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.

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.

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.

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.

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.

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.

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.

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.

簇毛麦染色体组特异性RAPD标记的筛选、定位和应用

以普通小麦中国春、中国春 簇毛麦二体附加系以及不同来源的簇毛麦为材料 ,用 10 0个 10碱基随机引物进行RAPD扩增。引物OPF0 2能在不同来源的簇毛麦及所有中国春 簇毛麦二体附加系中扩增出一条长约 75 0bp的片段OPF0 2 750 。普通小麦和硬粒小麦不能扩增出该片段。因此 ,OPF0 2 750 为分布于簇毛麦所有染色体上的一个簇毛麦染色体组特异片段。用引物OPF0 2对普通小麦 簇毛麦双二倍体、硬粒小麦 簇毛麦双二倍体以及几个普通小麦的簇毛麦二体代换系、二体附加系进行检测 ,发现NAU30 2已经丢失了其所附加的簇毛麦

簇毛麦1V染色体SSR标记的筛选

选用位于普通小麦 1A、1B、1D染色体上的 32对微卫星引物对普通小麦中国春、簇毛麦、6个中国春 簇毛麦二体附加系和 1个普通小麦 簇毛麦二体代换系进行SSR分析 ,发现引物Xgwm4 98在簇毛麦中扩增出 2条长分别为 110bp和190bp的片段 (即Xgwm4 98 110和Xgwm4 98 190 ) ,这两个片段仅在簇毛麦、中国春 簇毛麦 1Ha附加系中出现 ,其余 2Ha、4Ha、5Ha、6Ha、7Ha附加系、3V代换系和中国春中都缺少这两个片段。进一步研究表明 ,这两个片段与簇毛麦的居群无关。因此 ,Xgwm4 98 110和Xgwm4 98 190为簇毛麦 1V染色体所特有 ,可以用来快速跟踪导入普通小麦背景中的簇毛麦的1V染色体。

基于EST-PCR的簇毛麦染色体特异分子标记筛选及应用

为定位、转移和利用簇毛麦有益基因,通过花粉辐射,获得一批包括小麦-簇毛麦易位染色体的异染色体系。为了鉴定这批材料中的簇毛麦染色体身份,根据水稻、小麦的EST序列合成了240对STS引物,其中34对引物在普通小麦中国春与簇毛麦间存在多态性;进一步对亲本及簇毛麦二体异附加系进行PCR扩增分析,标记CINAU32-300可追踪簇毛麦1V染色体,标记CINAU33-280、CINAU34-510、CINAU35-1100、CINAU36-380和CINAU37-400可追踪簇毛麦2V染色体,标记CINAU38-250可追踪簇毛麦3V染色体,标记CINAU39-950和CINAU40-800可追踪簇毛麦4V染色体,标记CINAU41-745和CINAU42-1050可追踪簇毛麦5V染色体,标记CINAU44-765和CINAU45-495可追踪簇毛麦7V染色体。加上本室已开发的2个6V染色体特异标记,用这些簇毛麦特异分子标记鉴定辐射诱导材料的部分回交后代,选育出小麦背景中只包含单条簇毛麦染色体的整套1V至7V染色体系,同时有18条易位染色体的簇毛麦身份得到确定,表明这些标记可以用来快速检测普通小麦背景中的簇毛麦染色体或染色体片段。

利用离果山羊草3C染色体诱导簇毛麦2V染色体结构变异

【目的】簇毛麦是普通小麦的一个近缘物种,它具有许多抗病基因,在小麦育种中起重要作用。抗白粉病基因Pm21已被南京农业大学细胞遗传所成功地转移到小麦背景中,并被广泛地用于小麦育种实践。为了进一步转移和利用定位于簇毛麦2V染色体上的有用基因,如抗眼斑病基因、抗条锈基因和护颖颖脊刚毛基因,为小麦育种创造新种质。【方法】通过普通小麦农林26-离果山羊草3C二体异附加系与小麦-簇毛麦2V(2D)二体代换系杂交,综合运用染色体C-分带、基因组原位杂交、染色体构型分析和分子标记分析。【结果】从杂种F2和F3中鉴定出涉及簇毛麦2V结构变异的异染色体系7份,包括纯合缺失系1份(Del2VS·2VL-),易位系4份,其中纯合易位2份(初步推断为T3DS·2VL,T2VS·7DL)、小片段易位1份(T6BS·6BL-2VS)和中间插入易位1份(T2VS·2VL-W-2VL),等臂染色体1份(2VS·2VS)和单端体1份(Mt2VS)。利用可分别追踪2VS和2VL的分子标记Xwmc25-120和NAU/STSBCD135-1进行PCR分析,进一步证明这7份异染色体系中涉及簇毛麦2V染色体片段。【结论】涉及2V短臂的单端体Mt2VS,等臂染色体2VS·2VS和易位系T2VS·7DL在护颖颖脊上有簇状分布的刚毛,而涉及2V长臂的易位系T3DS·2VL无刚毛,进一步证实簇毛麦护颖颖脊刚毛基因位于2VS。离果山羊草3C染色体可有效诱发簇毛麦2V染色体结构变异。

簇毛麦6V染色体短臂特异分子标记的开发和应用

为开发簇毛麦6V染色体短臂特异的分子标记,并利用这些标记对缺失系进行鉴定,选用11个RGA和17对STS引物进行多态性分析,其中1个RGA引物和1对STS引物在对普通小麦扬麦5号、簇毛麦及普通小麦-簇毛麦6VS/6AL易位系进行多态性分析时,分别检测到一条约1000bp和约800bp的多态性片段,将这两个标记转化为稳定的特异性分子标记,分别命名为CINAU17-1086和CINAU18-723。运用这两对引物对一系列材料进行扩增,只有含6V染色体短臂的材料才能扩增出相应的特异条带,表明这两个标记均位于簇毛麦6VS上。进一步利用簇毛麦6VS缺失添加系、易位系将CINAU17-1086标记定位在簇毛麦6VSFL0.58与FL0.70之间,将CINAU18-723标记定位在簇毛麦6VSFL0.45与着丝粒之间。利用这两个特异标记对通过花粉辐射获得的部分簇毛麦6VS结构变异材料进行PCR鉴定,其结果与细胞学鉴定结果一致。CINAU17-1086和CINAU18-723标记可用来快速检测和追踪导入普通小麦背景中的簇毛麦6VS染色体片段,并对缺失系的断点进行了初步界定。

普通小麦近缘物种黑麦1R、簇毛麦1V及鹅观草1Rk^(#1)染色体特异分子标记的筛选

为筛选普通小麦近缘物种黑麦1R、簇毛麦1V及鹅观草1Rk#1染色体特异分子标记,根据已定位于普通小麦第一部分同源群的EST序列设计104对STS引物,对中国春、鹅观草、黑麦及簇毛麦进行多态性分析。在104对STS引物中,有53对在对照普通小麦中国春与鹅观草、黑麦及簇毛麦之间存在多态性。利用普通小麦-黑麦1R^7R二体异附加系筛选出5对黑麦1R染色体特异标记,分别是CINAU 19-500、CINAU20-950、CINAU21-1500、CINAU22-310和CINAU23-2000;利用普通小麦-簇毛麦1V^7V二体异附加系筛选出5对簇毛麦1V染色体特异标记,分别是CINAU23-1700、CINAU24-1050、CINAU25-1650、CINAU26-500和CINAU27-620;利用鹅观草二体异附加系DA1Rk#1、异代换系DS1Rk#1(1A)、端体系DA1Rk#1L、易位系T1Rk#1S.W、长臂缺失系Del1Rk#1L筛选出5对鹅观草1Rk#1特异标记,分别是CINAU27-960、CINAU28-1360、CINAU29-480、CINAU30-560和CINAU31-520。研究表明,可以利用普通小麦的EST序列设计PCR引物,转化成STS标记,筛选普通小麦近缘物种黑麦、簇毛麦及鹅观草等染色体特异的分子标记,快速检测和追踪导入普通小麦背景中的黑麦1R、簇毛麦1V及鹅观草1Rk#1染色体或染色体片段,为深入研究普通小麦远缘杂种材料提供新的工具。

具一对双随体染色体的硬粒小麦-簇毛麦双二倍体的合成、抗病性及细胞遗传学研究

通过幼胚培养和秋水仙碱处理，人工合成了具有一对双随体染色体的硬粒小麦——簇毛麦双二倍体（ＡＡＢＢＶＶ）。根尖细胞染色体数目２ｎ＝４２；花粉母细胞减数分裂中期Ⅰ，２ｎ＝２１″的细胞占６９．９４％，染色体构型为１．０′＋２０．４７″＋０．０２。天然和自交结实率分别为４９．０７％和３９．２３％。籽粒蛋白质含量为２０．９８％。抗白粉、条锈、叶锈和赤霉病。

簇毛麦染色体的改良C-分带

运用改良的C-分带技术,使簇毛麦(Haynaldia villosa Schur)V染色体组的7对染色体全部显示出特异性的丰富带纹,包括与N-带相似的近着丝粒带以及N-带所不具有的末端带和亚端带。这些带纹使V组7对染色体之间以及簇毛麦染色体与小麦染色体之间能明确地相区分。

硬粒小麦-簇毛麦双单倍体愈伤组织染色体加倍技术的研究

对硬粒小麦(Triticum durum)-簇毛麦(Haynaldia vtllosa)杂种幼胚和 F_1幼穗诱导的愈伤组织进行秋水仙素处埋染色体加倍试验,秋水仙素浓度分别为20、50、100和150mg/l;处理时间为8、10、12,14、16和19天;742块愈伤组织在20-27℃温度和每日10小时荧光灯照条件下进行处理。结果表明:不同处理的平均加倍率(结实株数/成活株数×100％)为78.8％,以秋水仙素浓度150mg/l 处理10天的加倍率最高为94.1％。平均处理效果(结实株数/处理愈伤组织数×100％)是15.8％。以秋水仙素浓度150mg/l 处理8天的处理效果最佳为34.1％。试验说明,利用生物技术——幼胚或幼穗培养,使远缘杂种后代快繁和染色体加倍同时进行是可行的。

普通小麦与华山新麦草、簇毛麦杂交后代染色体形态观察

对普通小麦(Tritcium aestivum)与华山新麦草(Psathyrostachys huashanica)杂交后代H9802-6、普通小麦与簇毛麦(Haynaldia villosa)杂交后代V9910-15-4花粉的减数分裂进行了细胞学观察。结果表明:H9802-6出现了异代换系和异附加系,因此该材料还不是很稳定;V9910-15-4后代个体中染色体数目、染色体构型多样复杂,染色体联会过程中出现了环状联会、顶端联会、单价体不联会等异常情况,其杂交后代的稳定及利用较困难。

小麦—簇毛麦染色体代换系、易位系特异蛋白组分的双向电泳比较分析(英文)

利用双向电泳技术,对栽培小麦(AABBDD)、染色体代换系(6V/6A)、易位系(6VS/6AL)、(6VS/6DL)和簇毛麦(VV)的叶片全蛋白进行了比较研究。在栽培小麦、代换系和两个易位系中检测到超过350个蛋白组分,它们的分子量范围是10-110 KD,等电点在4.5-8.6之间。栽培小麦、6V/6A、6VS/6AL、与6VS/6DL之间的双向电泳谱型极为相似,但与簇毛麦不同。在代换系、两个易位系和簇毛麦中检测到了特异蛋白组分16 KD/pI5.0,而在栽培小麦中未检测到该组分,这些结果表明16 KD/pI5.0蛋白可能定位于簇毛麦V染色体短臂上。