基因组原位杂交技术在植物研究中的应用

基因组原位杂交是以亲本之一的总基因组DNA做探针,另一亲本的基因组DNA做封阻,在荧光原位杂交技术的基础上发展起来的一种染色体/染色质检测技术。在其发展的十几年里,已在植物的基因组研究中发挥了重要的作用。应用这一技术可对多倍体中基因组之间的亲缘关系、基因组组成及起源进行研究;对杂交种中染色体组的组成进行分析;对代换系、附加系和易位系进行有效的鉴定,并对其中的外源染色体或染色体片段的来源、大小、数目及发生位点进行检测和定位。此外,利用基因组原位杂交技术还有助于确定物种间的同源性;研究杂交种中来源不同的染色质在核中的分布;探索B染色体的起源、染色体间的配对、重组、交换等现象。随着基因组荧光原位杂交技术体系的不断发展、完善和改进,其应用范围不断拓展,在植物基因组研究领域中发挥了越来越重要的作用。     

Development and characterization of common wheat-Thinopyrum intermedium translocation lines with resistance to barley yellow dwarf virus

We developed some wheat-Th. intermedium translocation lines,Yw642, Yw443 and Yw243, etc., showing good BYDV resistance from L1by induced homoeologous pairing using CS ph mutant. Characterization ofthese wheat lines was carried out by GISH and RFLP analysis. The resultsof GISH showed that the lines, YWw42, Yw443 and Yw243, etc., arehomozygous wheat-Th. intermedium translocation lines, in which thechromosome segments of Th. intermedium were transferred to thedistal end of a pair of wheat chromosomes. RFLP analysis indicated that thetranslocation chromosome of the wheat lines is T7DS · 7DL-7XL. Thebreakpoint of the translocation is located on the distal end of 7DL, betweenXpsr965 and Xpsr680 about 90–99 cm from the centromere. The BYDVgene is located on the distal end of 7XL around Xpsr680, Xpsr687 andXwg380. The RFLP markers of psr680, psr687 and wg380 werecosegregated with the BYDV resistance respectively and could be used formolecular assisted selection (MAS) in wheat breeding program for BYDVresistance.     

Designing grasses with a future – combining the attributes of Lolium and Festuca

Lolium species (considered the ideal grasses for European agriculture) are not sufficiently robust to meet many of the environmental challenges that face extensive agriculture in less favoured areas. Fortunately, adaptations to abiotic and biotic stresses exist amongst Festuca species related closely to Lolium. The complex of species has an enormous wealth of genetic variability and potentiality for genetic exchange, thus offering unique opportunities for the production of versatile hybrid varieties with new combinations of useful characters suited to modern grassland farming. The attributes of Lolium and Festuca can be combined into a single genotype by amphiploidy or alternatively, a limited number of characters can be selectively introgressed from Festucainto Lolium or vice versa. Androgenesis of the interspecific hybrids can generate genotypes combining characters that may not be recovered by sexual backcrossing. Genomic in situ hybridization(GISH) can differentially ‘paint’ the chromosomes of Lolium and Festuca and identify Lolium-Festuca recombinant chromosomes. GISH is valuable in the analysis of amphiploids, introgressions and androgenic genotypes and can be used to physically map introgressed traits. Introgression mapping is a powerful new approach to the mapping of traits and arises from a fusion of physical and genetic mapping. For example, in a diploidLolium introgression genotype with only one introgressed Festucasegment, the gene(s) for any Festucaderived trait expressed by the plant must be located within the segment. Using GISH and molecular markers, a dense but highly localised map of the Festuca segment is made in isolation of the Loliumgenome – this may simplify QTL analysis. This revised version was published online in July 2006 with corrections to the Cover Date.     

Characterization of a wheat–Thinopyron intermedium substitution line with resistance to powdery mildew

Among the progenies of a hybrid between common wheat Triticum aestivum L. cv. Yannong 15 and Thinopyron intermedium, plant E99018 was identified with the chromosome number 2n = 42 and stable agronomic traits. An analysis of the metaphase chromosome pairing indicated that it formed 21 bivalents but that 2 univalents were present in the F₁ hybrid of this plant with common wheat. Resistance verification by race 15 and with mixed races of Blumeria graminis f. sp. tritici at the seedling and adult stages showed that at both stages, the plant was immune to powdery mildew. In situ hybridization with the genomic Th. intermedium and the St genome DNAs as probes and wheat DNA as a block has shown that it contained a pair of Th. intermedium chromosomes. On the basis of the hybridization pattern of the St genome probe to the critical chromosome, a conclusion was reached that this pair of chromosomes belonged to the E genome. Therefore, plant E99018 was a spontaneously formed substitution line. An analysis by 116 SSR markers indicated that the substituted wheat chromosome was 2D and the most likely substitution in E99018 is 2E(2D).     

小麦–华山新麦草异代换系DH2322的分子细胞遗传学鉴定

利用分子标记、细胞学、基因组原位杂交(GISH)等技术,结合田间农艺性状调查,对小麦–华山新麦草七倍体材料H8911与硬粒小麦D4286杂交F4代分离群体中株系DH2322进行了综合鉴定。华山新麦草基因组特异SCAR标记鉴定表明,DH2322含有华山新麦草遗传物质;细胞遗传学观察显示,DH2322染色体构型为2n=42=21 II;有丝分裂和花粉母细胞减数分裂中期I基因组原位杂交(GISH)鉴定表明,DH2322的染色体由40条小麦染色体和2条华山新麦草Ns染色体构成,且2条Ns染色体能完全配对为一个二价体;SSR和STS分子标记分析表明,DH2322缺失小麦D基因组的2D染色体,而含有华山新麦草的2Ns染色体;农艺性状分析结果表明,DH2322具有双亲的形态学特征,结实性好,穗长和穗粒数显著大于亲本。     

Overcoming hybridization barriers between pea and some of its wild relatives

Pea would benefit from the plasticity and adaptability of its cross-incompatible relatives Pisum fulvum and Lathyrus sativus L., and we have tested reciprocal sexual crossings by manually cross-pollinating plants of genotypes of these three species. Studies of in situ germination of pollen grains on stigmata showed that pollen tubes were generally unable to germinate or could not reach the ovary. A few putative hybrid pods were nevertheless harvested, with one grain per pod germinated in vitro, then micropropagated for flow cytometry, isoenzyme, molecular (ribosomal ITS PCR-RFLP) and genomic in situ hybridization (GISH) studies. One such grain was recovered from an inter-generic cross of P. sativum x L. sativus and four from an inter-specific P. sativum x P. fulvumcross. A strong cross-incompatibility was shown between pea and grass pea, where the putative hybrid turned out to be pea. Conversely, with the interspecific, P. sativum x P. fulvum cross, flow cytometry and isoenzymes with leaf tissues strongly suggested hybridity, while molecular approaches and GISH confirmed the production of inter-specific hybrids, and without the need for a wild type P. sativum accession as a bridging cross.     

Analysis of wheat-Thinopyrum intermedium derivatives with BYDV-resistance

Chromosome compositions of seven lines, derived from hybrids between a wheat cultivar and the wheat-Thinopyrum intermedium addition line Z6, with barley yellow dwarf virus (BYDV) resistance, were determined by genomic in situ hybridization, cytogenetic and SSR assays. The results showed that line N522 was a disomic addition line, lines N420 and N439 were 2Ai-2(2B) chromosome substitution lines, lines N431 and N452 were 2Ai-2(2D) chromosome substitution lines, line N523 was a 2Ai-2S(2D) ditelosomic substitution line, and line N530 was a double ditelosomic line with the mitotic chromosome number of 2n = 40 + 4t. One pair of telosomes in line N530 lacked several proximal SSR markers of chromosome 2AS, but possessed certain terminal markers, which were consistent with an acrocentric structure, and the other pair of chromosome arms were presumably 2Ai-2S telosomes with BYDV-resistance. These wheat-Th. intermedium lines provide useful genetic resources for developing alien chromosome translocation lines.     

The long and winding road leading to the successful introgression of downy mildew resistance into onion

Downy mildew resistance originating from Allium roylei Stearn provides a complete resistance to onions and is based on one, dominant gene. Since A. roylei can successfully be hybridized with onion (A. cepa L.), a breeding scheme aimed at the introgression of this gene was initiated ca. 20 years ago. Several setbacks in this programme were encountered, firstly the identified molecular marker linked to the downy mildew resistance locus became increasingly difficult to use and finally lost its discriminating power and secondly the final step, making homozygous introgression lines (ILs), turned out to be more difficult then was hoped. GISH analysis showed that the chromosomal region harbouring the resistance locus was the only remaining piece of A. roylei in the nuclear background of onion and it also confirmed that this region was located on the distal end of chromosome 3. It was hypothesized that some factor present in the remaining A. roylei region was lethal when homozygously present in an onion genetic background. The identification of an individual with a smaller and more distally located introgression fragment and homozygous ILs in its progeny validated this hypothesis. With the help of these nearly isogenic lines four AFLP^® markers closely linked to the resistance gene were identified, which can be used for marker-aided selection. The introduction of downy mildew resistance caused by Peronospora destructor into onion is a significant step forward in the development of environmentally-friendly onion cultivars.     

受辐照窄颖赖草花粉DNA进入小麦胚囊的电镜自显影证据及杂种原位杂交鉴定

采用3H 胸腺嘧啶标记窄颖赖草 (Leymusangustus)花粉结合电镜自显影的方法证明受辐照窄颖赖草花粉DNA进入了小麦胚囊 ,运用基因组原位杂交技术证明所得杂种为真杂种 ,经辐照花粉获得的普通小麦J 1 1与窄颖赖草杂种中窄颖赖草染色体发生了数目和结构变异     

甘薯近缘野生种Ipomoea trifida(4x) GISH分析

以甘薯近缘野生种I. trifida (2x)为探针, 与I. trifida (4x) 2个株系“695104”和“697288”的体细胞染色体进行基因组荧光原位杂交, 结果显示, 2株系都与I. trifida (2x)有很近的亲缘关系, 但2株系的信号存在差异。“695104”几乎所有染色体整条都有均匀明亮的信号, 应为I. trifida (2x)基因组直接加倍而来；而 “697288”与“695104”不同, 虽然各条染色体也均有杂交信号, 但信号的区域与亮度有差异, 较为复杂, 可分为三种情况。第1种是整条染色体有均匀明亮的信号, 亮度与分布区域同“695104” , 有41条；第2种是几乎整条染色体有信号, 但亮度较第一种暗, 有14条；第3种为染色体部分区域有信号, 亮度较前二者更暗, 有5条。推测 “697288”是在加倍同时或之后又发生了基因组重组与部分变异。