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Development of Triticum aestivum-Leymus racemosus ditelosomic substitution line 7Lr#1S(7A) with resistance to wheat scab and its meiotic behavior analysis

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Chinese Science Bulletin 2008年第22期53卷 3522-3529页

作者： WANG LinSheng chen peidu State Key Laboratory of Crop Genetics and Germplasm Enhancement Cytogenetics Institute Nanjing Agricultural University Nanjing 210095 China College of Agronomy Henan University of Science and Technology Luoyang 471003 China

Leymus racemosus is highly resistant to wheat scab (Fusarum head bright). The transfer of scab resistant gene from L. racemosus to Triticum aestivum is of great significance for broadening the base of wheat resistance. In the present study, the pollen of T. aestivum-L. racemosus monosomic addition line with scab resistance was treated by irradiation with 1200 R 60Co-γ-rays prior to pollinating to emasculated wheat cv. Mianyang 85-45. Nine plants with a telocentric chromosome 7Lr#1S were observed in M1, and one ditelosomic substitution line 7Lr#1S was selected from selfcrossing progenies and confirmed by chromosome C-banding and GISH. Furthermore, a co-dominant EST-SSR marker CINAU 31 was employed to identify this substitution line. A pair of chromosome 7A of common wheat were found to be replaced by a pair of telocentric chromosome 7Lr#1S, and further investigation showed that chromosome configuration of the substitution line at MI of PMCs after GISH was 17.50○II W + 2.19 IIW + 0.42II7Lr#1S + 1.08 I7Lr#1S + 0.69 IW. Two telocentric chromosomes paired as a bivalent in 59.7% of PMCs. Abnormal chromosome behaviors of telocentric chromosomes were observed in part of PMCs at anaphase I and telophase I, including the moving of two telocentric chromosomes to the same pole, lagging and earlier separation of their sister chromatid. All these abnormal behaviors can be grouped into three distinct types of tetrads according to different numbers of 7Lr#1S in their daughter cells and various micronucleus in some tetrads. However, due to the high transmission frequency of the female and male gametes with a 7Lr#1S, 84% of the selfcrossing progeny plants had ditelosomic substitution. The substitution line showed high resistance to wheat scab in a successive two-year test both in the greenhouse and field; hence, the line will be particularly valuable for alien gene mapping, small fragment translocation induction and telosomic cytological behavior analysis.

关键词：小麦痂斑分裂现象病变

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Molecular cytogenetic analysis of Triticum aestivum-Leymus racemosus reciprocal chromosomal translocation T7DS·5LrL/T5LrS·7DL

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Chinese Science Bulletin 2010年第11期55卷 1026-1031页

作者： WANG LinSheng chen peidu WANG XiuE College of Agronomy Henan University of Science and Technology Luoyang 471003 China State Key Laboratory of Crop Genetics and Germplasm Enhancement Cytogenetics Institute Nanjing Agricultural University Nanfing 210095 China

In order to induce chromosome translocation between wheat chromosomes and chromosome 5Lr of Leymus racemosus, the mi- crosporocytes during meiosis of T. aestivum-L. racemosus disomic addition line DA5Lr were irradiated by 60Co γ-rays 800 R (100 R/min). Before flowering, the treated spikes were emasculated and bagged. After 2-3 d, the emasculated flowerets were pollinated using pollens from T. aestivum cv. Chinese Spring. One plant with two translocation chromosomes involved in both the long and short arm of 5Lr was detected in the M1 by GISH. The plant was crossed with line DA5Lr, and its progenies with one 5Lr and two translocation chromosomes were analyzed for chromosome pairing behavior in their pollen mother cells (PMCs). A cross-shaped configuration at diplonema and Z-shaped or ring-shaped quadrivalent configuration at metaphase I were observed, indicating that the two translocation chromosomes were reciprocal translocation. Chromosome C-banding indicated that the wheat chromosomes involved in the reciprocal translocation belonged to A- or D-genome. Fluorescence in situ hybridization using pSc119.2 and pAs1 as the probe found that only pAs1 signals were present in the wheat chromosome segments of the two translocation chromosomes. Combining these results, the reciprocal chromosomal translocation was designated as T7DS·5LrL/5LrS·7DL. The two transloca- tion chromosomes were found to be transmitted together in the gametes of heterozygous reciprocal translocation plants with the transmission ratios of 59.4% in the female gametes and 83.9% in the male gametes, revealing preferential pollen transmission. In the self-fertilized progenies of the heterozygous reciprocal translocation, a line with the homozygous translocation line with a pair of translocation chromosome T7DS·5LrL was identified. The T7DS·5Lr translocation line was highly resistant to wheat scab and can be used as a potential and new source in wheat improvement for scab resistance.

关键词：细胞遗传学分析染色体易位大赖草分子小麦互惠

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Induction and transmission of wheat-Haynaldia villosa chromosomal translocations

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Journal of Genetics and Genomics 2009年第5期36卷 313-320页

作者： Yaping Cao Tongde Bie Xiue Wang peidu chen National Key Laboratory of Crop Genetics and Germplasm Enhancement Cytogenetics Institute. Nanjing Agricultural University Nanjing 210095 China Wheat Research Institute Shanxi Academy of Agricultural Sciences Linfen 041000 China Yangzhou Academy of Agricultural Sciences Yangzhou 225007 China

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.

关键词： Triticum durum-Haynaldia villosa amphiploid pollen irradiation chromosomal translocation translocation transmission genomic in situ hybridization

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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

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Chinese Science Bulletin 2013年第33期58卷 4084-4091页

作者： YANG XueMing CAO AiZhong SUN YuLei chen peidu State Key Laboratory of Crop Genetics and Germplasm Enhancement Cytogenetics InstituteNanjing Agricultural University Provincial Key Laboratory of Agrobiology Institute of BiotechnologyJiangsu Academy of Agricultural Sciences

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 *** 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–*** alien chromosome lines carrying the Stpk-V ***,the precise physical location of the Stpk-V gene on chromosome 6VS is not *** 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 ***–*** 6V alien chromosome lines,including addition,substitution and translocation *** 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 *** 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.

关键词：异源染色体相关基因物理位置抗白粉病簇毛麦小麦族 TAC 跟踪

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Cytogenetic and molecular identification of three Triticum aestivum-Leymus racemosus translocation addition lines

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Journal of Genetics and Genomics 2009年第6期36卷 379-385页

作者： Le Wang Jianhua Yuan Tongde Bie Bo Zhou peidu chen National Key Laboratory of Crop Genetics and Germplasm Enhancement Cytogenetics Institute Nanjing Agricultural University Nanjing 210095 China Jiangsu Academy of Agricultural Sciences Nanjing 210014 China Yangzhou Academy of Agricultural Sciences Yangzhou 225007 China

Chromosome 2C from Aegilops cylindrica has the ability to induce chromosome breakage in common wheat （Tritivum aestivum）. In the BC1F3 generation of the T. aestivum cv. Chinese Spring and a hybrid between T. aestivum-Leymus racemosus Lr.7 addition line and T. aestivum-Ae, cylindrica 2C addition line, three disomic translocation addition lines （2n = 44） were selected by mitotic chromosome C-banding and genomic in situ hybridization. We further characterized these T. aestivum-L, racemosus translocation addition lines, NAU636, NAU637 and NAU638, by chromosome C-banding, in situ hybridization using the A- and D-genome-specific bacterial artificial chromosome （BAC） clones 676D4 and 9M13; plasmids pAsl and pSc119.2, and 45S rDNA; as well as genomic DNA of L. racemosus as probes, in combination with double ditelosomic test cross and SSR marker analysis. The translocation chromosomes were designated as T3AS-Lr7S, T6BS-Lr7S, and T5DS-Lr7L. The translocation line T3AS-Lr7S was highly resistant to Fusarium head blight and will be useful germplasm for resistance breeding.

关键词： wheat Leymus racemosus translocation line C-banding fluorescence in situ hybridization (FISH) test cross analysis

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Distribution of highly repeated DNA sequences in Haynaldia villosa and its application in the identification of alien chromatin

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Chinese Science Bulletin 2013年第8期58卷 890-897页

作者： ZHANG Wei ZHANG RuiQi FENG YiGao BIE TongDe chen peidu National Key Laboratory of Crop Genetics and Germplasm Enhancement Nanjing Agricultural University National Wheat Improvement Subcenter Yangzhou Academy of Agricultural Sciences

Haynaldia villosa (L.) is a wild relative species of common wheat that possesses many beneficial genes that can be used for wheat improvement. The accurate detection of H. villosa chromosomes in the genetic background of wheat is critical for transferring its beneficial genes to common wheat by chromosome engineering. The aim of the present study was to investigate the distribution patterns of two repeated DNA sequences, pSc119.2 and pAs1, as well as two rDNA multigene family sequences, 45S rDNA and 5S rDNA, in the individual chromosomes of H. villosa for the future precise identification of alien chromatin in germplasm development and breeding programs. A set of common wheat-H. villosa disomic addition 1V-7V lines was used to determine these specific signals on individual chromosomes of H. villosa. The results showed that two rDNA probes, pTa71 (45S rDNA) and pTa794 (5S rDNA), were located on 1VS and 5VS, respectively, and the signal could be discriminated exclusively in the common wheat background as effective markers of 1VS and 5VS. Furthermore, all seven chromosomes of H. villosa could be distinguished clearly by fluorescence in situ hybridization using pSc119.2 and pAs1 as probes in combination. The utilization of these cytogenetic markers of repetitive sequences, combined with other molecular markers sometimes, will make it possible for a precise identification of alien chromosomes with high efficiency.

关键词：染色体工程 DNA序列簇毛麦识别普通小麦 rDNA 应用荧光原位杂交

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Competitive Expression of Endogenous Wheat CENH3 May Lead to Suppression of Alien ZmCENH3 in Transgenic Wheat × Maize Hybrids

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Journal of Genetics and Genomics 2015年第11期42卷 639-649页

作者： Wei chen Qilin Zhu Haiyan Wang Jin Xiao Liping Xing peidu chen Weiwei Jin Xiu-E.Wang State Key Laboratory of Crop Genetics and Germplasm Enhancement Cytogenetics InstituteNanjing Agricultural University/JCIC-MCP National Maize Improvmental Center China Agricultural University

Uniparental chromosome elimination in wheat × maize hybrid embryos is widely used in double haploid production of wheat. Several explanations have been proposed for this phenomenon, one of which is that the lack of cross-species CENH3 incorporation may act as a barrier to interspecies hybridization. However, it is unknown if this mechanism applies universally. To study the role of CENH3 in maize chromosome elimination of wheat x maize hybrid embryos,？cos, maize ZmCENH3 and wheat aTaCENH3-B driven by the constitutive CaMV35S promoter were transformed into wheat variety Yangmai 158. Five transgenic lines for ZmCENH3 and six transgenic lines for ctTaCENH3-B were identified. RT-PCP analysis showed that the transgene could be transcribed at a low level in all ZmCENH3 transgenic lines, whereas transcription of endogenous wheat CENH3 was significantly up-regulated. Interestingly, the expression levels of both wheat CENH3 and ZmCENH3 in the ZmCENH3 transgenic wheat × maize hybrid embryos were higher than those in the non-transformed Yangmai 158 × maize hybrid embryos. This indicates that the alien ZmCENH3 in wheat may induce competitive expression of endogenous wheat CENH3, leading to suppression of ZmCENH3 over-expression. Eliminations of maize chromosomes in hybrid embryos of ZmCENH3 transgenic wheat ×maize and Yangmai 158 x maize were compared by observations on micronuelei presence, by marker analysis using maize SSRs （simple sequence repeats）, and by FISH （fluorescence in situ hybridization） using 45S rDNA as a probe. The results indicate that maize chromosome elimination events in the two crosses are not sigmficantly different. Fusion protein ZmCENH3- YFP could not be detected in ZmCENH3 transgenic wheat by either Western blotting or immnunostaining, whereas accumulation and loading of the αTaCENH3-B-GFP fusion protein was normal in aTaCENH3-B transgenic lines. As ZmCENH3-YFP did not accumulate after AM114 treatment, we speculate that low levels of ZmCENH3 protein in

关键词： CENH3 Transgenic wheat Uniparental chromosome elimation Triticum aestivura Zea mays

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Characterization of a PDR type ABC transporter gene from wheat (Triticum aestivum L.)

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Chinese Science Bulletin 2009年第18期54卷 3249-3257页

作者： SHANG Yi XIAO Jin MA LuLin WANG HaiYan QI ZengJun chen peidu LIU DaJun WANG XiuE State Key Laboratory of Crop Genetics and Germplasm Enhancement Cytogenetics Institute Nanjing Agricultural UniversityNanjing 210095 China Institute of Crop Research and Nuclear Technique Utilization Zhejiang Academy of Agricultural Science Hangzhou 310021 China

DON, as a virulence factor, plays an important role in the infection of Fusarium graminearum in wheat. The infection ability of F. graminearum depends on its capacity of producing DON. The production of DON by F. graminearum is significantly decreased in the wheat varieties with scab resistance. In this study, GeneChip analysis indicated that an EST encoding an ATP-binding cassette (ABC) transporter was up-regulated by 45 times in a wheat landrace Wangshuibai, which is resistant to DON accumulation. A pair of EST-derived primers were designed based on the EST sequence, and a clone was then isolated from a wheat genomic DNA TAC library. The TAC clone was sequenced using chromosome walking and gene prediction was conducted using Softberry. A cDNA clone of this gene was subsequently isolated from Wangshuibai induced by DON using gene-specific primers designed according to the untranslated sequence of the gene. The genome size of the gene is 7377 bp, consisting of 19 exons with coding sequences of 4308 bp. It encodes a protein with 1435 amino acid residues and the calculated molecular weight is about 161 kD. BLAST analysis indicated that the gene may belong to pleiotropic drug resistance (PDR) sub-family, and hence designated as TaPDR1 (Triticum aestivum pleiotropic drug resistance). TaPDR1 was located on chromosome 5A of wheat using nullisomic-tetrasomic lines of Chinese Spring. TaPDR1 was up-regulated by induction of both DON and F. graminearum. Expression patterns of TaPDR1 were different in wild-type Wangshuibai and the fast-neutron induced Wangshuibai mutant lacking FHB1, a major QTL of FHB resistance and DON resistance in chromosome arm 3BS. These results suggested that TaPDR1 might be a candidate gene responsible for DON ac-cumulation resistance. The expression profile showed that TaPDR1 expression was neither induced by hormones typically involved in biotic stress, such as JA and SA, nor by abiotic stresses, such as heat, cold, wounding and NaCl. However, TaPDR1

关键词：小麦多样性 PDR ABC

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Characterization of a Putative New Semi-Dominant Reduced Height Gene, Rht_NM9, in Wheat(Triticum aestivum L.)

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Journal of Genetics and Genomics 2015年第12期42卷 685-698页

作者： Yuan Lu Liping Xing Shujuan Xing Ping Hu Chaofan Cui Mingyi Zhang Jin Xiao Haiyan Wang Ruiqi Zhang Xiue Wang peidu chen Aizhong Cao National Key Laboratory of Crop Genetics and Germplasm Enhancement Cytogenetics Institute Nanjing Agricultural University

Plant height is an important agronomic trait in cereal crops, and can affect both plant architecture and grain yield. New dwarfing genes are required for improving the genetic diversity of wheat. In this study, a novel dwarf mutant, NM9, was created by treating seeds of the wheat variety NAU9918 with ethyl methanesulfonate （EMS）. NM9 showed obvious phenotypic changes, which were distinct from those caused by other dwarfing genes, especially the reduced plant height, increased effective tiller number, and elongated spike and grain length. The reduced plant height in NM9 was attributable to a semi-dominant dwarfing gene Rht__NM9, which was flanked by two closely linked SNP markers, SNP34 and SNP41, covering an 8.86-Mb region on the chromosome arm 2AS. The results of gibberellic acid （GA） sensitivity evaluation, comparative genomics analysis and allelism test indicated that RhtNM9 was neither allelic to Rht7 and Rht21 nor homoe- oallelic to Rht8, so Rht_NM9 was proposed to be a new dwarfing locus on the homoeologous group 2 chromosomes of wheat. Rht_NM9 has a negative effect on plant height and positive effects on effective tiller number and grain size, thus, Rht_NM9 could be used for elucidating the mechanisms underlying plant architecture and grain development.

关键词： Wheat Mutant Plant architecture Gene mapping

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Inducement of chromosome translocation with small alien segments by irradiating mature female gametes of the whole arm translocation line

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Science China(Life Sciences) 2008年第4期51卷 346-352页

作者： chen ShengWei1,2, chen peidu1 & WANG XiuE1 1 National Key Laboratory of Crop Genetics and Germplasm Enhancement, Nanjing Agricultural University, Nanjing 210095, China 2 College of Agronomy and Biotechnology, Yunnan Agricultural University, Kunming 650201, China National Key Laboratory of Crop Genetics and Germplasm Enhancement Nanjing Agricultural University Nanjing China College of Agronomy and Biotechnology Yunnan Agricultural University Kunming China

Haynaldia villosa Schur. (syn. Dasypyrum villosum Candargy, 2n=14, VV) has been proved to be an important genetic resource for wheat improvement. The development of translocation with small alien chromosome segments, especially interstitial translocation, will be helpful for better utilization of its useful genes. Up to now, most of the reported Triticum aestivum – H. villosa translocation lines are involved in a whole arm or large alien fragments. In this paper, we report a highly efficient approach for the creation of small chromosome segment translocation lines. Before flowering, the female gametes of wheat-H. villosa 6VS/6AL translocation line were irradiated by 60CO-γ ray at 160 Rad/M dosage rate and three dosages (1600, 1920, 2240 Rad). Anthers were removed from the irradiated florets on the same day and the florets were pollinated with normal fresh pollens of T. aestivum cv. Chinese Spring after 2-3 days. Genomic in situ hybridization (GISH) at mitosis metaphase of root-tip cell of M1 plants was used to detect the chromosome structural changes involving 6VS of H. villosa. Among the 534 M1 plants screened, 97 plants contained small segment chromosome structural changes of 6VS, including 80 interstitial translocation chromosomes, 57 terminal translocation chromosomes and 55 deletion chromosomes. For the 2240 Rad dosage treatment, the inducement frequencies of interstitial translo-cation, terminal translocation and deletion were 21.02%, 14.01%, and 14.65%, respectively, which were much higher than those previously reported. The M2 seeds were obtained by backcrossing of 74 M1 plants involving 146 chromosomes structural changes of 6VS, and it was found that the structural aberrations in the M1 plants could be transmitted to their progenies. Irradiating mature female gametes of whole arm translocation is a new and highly efficient approach for creation of small segment chromosome struc-tural changes, especially for interstitial translocations.

关键词： irradiation mature female gametes small fragment translocation chromosome wheat Haynaldia villosa genomic in situ hybridization

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