4S·6AL and T6V#2-6V#4S·6AL translocations were identified by molecular markers and confirmed by genomic in situ hybridization (GISH). Individuals that were heterozygous or homozygous for the translocation chromosome in Ph1 and ph1b backgrounds were obtained. The ratio of PM resistance vs. susceptibility in the self-pollinated heterozygous plants was 3:1, and the phenotype was completely consistent with the KASP genotyping. Thus, the new translocation chromosomes had higher transmission rate than the original T6V#4S·6DL, and so can be effectively applied in breeding programs.

Impact of serum uric acid on subclinical myocardial injury in general population.

BACKGROUND AND AIMS: Hyperuricemia is widely thought as a risk factor for myocardial infarction (MI) and all-cause mortality; however, the relation of serum uric acid (sUA) and subclinical myocardial injury (SCeMI) remains unclear. We hypothesize that sUA is associated with subclinical myocardial injury. METHODS AND RESULTS: A total of 5880 adult individuals (57.9 ± 13.0 years, 54.23% women) without known cardiovascular disease from National Health and Nutrition Examination Survey (NHANES) III were included. Determined by Cardiac Infarction Injury Score (CIIS) from 12-lead electrocardiogram, SCeMI was defined by CIIS ≥10 units. The relationship between sUA and SCeMI was analyzed by using logistic regression models and the smooth curve fitting. Subgroup analyses were conducted. After adjusting for potential confounding variables, the smooth curve fitting revealed a non-linear relationship between sUA level and SCeMI. When sUA was above the inflection point 266.5 µmol/L, each 100 unit increase in sUA increase the risk of SCeMI by 15%. In women group, when sUA>340.3 µmol/L, each 100 unit increase in sUA increase the risk of SCeMI by 71%, but no significant correlation was observed in men group. CONCLUSIONS: Our findings confirm that sUA is an independent risk factor for subclinical myocardial injury after adjusting for potential confounding variables, and existence of such an association in women only, which require more random control trials to confirm the strategy of cardiovascular disease prevention based on sUA reduction in female.

High uric acid promotes mitophagy through the ROS/CaMKIIδ/Parkin pathway in cardiomyocytes in vitro and in vivo.

BACKGROUND: Increasing evidence has suggested that high uric acid (HUA) is closely related to cardiovascular disease (CVD). Mitophagy abnormalities have been reported to participate in multiple pathogenic processes of CVD. However, the potential molecular mechanisms remain unclear. Herein, we investigated the effect of HUA-induced mitophagy and its potential molecular mechanism in cardiomyocytes. METHODS: We established a model of cardiomyocytes induced by HUA in vitro and in vivo. Mitochondrial membrane potential (MMP), reactive oxygen species (ROS) production and adenosine triphosphate (ATP) content were measured. The mitophagy-related protein expression of LC3B-II, Parkin, Ca2+/calmodulin-dependent protein kinase II δ (CaMKIIδ) and P62 was measured by Western blot. Based on the colocalization of lysosomes and mitochondria, a confocal microscope was used to detect mitophagy. Additionally, we established a mitophagy inhibitor group (3-MA) and CaMKIIδ inhibitor group (KN-93) to verify the pathway. RESULTS: In the HUA stimulation model, ROS production was increased, and mitochondrial injury indexes (MMP and ATP contents) were decreased. Moreover, these indicators were reversed by 3-MA and KN-93. Under HUA stimulation, the expression of LC3B-II, Parkin, CaMKIIδ and P62 increased significantly. Furthermore, these protein levels were reduced by 3-MA and KN-93. CONCLUSION: HUA can promote cardiomyocyte mitophagy activation through the ROS/CaMKIIδ/parkin pathway axis. This study may provide a new target and theoretical basis for the prevention and treatment of HUA-related metabolic heart disease in the future.

Screening and functional characterization of candidate resistance genes to powdery mildew from Dasypyrum villosum#4 in a wheat line Pm97033.

KEY MESSAGE: Three genes designated DvLox, Pm21#4, and Pm21#4-H identified in a wheat-Dasypyrum villosum#4 T6V#4S·6DL translocation line Pm97033 conferred wheat for powdery mildew resistance. Powdery mildew (PM) caused by Blumeria graminis f. sp. tritici (Bgt) is one of the most devastating diseases in wheat. To date, only a few genes conferring resistance to wheat PM are cloned. Dasypyrum villosum is a wild relative of wheat, which provides Pm21 conferring wheat immunity to PM. In this study, we obtained many differentially expressed genes (DEGs) from a wheat-D. villosum#4 T6V#4S·6DL translocation line Pm97033 using RNA-sequencing. Among them, 7 DEGs associated with pathogen resistance were up-regulated in front of Bgt infection. Virus-induced gene silencing and transformation assays demonstrated that two of them, DvLox and Pm21#4 encoding a lipoxygenase and a encoding coiled-coil/nucleotide-binding site/leucine-rich repeat resistance protein, conferred wheat PM resistance. The transgenic wheat plants expressing DvLox enhanced PM resistance, and the transgenic wheat plants expressing Pm21#4 showed PM immunity. The Pm21#4-silenced Pm97033 plants by the cluster regularly interspaced short palindromic repeats-associated endonuclease (CRISPR/Cas9) system were susceptible to PM. Thus, Pm21#4 is a key gene contributing PM immune resistance in Pm97033. Constitutively expression of Pm21#4-H, which is silenced in Pm97033 and D. villosum#4, endowed a PM-susceptible wheat variety Fielder with PM immune resistance.

Efficient induction of haploid plants in wheat by editing of TaMTL using an optimized Agrobacterium-mediated CRISPR system.

The use of CRISPR/LbCpf1 and CRISPR/xCas9 systems in wheat have not yet been reported. In this study, we compared the efficiencies of three CRISPR editing systems (SpCas9, LbCpf1, and xCas9), and three different promoters (OsU6a, TaU3, and TaU6) that drive single-guide (sg)RNA, which were introduced into wheat via Agrobacterium-mediated transformation. The results indicated that TaU3 was a better choice than OsU6a or TaU6. The editing efficiency was higher using two sgRNAs than one sgRNA, and mutants with a large fragment deletion between the two sgRNAs were produced. The LbCpf1 and xCas9 systems could both be used successfully. Two endogenous genes, TaWaxy and TaMTL, were edited with high efficiency by the optimized SpCas9 system, with the highest efficiency (80.5%) being achieved when using TaU3 and two sgRNAs to target TaWaxy. Rates of seed set in the TaMTL-edited T0 transgenic plants were much lower than that of the wild-type. A haploid induction rate of 18.9% was found in the TaMTL-edited T1 plants using the CRISPR/SpCas9 system. Mutants with reverse insertion of the deleted sequences of TaMTL and TaWaxy between the two sgRNAs were identified in the edited T0 plants. In addition, wheat grains lacking embryos or endosperms were observed in the TaMTL-edited T1 generation.

Pairing and Exchanging between Daypyrum villosum Chromosomes 6V#2 and 6V#4 in the Hybrids of Two Different Wheat Alien Substitution Lines.

Normal pairing and exchanging is an important basis to evaluate the genetic relationship between homologous chromosomes in a wheat background. The pairing behavior between 6V#2 and 6V#4, two chromosomes from different Dasypyrum villosum accessions, is still not clear. In this study, two wheat alien substitution lines, 6V#2 (6A) and 6V#4 (6D), were crossed to obtain the F1 hybrids and F2 segregating populations, and the testcross populations were obtained by using the F1 as a parent crossed with wheat variety Wan7107. The chromosomal behavior at meiosis in pollen mother cells (PMCs) of the F1 hybrids was observed using a genomic in situ hybridization (GISH) technique. Exchange events of two alien chromosomes were investigated in the F2 populations using nine polymerase chain reaction (PCR) markers located on the 6V short arm. The results showed that the two alien chromosomes could pair with each other to form ring- or rod-shaped bivalent chromosomes in 79.76% of the total PMCs, and most were pulled to two poles evenly at anaphase I. Investigation of the F2 populations showed that the segregation ratios of seven markers were consistent with the theoretical values 3:1 or 1:2:1, and recombinants among markers were detected. A genetic linkage map of nine PCR markers for 6VS was accordingly constructed based on the exchange frequencies and compared with the physical maps of wheat and barley based on homologous sequences of the markers, which showed that conservation of sequence order compared to 6V was 6H and 6B > 6A > 6D. In the testcross populations with 482 plants, seven showed susceptibility to powdery mildew (PM) and lacked amplification of alien chromosomal bands. Six other plants had amplification of specific bands of both the alien chromosomes at multiple sites, which suggested that the alien chromosomes had abnormal separation behavior in about 1.5% of the PMCs in F1, which resulted in some gametes containing two alien chromosomes. In addition, three new types of chromosome substitution were developed. This study lays a foundation for alien allelism tests and further assessment of the genetic relationship among 6V#2, 6V#4, and their wheat homoeologous chromosomes.

Development of PCR markers specific to Dasypyrum villosum genome based on transcriptome data and their application in breeding Triticum aestivum-D. villosum#4 alien chromosome lines.

BACKGROUND: Dasypyrum villosum is an important wild species of wheat (Triticum aestivum L.) and harbors many desirable genes that can be used to improve various traits of wheat. Compared with other D. villosum accessions, D. villosum#4 still remains less studied. In particular, chromosomes of D. villosum#4 except 6V#4 have not been introduced into wheat by addition or substitution and translocation, which is an essential step to identify and apply the alien desired genes. RNA-seq technology can generate large amounts of transcriptome sequences and accelerate the development of chromosome-specific molecular markers and assisted selection of alien chromosome line. RESULTS: We obtained the transcriptome of D. villosum#4 via a high-throughput sequencing technique, and then developed 76 markers specific to each chromosome arm of D. villosum#4 based on the bioinformatic analysis of the transcriptome data. The D. villosum#4 sequences containing the specific DNA markers were expected to be involved in different genes, among which most had functions in metabolic processes. Consequently, we mapped these newly developed molecular markers to the homologous chromosome of barley and obtained the chromosome localization of these markers on barley genome. Then we analyzed the collinearity of these markers among D. villosum, wheat, and barley. In succession, we identified six types of T. aestivum-D. villosum#4 alien chromosome lines which had one or more than one D. villosum#4 chromosome in the cross and backcross BC3F5 populations between T. durum-D. villosum#4 amphidiploid TH3 and wheat cv. Wan7107 by employing the selected specific markers, some of which were further confirmed to be translocation or addition lines by genomic in situ hybridization (GISH). CONCLUSION: Seventy-six PCR markers specific to chromosomes of D. villosum#4 based on transcriptome data were developed in the current study and their collinearity among D. villosum, wheat, and barley were carried out. Six types of Triticum aestivum-D. villosum#4 alien chromosome lines were identified by using 12 developed markers and some of which were further confirmed by GISH. These novel T. aestivum-D. villosum#4 chromosome lines have great potential to be used for the introduction of desirable genes from D. villosum#4 into wheat by chromosomal translocation to breed new wheat varieties.

Development of a set of PCR markers specific to Aegilops longissima chromosome arms and application in breeding a translocation line.

KEY MESSAGE: Transcriptome data were used to develop 134 Aegilops longissima specific PCR markers and their comparative maps were constructed by contrasting with the homologous genes in the wheat B genome. Three wheat- Ae. longissima 1BL·1S l S translocation lines were identified using the correspondence markers. Aegilops longissima is an important wild species of common wheat that harbors many genes that can be used to improve various traits of common wheat (Triticum aestivum L.). To efficiently transfer the traits conferred by these Ae. longissima genes into wheat, we sequenced the whole expression transcript of Ae. longissima. Using the transcriptome data, we developed 134 specific polymerase chain reaction markers located on the 14 chromosome arms of Ae. longissima. These novel molecular markers were assigned to specific chromosome locations based on a comparison with the homologous genes in the B genome of wheat. Annotation of these genes showed that most had functions related to metabolic processes, hydrolase activity, or catalytic activity. Additionally, we used these markers to identify three wheat-Ae. longissima 1BL·1SlS translocation lines in somatic variation populations resulting from a cross between wheat cultivar Westonia and a wheat-Ae. longissima substitution line 1Sl(1B). The translocation lines had several low molecular weight glutenin subunits encoding genes beneficial to flour processing quality that came from Ae. longissima 1SlS. The three translocation lines were also confirmed by genomic in situ hybridization. These translocation lines will be further evaluated for potential quality improvement of bread-making properties of wheat.

Development and comparative genomic mapping of Dasypyrum villosum 6V#4S-specific PCR markers using transcriptome data.

KEY MESSAGE: Twenty-five Dasypyrum villosum 6V#4S-specific PCR markers were developed using transcriptome data and further assigned to comparative genomic maps of wheat chromosome 6A, 6B, and 6D and barley chromosome 6H contrasting their homologous genes in these genomes. Two Dasypyrum villosum accessions, D.v#2 and No. 1026 from England and Russia, respectively, contain Pm21 on chromosome 6V#2S and PmV on chromosome 6V#4S. Both genes confer high resistance to powdery mildew (PM) in wheat. Even though several molecular markers have been developed to detect Pm21 and PmV, only the MBH1 marker can simultaneously detect both Pm21 and PmV. In this study, we first used a high-throughput sequencing technique to obtain the transcriptome sequences of a wheat-D. villosum translocation line, Pm97033-which contains chromosome 6V#4S carrying the PmV locus, under wheat PM pathogen induction. Twenty-five 6V#4S chromosome-specific markers were developed. Three of them were able to clearly distinguish chromosomes 6V#4S and 6V#2S by product size, four amplified the product specific for chromosome 6V#4S only, and the remaining 18 markers identified chromosome 6VS in wheat backgrounds. Two different D. villosum accessions, their derived translocation lines and wheat varieties carrying different chromosome 6VS were identified using these specific markers. The 25 newly developed markers together with the known PM resistance gene Stpk-V were used to construct comparative genomic maps with the homoeologous chromosome arms of wheat and barley. The colinearity of the identified gene sequences amplified by the 25 markers among wheat chromosomes 6A, 6B, and 6D and barley chromosome 6H was not very conserved and interrupted frequently by inversion and insertion. Our markers have potential in marker assisted selection for PM resistance breeding, and for locating other potential important genes and cloning the PmV gene on chromosome 6V#4S.

Cloning and characterization of TaVIP2 gene from Triticum aestivum and functional analysis in Nicotiana tabacum.

Wheat is recalcitrant to genetic transformation. A potential solution is to manipulate the expression of some host proteins involved in T-DNA integration process. VirE2 interacting protein 2 (VIP2) plays an important role in T-DNA transport and integration. In this study, a TaVIP2 gene was cloned from common wheat. Southern blot and allele-specific polymerase chain reaction (AS-PCR) combined with an online chromosomal location software tool revealed that three TaVIP2 genes were located on wheat chromosomes 1AL, 1BL, and 1DL. These three homoeoallelic TaVIP2 genes all contained 13 exons and 12 introns, and their coding sequences were the same; there were a few single nucleotide polymorphisms (SNPs) among the three genes. The heterologous expression of the TaVIP2 gene in tobacco led to enhancement of the Agrobacterium-mediated transformation efficiency up to 2.5-fold. Transgenic tobacco plants expressing TaVIP2 showed enhanced resistance to powdery mildew. Further quantitative real-time PCR (qRT-PCR) revealed that overexpression of TaVIP2 in transgenic tobacco up-regulated the expression of an endogenous gene, NtPR-1, which likely contributed to powdery mildew resistance in transgenic tobacco. Our study indicates that the TaVIP2 gene may be highly useful in efforts to improve Agrobacterium-mediated transformation efficiency and to enhance powdery mildew resistance in wheat.

Production and identification of haploid dwarf male sterile wheat plants induced by corn inducer.

BACKGROUND: Using the cross of wheat and maize is a very useful way to produce wheat haploid plants by chromosome elimination. Dwarf male sterile wheat (DMSW) and corn inducer are potential important germplasm for wheat breeding by recurrent selection and doubled haploid strategies. There is no report yet to achieve the haploid plants from DMSW induced by maize inbred line and especially the corn inducer. RESULTS: Haploid plants of DMSW were successfully obtained in this study induced by both maize pollens of inducer line and normal inbred line. The efficiencies for wheat embryos formation and plantlets production induced by the two corn lines had no significant difference. All the eleven haploid wheat plants derived from the male sterile material were identified by botanic appearance, cytology, cytogenetics, and molecular markers. They were all haploid based on their guard cell length of 42.78-42.90 µm compared with the diploid control of 71.52 µm, and their chromosome number of 21 compared with the diploid control of 42. In addition, according to anthers, plant height, and molecular markers, the haploid plants were divided into two types. Eight of them showed dwarf, having no anthers, and the special band of Rht10, and the other three plants displayed normal plant height, having anthers, and not containing the special band of Rht10, indicating that they were originated from the MS2/Rht10 and ms2/rht10 female gametes, respectively. CONCLUSIONS: MS2/Rht10 haploid plants were successfully obtained in this study by using corn inducer and inbred line, and will be employed as candidate materials for the potential cloning of MS2 dominant male gene.

Global analysis of differentially expressed genes and proteins in the wheat callus infected by Agrobacterium tumefaciens.

Agrobacterium-mediated plant transformation is an extremely complex and evolved process involving genetic determinants of both the bacteria and the host plant cells. However, the mechanism of the determinants remains obscure, especially in some cereal crops such as wheat, which is recalcitrant for Agrobacterium-mediated transformation. In this study, differentially expressed genes (DEGs) and differentially expressed proteins (DEPs) were analyzed in wheat callus cells co-cultured with Agrobacterium by using RNA sequencing (RNA-seq) and two-dimensional electrophoresis (2-DE) in conjunction with mass spectrometry (MS). A set of 4,889 DEGs and 90 DEPs were identified, respectively. Most of them are related to metabolism, chromatin assembly or disassembly and immune defense. After comparative analysis, 24 of the 90 DEPs were detected in RNA-seq and proteomics datasets simultaneously. In addition, real-time RT-PCR experiments were performed to check the differential expression of the 24 genes, and the results were consistent with the RNA-seq data. According to gene ontology (GO) analysis, we found that a big part of these differentially expressed genes were related to the process of stress or immunity response. Several putative determinants and candidate effectors responsive to Agrobacterium mediated transformation of wheat cells were discussed. We speculate that some of these genes are possibly related to Agrobacterium infection. Our results will help to understand the interaction between Agrobacterium and host cells, and may facilitate developing efficient transformation strategies in cereal crops.

Synthesis, characterization, and DNA binding of a novel ligand and its Cu (II) complex.

A novel naphthalene-2,3-diamine-2-salicylaldehyde (NS) ligand and its mononuclear copper(II) complex (CuNS) have been synthesized and structurally characterized. The UV­vis absorption and emission spectra of NS showed obvious changes on addition of Cu2+ solution. The interaction of the compounds with calf thymus DNA and G-quadruplex DNA were investigated by spectroscopic methods and thermal melting assay. The nucleolytic cleavage activity of the compounds was investigated on double-stranded circular pBR322 plasmid DNA and G-quadruplex DNA by electrophoretic mobility shift assay. The results show that CuNS has a greater ability to stabilize G-quadruplex DNA over calf-thymus DNA. The cytotoxicity of the compounds toward HpeG2 cancer cells was also studied, and they showed significant potential for antineoplastic effects.

Combinational transformation of three wheat genes encoding fructan biosynthesis enzymes confers increased fructan content and tolerance to abiotic stresses in tobacco.

KEY MESSAGE: Seven kinds of transgenic tobacco plants transformed with combinations of three FBE genes were obtained. The transgenic plants transformed with Ta1-SST + Ta6-SFT genes appeared to have the highest fructan or soluble sugar content and the strongest salt tolerance. Fructan is thought to be one of the important regulators involved in plant tolerance to various abiotic stresses. In this study, wheat-derived genes, Ta1-SST, Ta6-SFT, and Ta1-FFT, encoding fructan biosynthesis enzymes (FBE) were isolated and cloned into vectors modified pBI121 or pZP211. Seven different combinations of the three target genes were transformed into tobacco plants through an Agrobacterium-mediated approach, and transgenic tobacco plants were identified by PCR, ELISA, and Southern blotting. Compared with tobacco plants transformed with other six combinations of the three target genes and with wild-type plants, the transgenic plants transformed with Ta1-SST + Ta6-SFT genes contained the highest fructan and soluble sugar content. All seven types of transgenic tobacco plants displayed a much higher level of tolerance to drought, low temperature, and high salinity compared with the wild type. Differences of drought and low temperature tolerance between the transgenic plants containing a single FBE gene and those harboring two or three FBE genes were not significant, but the salt tolerance level of the transgenic plants with different FBE gene combinations from high to low was: Ta1-SST + Ta6-SFT > Ta1-SST + Ta6-SFT + Ta1-FFT > Ta1-SST + Ta1-FFT > Ta1-SFT + Ta1-FFT > single FBE gene. These results indicated that the tolerances of the transgenic tobacco plants to various abiotic stresses were associated with the transformed target gene combinations and the contents of fructan and soluble sugar contained in the transgenic plants.

Research progress in BYDV resistance genes derived from wheat and its wild relatives.

Barley yellow dwarf virus (BYDV) may cause a serious disease affecting wheat worldwide. True resistance to BYDV is not naturally found in wheat. BYDV resistance genes are found in more than 10 wild relative species belonging to the genera of Thinopyrum, Agropyron, Elymus, Leymus, Roegneria, and Psathyrostachy. Through wide crosses combining with cell culture, use of ph mutants, or irradiation, 3 BYDV resistance genes in Th. intermedium, including Bdv2, Bdv3 and Bdv4, were introgressed into common wheat background. Various wheat-Th. intermedium addition and substitution, translocation lines with BYDV-resistance were developed and characterized, such as 7D-7Ai#1 (bearing Bdv2), 7B-7Ai#1, 7D-7E (bearing Bdv3), and 2D-2Ai-2 (bearing Bdv4) translocations. Three wheat varieties with BYDV resistance from Th. intermedium were developed and released in Australia and China, respectively. In addition, wheat-Agropyron cristatum translocation lines, wheat-Ag. pulcherrimum addition and substitution lines, and a wheat-Leymus multicaulis addition line (line24) with different resistance genes were developed. Cytological analysis, morphological markers, biochemical markers, and molecular markers associated with the alien chromatin carrying BYDV resistance genes were identified and applied to determine the presence of alien, chromosomes or segments, size of alien chromosome segments, and compositions of the alien chromosomes. Furthermore, some resistance-related genes, such as RGA, P450, HSP70, protein kinases, centrin, and transducin, were identified, which expressed specifically in the resistance translocation lines with Bdv2. These studies lay the foundations for developing resistant wheat cultivars and unraveling the resistance mechanism against BYDV.

[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.