Microdissection of additional chromosome in common wheat-Th. intermedium TAI-27 and screening of its special probe

It was identified that there were 4 St chromosomes derived from Th, intermedium in common wheat-Th. intermedium alien additional line TAI-27 by in situ hybridization. Two St chromosomes added to wheat chromosome in TAI-27 as well as two of them replaced two of 42 in wheat chromosomes. This indicates that TAI-27 is not only an alien additional line, but also a replacing line. The additional chromosomes in TAI-27 were microdissected and a microcloning library was constructed. A special probe of Th. intermedium was obtained from a microcloning library. The sequence analysis indicated that there were no homology with Genebank data. This makes it possible to screen genes with the disease-resistance, adversity-tolerance and fine character from Th.intermedium.     

Microdissection of additional chromosome in common wheat-Th.intermedium TAI-27 and screening of its special probe

It was identified that there were 4 St chromosomes derived from Th.intermedium in common wheat-Th.intermedium alien additional line TAI-27 by in situ hybridization.Two St chromosomes added to wheat chromosome in TAI-27 as well as two of them replaced two of 42 in wheat chromosomes.This indicates that TAI-27 is not only an alien additional line,but also a replacing line.The additional chromosomes in TAI-27 were microdissected and a microcloning library was constructed.A special probe of Th.intermedium was obtained from a microcloning library.The sequence analysis indicated that there were no homology with Genebank data.This makes it possible to screen genes with the disease-resistance,adversity-tolerance and fine character from Th.intermedium.     

Development of EST-PCR markers for <Emphasis Type="Italic">Thinopyrum intermedium</Emphasis> chromosome 2Ai#2 and their application in characterization of novel wheat-grass recombinants

A series of expressed sequence tags-derived polymerase chain reaction (EST-PCR) markers specific to chromosome 2Ai#2 from Thinopyrum intermedium were developed in this study using a new integrative approach. The target alien chromosome confers high resistance to barley yellow dwarf virus (BYDV), which is a severe virus disease in wheat. To generate markers evenly distributed on 2Ai#2, a total of 105 primer pairs were designed based on mapped ESTs from 8 bins of wheat chromosome 2B with intron-prediction by aligning ESTs with genomic sequences of the new model plant Brachypodium distachyon. Eight and seven polymorphic markers on the short arm and the long arm of chromosome 2Ai#2, respectively, were obtained with a polymorphism rate of 14.3%. These chromosome 2Ai#2-specific EST-PCR markers were then used in tracing and exploring the structural variation of the alien chromosome in the population derived from the immature embryo culture of the cross between N452, a 2Ai#2(2D) substitution line, and common wheat CB037. Two centric fusion of translocations involving 2Ai#2 short or long arm with wheat chromosome 2D and some new genetic stocks including telosomes with the alien chromosome short or long arm were identified in the SC₃ generations, which provided basic materials to further study the mechanism of the BYDV resistance. BYDV tests in two field seasons suggest that the BYDV resistance was mainly conferred by the short arm, gene interaction on both arms of the alien chromosome was discussed.     

Introgression and characterization of barley yellow dwarf virus resistance from Thinopyrum intermedium into wheat.

Wheatgrasses (species of Agropyron complex) have previously been reported to be resistant to barley yellow dwarf virus (BYDV). To introgress this resistance into wheat, Triticum aestivum x Thinopyrum (Agropyron) intermedium hybrids were advanced through a backcrossing program and reaction to BYDV, as determined by enzyme-linked immunosorbent assay (ELISA), is reported for the first time in backcross populations of wide hybrids between wheat and wheatgrasses. ELISA values revealed highly resistant to highly susceptible segregants in backcrosses. BYDV resistance was expressed in some backcross derivatives. Continued selection, based on cytology and ELISA in each generation, eliminated most of the unwanted wheatgrass chromosomes and produced self-fertile BYDV resistant wheat lines. The BYDV resistant lines with 2n = 42 had normal chromosome pairing similar to wheat, and their F1 hybrids with wheat had two univalents. DNA analyses showed that the source of alien chromatin in these BYDV resistant wheat lines is distinguishable from that in other Th. intermedium derived BYDV resistant wheat lines. Chromosome pairing and restriction fragment length polymorphism analyses indicated that the 42 chromosome resistant Purdue wheat lines are substitution lines in which chromosome 7D was replaced by a chromosome from Th. intermedium that was carrying gene(s) for BYDV resistance.     

Structural organization of an alien Thinopyrum intermedium group 7 chromosome in U.S. soft red winter wheat (Triticum aestivum L.).

Barley yellow dwarf virus (BYDV) resistance in soft red winter wheat (SRWW) cultivars has been achieved by substituting a group 7 chromosome from Thinopyrum intermedium for chromosome 7D. To localize BYDV resistance, a detailed molecular genetic analysis was done on the alien group 7 Th. intermedium chromosome to determine its structural organization. Triticeae group 7 RFLP markers and rye specific repetitive sequences used in the analysis showed that the alien chromosome in the P29 substitution line has distinguishing features. The 350-480 bp rye telomeric sequence family was present on the long arm as determined by Southern and fluorescence in situ hybridization. However, further analysis using a rye dispersed repetitive sequence indicated that this alien chromosome does not contain introgressed segments from the rye genome. The alien chromosome is homoeologous to wheat chromosomes 7A and 7D as determined by RFLP analysis. Presence of the waxy gene on chromosomes 7A, 7B, and 7D but its absence on the alien chromosome in P29 suggests some internal structural differences on the short arm between Th. intermedium and wheat group 7 chromosomes. The identification of rye telomeric sequences on the alien Thinopyrum chromosome and the homoeology to wheat chromosomes 7A and 7D provide the necessary information and tools to analyze smaller segments of the Thinopyrum chromosome and to localize BYDV resistance in SRWW cultivars.     

Cloning of resistance gene analogs located on the alien chromosome in an addition line of wheat-Thinopyrum intermedium

Homology-based gene/gene-analog cloning method has been extensively applied in isolation of RGAs (resistance gene analogs) in various plant species. However, serious interference of sequences on homoeologous chromosomes in polyploidy species usually occurred when cloning RGAs in a specific chromosome. In this research, the techniques of chromosome microdissection combined with homology-based cloning were used to clone RGAs from a specific chromosome of Wheat-Thinopyrum alien addition line TAi-27, which was derived from common wheat and Thinopyrum intermedium with a pair of chromosomes from Th. intermedium. The alien chromosomes carry genes for resistance to BYDV. The alien chromosome in TAi-27 was isolated by a glass needle and digested with proteinase K. The DNA of the alien chromosome was amplified by two rounds of Sau3A linker adaptor-mediated PCR. RGAs were amplified by PCR with the degenerated primers designed based on conserved domains of published resistance genes (R genes) by using the alien chromosome DNA, genomic DNA and cDNA of Th. intermedium, TAi-27 and 3B-2 (a parent of TAi-27) as templates. A total of seven RGAs were obtained and sequenced. Of which, a constitutively expressed single-copy NBS-LRR type RGA ACR3 was amplified from the dissected alien chromosome of TAi-27, TcDR2 and TcDR3 were from cDNA of Th. intermedium, AcDR3 was from cDNA of TAi-27, FcDR2 was from cDNA of 3B-2, AR2 was from genomic DNA of TAi-27 and TR2 was from genomic DNA of Th. intermedium. Sequence homology analyses showed that the above RGAs were highly homologous with known resistance genes or resistance gene analogs and belonged to NBS-LRR type of R genes. ACR3 was recovered by PCR from genomic DNA and cDNA of Th. intermedium and TAi-27, but not from 3B-2. Southern hybridization using the digested genomic DNA of Th. intermedium, TAi-27 and 3B-2 as the template and ACR3 as the probe showed that there is only one copy of ACR3 in the genome of Th. intermedium and TAi-27, but it is absent in 3B-2. The ACR3 could be used as a specific probe of the R gene on the alien chromosome of TAi-27. Results of Northern hybridization suggested that ACR3 was constitutively expressed in Th. intermedium and TAi-27, but not 3B-2, and expressed higher in leaves than in roots. This research demonstrated a new way to clone RGAs located on a specific chromosome. The information reported here should be useful to understand the resistance mechanism of, and to clone resistant genes from, the alien chromosome in TAi-27.     

Variation of wheatgrass chromosomes in wheat-wheatgrass alien addition line "TAI-27"revealed by fluorescence in situ hybridization (FISH)

The Karyotyp of the primary wheat-whastgrass alien addition line TAI-27 was 2n = 44 in which all d the chromosomes were metacentric and subrmetacentric. However, in the progeny of TAI-27 a pair of chromosomes had become small chromosomea in the two morphologically different plants. Fluorescence in situ hybridizstionm (FISH) technique was used to analyze the two different plants. The observations indicate that a pair of small chromosomes in one varietion line are from wheatgrass. In another variation line, a pair of small chromosomes are also from whest-grass, while another pair of wheatgrass chromosomes have substituted the wheat chromosomes. TAI-27 and its variant lines showed a high level of resistance to barley yellow dwarf virus (BYDV). The pessible explanation for such a variation and the potential use of the variant lines were discussed briefly.     

天兰冰草染色体对小冰麦外植体再生能力的影响及基因的染色体定位

本文研究了天兰冰草（Ａｇｒｏｐｙｒｏｎｉｎｔｅｒｍｅｄｉｕｍ２ｎ＝４２）染色体导入小麦后对小冰麦外植体再生能力的影响,从而分析调控再生能力的基因所在天兰冰草染色体的定位。对异源八倍体小冰麦的研究表明,与天兰冰草强再生能力有关的基因主要分布在附加到中２的冰草染色体组上;对７个异附加系小冰麦的研究进一步表明,冰草强再生能力的性状由多基因调控,这些基因主要分布在附加到ＴＡＩ－１１、ＴＡＩ－１２、ＴＡＩ－１３的冰草染色体上。此外,小冰麦杂种Ｆ１的研究表明,在组织培养中同样能够表现出杂种优势。     

Variation of wheatgrass chromosomes in wheat-wheatgrass alien addition line “TAI-27” revealed by fluorescencein situ hybridization (FISH)

The Karyotyp of the primary wheat-whastgrass alien addition line TAI-27 was 2n = 44 in which all d the chromosomes were metacentric and subrmetacentric. However, in the progeny of TAI-27 a pair of chromosomes had become small chromosomea in the two morphologically different plants. Fluorescence in situ hybridizstionm (FISH) technique was used to analyze the two different plants. The observations indicate that a pair of small chromosomes in one varietion line are from wheatgrass. In another variation line, a pair of small chromosomes are also from whest-grass, while another pair of wheatgrass chromosomes have substituted the wheat chromosomes. TAI-27 and its variant lines showed a high level of resistance to barley yellow dwarf virus (BYDV). The pessible explanation for such a variation and the potential use of the variant lines were discussed briefly.     

The use of cell culture for subchromosomal introgressions of barley yellow dwarf virus resistance from Thinopyrum intermedium to wheat.

Barley yellow dwarf virus (BYDV) resistance has been transferred to wheat from a group 7 chromosome of Thinopyrum (Agropyron) intermedium. The source of the resistance gene was the L1 disomic addition line, which carries the 7Ai-1 chromosome. The resistance locus is on the long arm of this chromosome. BYDV resistant recombinant lines were identified after three or more generations of selection against a group 7 Th. intermedium short arm marker (red coleoptile) and selection for the presence of BYDV resistance. One recombinant line produced by ph. mutant induced homoeologous pairing and 14 recombinant lines induced by cell culture have been identified. Resistance in seven of the cell culture induced recombinants has been inherited via pollen according to Mendelian segregation ratios for up to eight generations. Meiotic analysis of heterozygotes indicates that the alien chromatin in the cell culture induced recombinants is small enough to allow regular meiotic behaviour. The ph-induced recombinant was less regular in meiosis. A probe, pEleAcc2, originally isolated from Th. elongatum and that hybridizes to dispersed repeated DNA sequences, was utilised to detect Th. intermedium chromatin, which confers resistance to BYDV, in wheat backgrounds. Quantification of these hybridization signals indicated that the translocations involved a portion of alien chromatin that was smaller than the complete long arm of 7Ai-1. Restriction fragment length polymorphism analysis confirmed the loss of the short arm of 7Ai-1 and indicated the retention of segments of the long arm of 7Ai-1. Two 7Ai-1L DNA markers always assorted with the BYDV resistance. A third 7Ai-IL DNA marker was also present in seven of eight recombinants. In all recombinants except TC7, the 7Ai-1L markers replaced the 7DL markers. None of the wheat group 7 markers was missing from TC7. It is concluded that all the resistant lines are the result of recombination with wheat chromosome 7D, except line TC7, which is the result of recombination with an unidentified nongroup 7 chromosome.     

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 ofph 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-TAi#1 （bearing Bdv2）, 7B-7Ai#1, 7D-7E （beating 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.     

小冰麦异附加系中天兰冰草染色体的变异

通过对两套14种小冰麦异附加系的体细胞染色体观察,发现3个异附加系各有1对染色体发生了显著变异。其中TAl-14t有1对端着丝点染色体,TAl-22s和TAl-27s各有1对近中着丝点的小染色体。通过组胞遗传学分析,从3个方面证明了发生变异的染色体均来自天兰冰草。此外还发现在其中的两个异附加系(TAI-14t和TAl-27s)中可能产生了自发易位。     

应用原位杂交及RAPD技术标记抗黄矮病小麦一中间偃麦草染色体异附加系

以生物素（Ｂｉｏｔｉｎ－１６－ｄＵＴＰ）标记中间偃麦草基因组 ＤＮＡ为探针,与抗黄矮病小麦－中间偃麦草染色体异附加系Ｚ６进行原位杂交,鉴定出附加的１对中间偃麦草染色体。对异附加系 Ｚ６和 Ｌ１及它们的小麦亲本进行了 ＲＡＰＤ分析,从 １２０个随机引物中,筛选出 ２个引物可以扩增出附加染色体的特异ＤＮＡ片段,可作为鉴定寻人小麦的中间偃麦草染色质的分子标记。     

人参植物与皂苷生物合成相关的差减cDNA文库构建及基因差异表达分析

应用抑制差减杂交技术,分别以源于4年和1年生人参根组织cDNA群体作为检测子(tester)与驱赶子(driver),成功构建了与人参植物皂苷生物合成相关的差减cDNA文库,并时从中筛选的阳性cDNA克隆进行DNA测序及其序列分析、PCR及Northern印迹杂交鉴定．结果显示,获得的13个克隆为新基因序列．其中6个差减克隆系人参植物根生长发育阶段差异表达基因．目前,6个差异表达新基因的结构与功能仍在进一步研究中．     

Disomic Thinopyrum intermedium addition lines in wheat with barley yellow dwarf virus resistance and with rust resistances.

Zhong 5 is a partial amphiploid (2n = 56) between Triticum aestivum (2n = 42) and Thinopyrum intermedium (2n = 42) carrying all the chromosomes of wheat and seven pairs of chromosomes from Th. intermedium. Following further backcrossing to wheat, six independent stable 2n = 44 lines were obtained representing 4 disomic chromosome addition lines. One chromosome confers barley yellow dwarf virus (BYDV) resistance, whereas two other chromosomes carry leaf and stem rust resistance; one of the latter also confers stripe rust resistance. Using RFLP and isozyme markers we have shown that the extra chromosome in the Zhong 5-derived BYDV resistant disomic addition lines (Z1, Z2, or Z6) belongs to the homoeologous group 2. It therefore carries a different locus to the BYDV resistant group 7 addition, L1, described previously. The leaf, stem, and stripe rust resistant line (Z4) carries an added group 7 chromosome. The line Z3 has neither BYDV nor rust resistance, is not a group 2 or group 7 addition, and is probably a group 1 addition. The line Z5 is leaf and stem rust resistant, is not stripe rust resistant, and its homoeology remains unknown.     

Cloning and partial characteristics of the barley D-hordein sequence]

A number of clones containing major endosperm-specifically transcribed gene copies were selected from a cDNA library developed on the basis of barley endosperm mRNA. Approx. 30% of the recombinant clones carried sequences homologous to mRNA of various cereal storage proteins. Some of them appeared to be related to cDNA clones of wheat and barley storage proteins. The typical insert length ranged from 0.3 to 1.7 kB. A couple of clones among them were selected which revealed positive hybridization with all probes used. The positive signals disappeared after stringent washing of the filters. The nucleotide sequences of two representatives of the group were determined and corresponding amino acid sequence deduced after subsequent computer analysis. The comparison with known cereal storage protein genes revealed relatively high homology level with the central part of wheat high molecular weight (HMW) glutenine subunit genes. The fact suggests the cloned gene to belong to barley D-hordein family.     

白粉病菌诱导的小麦表达序列标签(EST)研究

白粉病是我国小麦的主要病害之一.尝试用表达序列标签(expressed sequence tags, EST)技术,研究了经白粉病菌诱导后的小麦基因表达.从构建的普通cDNA文库中随机挑取约1 500个阳性克隆并进行测序, 获不重复ESTs序列387条.不重复序列均获GenBank的存储号.其中49.4%的序列与已知基因同源,196条序列功能未知, 84条序列为新ESTs.将不重复序列制备成高密度点阵膜,用差示杂交法筛选到几个抗病相关序列.     

小偃麦附加系Z1和Z2中外源染色体2Ai-2的结构组成

小偃麦附加系Z1和Z2中外源染色体2Ai-2的结构组成@张增燕$中国农业科学院作物育种栽培研究所!北京100081@辛志勇$中国农业科学院作物育种栽培研究所!北京100081@陈孝$中国农业科学院作物育种栽培研究所!北京100081小偃麦;;附加系;;染色体     

Characterization of derivatives from wheat-Thinopyrum wide crosses

Partial amphiploids are lines that contain 42 (38-42) wheat and 14 (14-18) alien chromosomes. They are derived by backcrossing wheat onto hybrids between wheat and either Thinopyrum intermedium (6x) or Th. ponticum (10x). GISH analysis has shown that, with possibly one exception, the alien genomes (chromosome sets) in partial amphiploids are found to be hybrids i.e. composed of chromosomes from more than one alien genome. The individual partial amphiploids are meiotically stable and nearly perfectly fertile, but hybrids between different lines were characterized by varying numbers of unpaired chromosomes and consequently variable degrees of sterility. Translocated chromosomes involving different Thinopyrum genomes or Thinopyrum and wheat genomes were found in partial amphiploids and consequently in the addition lines derived from them. Partial amphiploids have proven to be an excellent tertiary gene pool for wheat improvement, containing resistance to biotic stresses not present in wheat itself. Resistance to Barley Yellow Dwarf Virus (BYDV) and Wheat Streak Mosaic Virus (WSMV) have been found in partial amphiploids and addition lines derived from both Th. intermedium and Th. ponticum. Excellent resistance to Fusarium head blight has been found on a Th. intermedium chromosome that had substituted for chromosome 2D in wheat. Genes for resistance to leaf rust and stem rust have already been incorporated into wheat and tagged with molecular markers.