Microdissection of Haynaldia villosa Telosome 6VS and Cloning of Species-specific DNA Sequences

The material T240_6 derived from SC 2 young embryo of the combination CA9211/RW15 (6D/6V alien substitution) was telosomic substitution line of 6VS identified by GISH (genomic in situ hybridization) analysis. The 6VS was microdissected with a needle and transferred into a 0.5 mL Ep tube. In the 'single tube', all the subsequence steps were conducted. After two round of LA (Linker adaptor)_PCR amplification, the size of PCR bands ranged from 100 to 3 000 bp, with predominate bands 600-1 500 bp. The products were confirmed by Southern blotting analysis using Haynaldia villosa (L.) Schur. genomic DNA labeled with 32 P as probe. The PCR products were purified and ligated into clone vector-pGEM_T easy vector. Then, the plasmids were transformed into competence E. coli JM109 with cool CaCl 2. It was estimated that there were more than 17 000 white clones in the library. The size of insert fragments distributed from 100-1 500 bp, with average of 600 bp. Using H. villosa genomic DNA as probe, dot blotting results showed that 37% clones displayed strong and medium positive signals, and 63% clones had faint or no signals. It is demonstrated that there were about 37% repeat sequence clones and 67% single/unique sequence clones in the library. Eight H. villosa_specific clones were screened from the library, and two clones pHVMK22 and pHVMK134 were used for RFLP analysis and sequencing. Both of them were H. villosa specific clones. The pHVMK22 was a unique sequence clone, and the pHVMK134 was a repeat sequence clone. When the pHVMK22 was used as a probe for Southern hybridization, all the powdery mildew resistance materials showed a special band of 2 kb, while all the susceptible ones not. The pHVMK22 may be applied to detect the existence of Pm21.

Establishment of 6VS Telocentric Lines of Haynaldia villosa Resistant to Powdery Mildew Induced by Immature Embryo Culture

The line of T240-6 was selected from 32 SC 2 lines of immature embryo culture of T240 (common wheat (Triticum aestivum L.)× Wheat-Haynaldia villosa (L.) Schur. 6D/6V substitution line) through powdery mildew characterization, glutamate oxaloacetate transaminase (GOT) enzyme and gliadin (Gli) analyses and in situ hybridization. All of the individual plants resistant to powdery mildew lacked the locus of GOT at 6VL arm (GOT-V 2) and had gliadin locus at 6VS arm (Gli-V 2) of Haynaldia villosa. All the plants resistant to powdery mildew had one or two telocentric chromosomes that did not pair with wheat chromosomes but paired between themselves. T240-6 was identified as a telocentric line through in situ hybridization.

Production of Somatic Hybrid Plants Between Two Types of Wheat Protoplasts and the Protoplasts of Haynaldia villosa

小麦 (TriticumaestivumL .)济南 177的两种原生质体 ,一种来自快速生长的悬浮细胞 ,它们因长期继代而丧失分化能力 ,其染色体只有 2n =2 4～ 2 8;另一种来自可以再生的愈伤组织 ,其原生质体不能持续分裂。它们中任一种与UV照射过的簇毛麦原生质体融合均不能再生植株。然而当它们混合在一起作为受体时 ,能够获得再生绿色植株。细胞核基因和胞质基因的分析证明这些绿色植株是杂种。以上事实说明这两种原生质体在融合时存在某些互补的关系 ,讨论了这种融合方式的可能作用及重要性。

Identification of Triticum aestivum-Haynaldia villosa Translocation Line T6BS·6BL-2VS

[Objective] The aim of experiment was to provide a new germplasm for wheat breeding by further using desirable genes in 2V chromosome of Haynaldia villosa.[Method] Through hybridization between common wheat(Triticum aestivum)-Haynaldia villosa disomic substitution line and common wheat Nonglin26-3C chromosome of Aegilops triuncialis disomic addition line,the analysis methods such as chromosome C-banding,genomic in situ hybridization and molecular marker technique were comprehensively applied and combined characters investigation.[Result] The wheat-Haynaldia villosa translocation line(T6BS·6BL-2VS)was selected from hybrid progenies to conduct characters investigation,which found some bristles on glume ridge of T6BS·6BL-2VS.[Conclusion] The translocation line induced by gametocidal chromosome was a small segment translocation line and the gene of bristle on glume ridge of Haynaldia villosa was located between the middle and the terminal of 2VS.

Development of EST-PCR Markers for the Chromosome 4V of Haynaldia villosa and Their Application in Identification of 4V Chromosome Structural Aberrants

EST-PCR based molecular markers specific for alien chromosomes are not only useful for the detection of the introgressed alien chromatin in the wheat background, but also provide evidence of the syntenic relationship between homoeologous chromosomes. In the present study, in order to develop high density and evenly distributed molecular markers on chromosome 4V of Haynaldia villosa, a total of 607 primer pairs were designed according to the EST sequences, which were previously located in 23 different bins of wheat chromosomes 4A, 4B and 4D. By using the Triticum durum-H, villosa amphiploid and T. aestivum-H, villosa alien chromosome lines involving chromosome 4V, it was found that 9.23% of the tested primers could amplify specific bands for chromosome 4V. Thirty and twenty-six specific markers could be assigned to chromosome arms 4VS and 4VL, respectively. These 4V specific markers provided efficient tools for the characterization of structural variation involving the chromosome 4V as well as for the selection of useful genes located on chromosome 4V in breeding programs.

Genetic Analysis and Molecular Mapping of an All-Stage Stripe Rust Resistance Gene in Triticum aestivum-Haynaldia villosa Translocation Line V3

Triticum aestivum-Hayaldia villosa translocation line V3 has shown effective all-stage resistance to the seven dominant pathotypes of Puccinia striiforms f.sp.tritici prevalent in China.To elucidate the genetic basis of the resistance,the segregating populations were developed from the cross between V3 and susceptible genotype Mingxian 169,seedlings of the parents and F 2 progeny were tested with six prevalent pathotypes,including CYR29,CYR31,CYR32-6,CYR33,Sun11-4,and Sun11-11,F 1 plants and F 3 lines were also inoculated with Sun11-11 to confirm the result further.The genetic studied results showed that the resistance of V3 against CYR29 was conferred by two dominant genes,independently,one dominant gene and one recessive gene conferring independently or a single dominant gene to confer resistance to CYR31,two complementary dominant genes conferring resistance to both CYR32-6 and Sun11-4,two independently dominant genes or three dominant genes（two of the genes show cumulative effect） conferring resistance to CYR33,a single dominant gene for resistance to Sun11-11.Resistance gene analog polymorphism（RGAP） and simple-sequence repeat（SSR） techniques were used to identify molecular markers linked to the single dominant gene（temporarily designated as YrV3） for resistance to Sun11-11.A linkage map of 2 RGAP and 7 SSR markers was constructed for the dominant gene using data from 221 F 2 plants and their derived F 2：3 lines tested with Sun11-11 in the greenhouse.Amplification of the complete set of nulli-tetrasomic lines of Chinese Spring with a RGAP marker RG1 mapped the gene on the chromosome 1B,and then the linked 7 SSR markers located this gene on the long arm of chromosome 1B.The linkage map spanned a genetic distance of 25.0 cM,the SSR markers Xgwm124 and Xcfa2147 closely linked to YrV3 with genetic distances of 3.0 and 3.8 cM,respectively.Based on the linkage map,it concluded that the resistance gene YrV3 was located on chromosome arm 1BL.Given chromosomal location,the reaction patterns and pedigree analysis,YrV3 should be a novel gene for resistance to stripe rust in wheat.These closely linked markers should be useful in stacking genes from different sources for wheat breeding and diversification of resistance genes against stripe rust.

Development of oligonucleotide probes for FISH karyotyping in Haynaldia villosa,a wild relative of common wheat

Haynaldia villosa is a wild relative of wheat and a valuable gene resource for wheat improvement.Owing to the limited number of probes available for fluorescence in situ hybridization(FISH),the resolution at which the karyotype of H.villosa can be characterized is poor,hampering accurate characterization of small segmental alien introgressions.We designed ten oligonucleotide probes using tandem repeats in DNA sequences derived from the short arm of H.villosa chromosome 6 V(6 VS).FISH with seven of them resulted in clear signals on H.villosa chromosomes.Using these,we constructed FISH karyotypes for H.villosa using oligo-6 VS-1 and oligo-6 VS-35 oligonucleotides and characterized the distribution of the two probes in five different H.villosa accessions.The new FISH probes can efficiently characterize H.villosa introgressions into wheat.

Heterologous expression of the Haynaldia villosa pattern-recognition receptor CERK1-V in wheat increases resistance to three fungal diseases

Wheat production is under continuous threat by various fungal pathogens.Identification of multipledisease resistance genes may lead to effective disease control via the development of cultivars with broad-spectrum resistance.Plant Lysin-motif(LysM)-type pattern-recognition receptors,which elicit innate immunity by recognizing fungal pathogen associated molecular patterns such as chitin,are potential candidates for such resistance.In this study,we cloned a LysM receptor-like kinase gene,CERK1-V,from the diploid wheat relative Haynaldia villosa.CERK1-V expression was induced by chitin and Blumeria graminis f.sp.tritici,the causal agent of wheat powdery mildew.Heterologous overexpression of CERK1-V in wheat inhibited the development of three fungal pathogens,thereby increased resistance to powdery mildew,yellow rust,and Fusarium head blight.CERK1-V physically interacted with the wheat Lys M protein Ta CEBi Ps.CERK1-V/Ta CEBi Ps interaction promoted chitin recognition and activated chitin signal transduction in wheat.Transgenic plants with excessively high CERK1-V expression showed high resistance but abnormal plant growth,whereas plants with moderate expression level showed adequate resistance level with no marked impairment of plant growth.In transgenic lines,RNA-seq showed that gene expression involved in plant innate immunity was activated.Expression of genes involved in photosynthesis,ER stress and multiple phytohormone pathways was also activated.Optimized expression of CERK1-V in wheat can confer disease resistance without compromising growth or defense fitness.

The Meiotic Behavior of an Alien Chromosome in Triticum aestivum-Haynaldia villosa Monosomic Addition Lines

By the combination of cytological analysis and using genomic in situ hybridization technique to identify an alien chromosome in wheat-Haynaldia villosa monosomic addition lines, we studied the meiotic behavior of the alien chromosome. The results indicated that the frequency of bivalent pairing was lower than the value expected in PMCs of two monosomic addition lines, the frequency of wheat chromosomes unpairing increased, and the wheat homologous chromosome pairing was interfered with by the added chromosome 6V at metaphase I. The chromosome 6V lagged in 20.3% -29.3% of PMCs, sister chromatids 6V early divided in 29.0% - 34.1% of PMCs, the single chromosome 6V in 18.2% - 26.1% of PMCs went to a pole randomly, the breakage frequency of chromosome 6V was 1.2% - 2.9%. Meanwhile, it was also found that several wheat chromosomes showed earlier division, lagging and breakage in a few PMCs. It revealed that the added chromosome 6V influenced the behavior of wheat chromosomes at anaphase. It was also found that the translo-cation was produced between 6V and wheat chromosomes in 1.2% of PMCs. It offered evidence for transloca-tion between wheat and Haynaldia villosa 6V chromosomes.

Structural Changes of 2V Chromosome of Haynaldia villosa Induced by Gametocidal Chromosome 3C of Aegilops triuncialis

Haynaldia villosa （2n=2X= 14, VV）, a relative of wheat, plays important roles in wheat improvement mainly owing to its disease resistance. Powdery mildew resistance gene Pm21 has been successfully transferred into wheat by Cytogenetic Institute, Nanjing Agricultural University, China, and is widely used in the current wheat breeding programs. In this research, our objective is to further transfer and utilize the beneficial genes such as eye-spot resistance, yellow rust resistance, and gene of the tufted bristles on the glume ridge （a remarkable morphology） mapped on 2V of Haynaldia villosa. A disomic addition line with gametocidal chromosome 3C ofAegilops triuncialis added in Norin-26 was crossed to the wheat-H, villosa disomic substitution 2V（2D） and the hybrid F1 was then self-crossed. Chromosome C-banding, genomic in situ hybridization （GISH）, and meiotic analysis in combination with molecular markers were applied to detect the chromosome variations derived from hybrids Fz and F3. To date, four translocations including one small segmental translocation T6BS·6BL-2VS, two whole arm translocations （preliminarily designed as T3DS·2VL and T2VS.7DL） and one intercalary translocation T2VS·2VL-W-2VL, one deletion Del. 2VS·2VL-, one monotelosomic Mt2VS, and one isochromosome 2VS·2VS line have been developed and characterized. One wheat SSR marker Xwmc25.120 tagging 2VS and one wheat STS marker NAU/STSBCD135-1 （2BL） tagging 2VL were successfully used to confirm the alien chromosome segments involved in the seven lines. The tufted bristles on the glume ridge appeared in lines T2VS-7DL, Mt2VS, 2VS-2VS as well as the parent DS2V（2D）, whereas in T3DS·2VL, this trait did not appear. The gene controlling the tufted bristles was located on 2VS. Gametocidal chromosome 3C ofAegilops triuncialis could successfully induce chromosome 2V structural changes.

Molecular Characterization of a Triticum durum-Haynaldia villosa Amphiploid and Its Derivatives for Resistance to Gaeumannomyces graminis var. tritici

Take-all is a serious disease found in wheat across the world. Haynaldia villosa is considered to be resistant to take-all at a high level. TH3 was an amphiploid （2n =42, AABBVV） between Triticum durum and Haynaldia viUosa with significant resistance to take-all fungus isolated from China. In greenhouse experiment, the derivatives of the hybrid between wheat and TH3 showed better resistance to take-all than that of the wheat control. One of the derivatives named HW918-5 was selected for further analysis. Cytological and genomic in situ hybridization （GISH） analysis indicated that a monotelosome originated from H. villosa existed in the genome of the offspring of the line HW918-5. The monotelosome with promising resistant gene for take-all was located on the 3V chromosome of H. villosa in the further PCR-based molecular analysis.

Cloning and Characterization of a Family of Disease Resistance Gene Analogs from 6VS of Haynaldia villosa

In the present study, microdissection of 6VS and the cloning of the resistance gene analogs(RGA)from them were reported. The 6VS were microdissected with needle and 10 types of resistance gene analogs were obtained by PCR with degenerate oligonucleotide primer designed according to resistance genes. They were designated as Hvrgak1-Hvrgak10, GenBank accession numbers are AF387113-AF387121, AY040671- AY040672. Identity among RGAs was about 10-50%, and identity with cloned R gene from plants was 5-20%. Southern hybridization analysis results showed 3 RGAs, Hvrgak2, Hvrgak4, and Hvr-gak5 were linked with wheat powdery mildew resistance. These RGAs may be used as direct entrance or probes for cloning the disease resistance genes.

簇毛麦（Haynaldia Villosa）的原生质体培养

簇毛麦的幼胚在含有９μｍｏｌ／Ｌ２，４－Ｄ的ＭＢ固体培养基上诱导产生愈伤组织，在相同的培养基上继代培养两个月后出现淡黄色的致密瘤状愈伤组织．在继代培养过程中，愈伤组织的分化能力丧失较快．继代培养一年左右，改变培养条件，可得到颗粒状及粉粒状愈伤组织．分别用这两种类型的愈伤组织建立悬浮培养细胞系．愈伤组织的形态结构与悬浮系的建立及原生质体培养密切相关．来源于颗粒悬浮系的细胞团块产生的原生质体分裂后可产生体细胞胚，而用粉粒悬浮系分离的原生质体不能持续分裂．

Mass Production of Intergeneric Chromosomal Translocations through Pollen Irradiation of Triticum durum-Haynaldia villosa Amphiploid

Haynaldia villosa possesses a lot of important agronomic traits and has been a powerful gene resource for wheat improvement. However, only several wheat-H, villosa translocation lines have been reported so far. In this study, we attempted to develop an efficient method for inducing wheat-H, villosa chromosomal translocations. Triticum durum- Haynaldia villosa amphiploid pollen treated with 1 200 rad ^60Co-y-rays was pollinated to Triticum aestivum cv. ＇Chinese Spring＇. Ninety-eight intergeneric translocated chromosomes between T. durum and H. villosa were detected by genomic in situ hybridization in 44 of 61 M1 plants, indicating a translocation occurrence frequency of 72.1%; much higher than ever reported. There were 26, 62 and 10 translocated chromosomes involving whole arm translocations, terminal translocations, and intercarlary translocations, respectively. Of the total 108 breakage-fusion events, 79 involved interstitial regions and 29 involved centric regions. The ratio of small segment terminal translocations （W.W-V） was much higher than that of large segment terminal translocations （W-V.V）. All of the M1 plants were self-sterile, and their backcross progeny was all obtained with ＇Chinese Spring＇ as pollen donors. Transmission analysis showed that most of the translocations were transmittable. This study provides a new strategy for rapid mass production of wheat-alien chromosomal translocations, especially terminal translocations that will be more significant for wheat improvement.

Comparative study of symmetric and asymmetric somatic hybridization between common wheat and Haynaldia villosa

Symmetric and asymmetric protoplast fusion between long term cell suspension-derived protoplasts of Triticum aestivum (cv. Jinan 177) and protoplasts of Haynaldia villosa prepared from one-year-old embryogeneric calli was performed by PEG method. In asymmetric fusion, donor calli were treated with gamma ray at a dose of 40, 60, 80 Gy (1.3 Gy/min) respectively and then used to isolate protoplasts. Results of morphological, cytological, biochemical (isozyme) and 5S rDNA spacer sequence analysis revealed that we obtained somatic hybrid lines at high frequency from both symmetric and asymmetric fusion. Hybrid plants were recovered from symmetric and low dose γ-fusion combinations. GISH (genomic in situ hybridization) analysis proved exactly the existence of both parental chromosomes and the common occurrence of several kinds of translocation between them in the hybrid clones regenerated from symmetric and asymmetric fusion. And the elimination of donor DNA in hybrid clones regenerated from asymmetric fusion combinations was found to increase with the increasing gamma doses. It is concluded that transference and recombination of nuclear DNA can be achieved effectively by symmetric and asymmetric fusion, hybrids with small fragment translocation which are valuable in plant breeding can be obtained directly by asymmetric fusion.

Induction and transmission of wheat-Haynaldia villosa chromosomal translocations

In order to develop more wheat-Haynaldia villosa translocations involving different chromosomes and chromosome segments of H. villosa, T. durum-H, villosa amphiploid was irradiated with ^60Co γ-rays at doses of 800, 1,200, and 1,600 rad. Pollen collected from the spikes 1, 2, and 3 days after irradiation were transferred to emasculated spikes of the common wheat cv. ‘Chinese Spring＇. Genomic in situ hybridization was used to identify wheat-H, villosa chromosome translocations in the M1 generation. Transmission of the identified translocation chromosomes was analyzed in the BC1, BC2, and BC3 generations. The results indicated that all three irradiation doses were highly efficient for inducing wheat-alien translocations without affecting the viability of the M1 seeds. Within the range of 800-1,600 rad, both the efficiency of translocation induction and the frequency of interstitial chromosome breakage-fusion increased as the irradiation dosage increased. A higher translocation induction frequency was observed using pollen collected from the spikes 1 day after irradiation over that of 2 or 3 days after irradiation. More than 70% of the translocations detected in the M1 generation were transmitted to the BC1 through the female gametes. All translocations recovered in the BC1 generation were recovered in the following BC2, and BC3 generations. The transmission ability of different translocation types in different genetic backgrounds showed an order of ‘whole-arm translocation 〉 small alien segment translocation 〉 large alien segment translocation＇, through either male or female gametes, In general, the transmission ability through the female gametes was higher than that through the male gametes. By this approach, 14 translocation lines that involved different H. villosa chromosomes have been identified in the BC3 using EST-STS markers, and eight of them were homozygous.

Construction and application of oligo-based FISH karyotype of Haynaldia villosa

Haynaldia villosa （L.） Schur （syn. Dasypyrurn villosum （L.） Can- dargy） （2n - 14, genome VV）, a wild relative of wheat, is an impor- tant gene pool for improving wheat quality and disease resistance. Several genes found in H. villosa have been transferred into wheat to improve wheat resistance by the development of alien transloca- tion lines. The seed storage protein loci on chromosome 1V contribute to grain quality （Zhang et al., 2014）.

Asymmetric somatic hybridization between Triticum aestivum L. and Haynaldia villosa Schur

Suspension cell-derived protoplasts of wheat, inactivated with different concentrations (0-2.5mol/L) of IOA, were fused by PEG method with the Haynaldia villosa protoplasts which originated from the calli 4-5d after subculture and were irradiated with 60Co-γ ray. Cell colonies, calli or regenerated plants were obtained from different combinations of fusion. The calli and plants were verified to be hybrids by chromosome counting, isozyme analysis and morphological inspection.

Tracing the location of powdery mildew resistance-related gene Stpk-V by FISH with a TAC clone in Triticum aestivum–Haynaldia villosa alien chromosome lines

Bacterial artificial chromosomes(BACs)or yeast artificial chromosomes(YACs)containing large inserts as probes for fluorescence in situ hybridization(FISH)have been used in the physical mapping of specific DNA sequences,especially for single-or low-copy sequences.Our earlier study identified Stpk-V,a powdery mildew resistance-related gene located on the 6VS chromosome arm of the wild grass Haynaldia villosa(tribe Triticeae),and obtained several Triticum aestivum–H.villosa alien chromosome lines carrying the Stpk-V gene.However,the precise physical location of the Stpk-V gene on chromosome 6VS is not known.In this study,we used TAC-FISH with TAC15 as the probe coupled with sequential genomic in situ hybridization(GISH)to determine the physical location of the Stpk-V gene in different T.aestivum–H.villosa 6V alien chromosome lines,including addition,substitution and translocation lines.The result indicated that the fraction length of the Stpk-V locus is 0.575±0.035 on the 6V chromosome short arm and this was confirmed by FISH using TAC15 as the probe for tracing the Stpk-V gene in other genetic stocks.The cytological mapping strategies used in this study will be of benefit for tracing the alien gene location in the course of introducing desirable traits from wild species.

Characterization of RAPD Markers,and the RFLP Marker Linked to Powdery Mildew Resistance Gene Derived from Different Accessions of H.villosa

The analysis was carried out on performance of the resistance gene from Haynaldia villosa accession of the former Soviet Union to different isolates of Bluemerie graminis. Polymorphisms were revealed between 6D/6V substitution line Pm930640 and its pedigree parents using five RAPD markers of OPAN031700, OPAI01700, OPAL03750, OPAD07480 and OPAG15580 screened out from 120 random 10-mers primers. Three RAPD markers of OPAN03, OPAI01 and OPAL03 were linked with the resistance gene by analysis of F2 population of Chancellor×Pm930640. Analysis of 29 wheat lines including part of lines conferring the known genes from Pm1 to Pm20 respectively, lines conferring resistance gene from two H. villosa accessions and the related wheat parents, were analyzed and the results showed that these markers not only linked to the gene resistant to powdery mildew from H. villosa, but also detected different genetic backgrounds. OPAL03750 can be used as the marker to distinguish the different resistant lines from two H. villosa accessions because it was only observed in the materials from H. villosa of the former Soviet Union. RFLP analysis also showed the polymorphisms between two H. villosa accessions and their derived resistant lines.