Herrmann R G, Martin R, Busch W, Wanner G, Hohmann U
Botanisches Institut der Ludwig-Maximilians-Universität München, Germany.
Symp Soc Exp Biol. 1996;50:25-30.
Three principal approaches have been used in our laboratory to analyze Triticeae genomes. (i) Synteny analysis: synteny among different Gramineae genomes was studied employing the elegant system of the Agropyron chromosome-induced deletion lines of wheat. Deletion mapping, predominantly of the homoeologous group 7 chromosomes, has led to the construction of a high density physical consensus map of wheat. The integration of wheat, barley and oat RFLP markers proves the colinearity between the wheat A-, B- and D-genomes, the H-genome of barley, and the E-genome of Agropyron. (ii) Light microscopic in situ techniques: the recent improvement of a drop technique for plant protoplasts was crucial for the sensitivity enhancement of fluorescence in situ hybridization (FISH), the efficient preparation of plant chromosomes for high resolution scanning electron microscopy, mapping of low-copy sequences, and comparative in situ hybridization. A tandemly amplified repetitive sequence element from microdissected barley chromosomes has enabled the karyotyping of Gramineae genomes in a single step. We have isolated and characterized members of this element family from other Triticeae species using PCR. The significant interspecific sequence differences were useful to identify single plant genomes, chromosomes and chromosome segments via post-hybridization washes under different stringency conditions. These sequences are also useful for simultaneous double or triple hybridization experiments in an attempt to localize new sequences on specific chromosomes or chromosome segments. The physical mapping of the Sec-1 locus has been refined on the satellite of chromosome 1R of rye, and the syntenic locus on barley chromosome 1H was identified. (iii) Physical mapping of rDNA sequences by high resolution electron microscopy: a method was developed for in situ hybridization and signal detection using high resolution field emission scanning electron microscopy and a backscattered electron detector. Colloidal gold particles were localized on chromosome structures resembling the 30 nm fibre. An rDNA probe was located in the secondary constriction and the highly compact adjacent regions of barley chromosomes.
我们实验室采用了三种主要方法来分析小麦族基因组。（i）共线性分析：利用小麦冰草染色体诱导缺失系这一精妙系统研究了不同禾本科基因组间的共线性。缺失作图，主要是对第7部分同源群染色体进行缺失作图，已促成构建了一张高密度的小麦物理一致性图谱。小麦、大麦和燕麦RFLP标记的整合证明了小麦A、B和D基因组、大麦H基因组以及冰草E基因组之间的共线性。（ii）光学显微镜原位技术：植物原生质体滴片技术的近期改进对于增强荧光原位杂交（FISH）的灵敏度、高效制备用于高分辨率扫描电子显微镜观察的植物染色体、低拷贝序列作图以及比较原位杂交至关重要。来自显微切割大麦染色体的串联扩增重复序列元件使得能够一步完成禾本科基因组的核型分析。我们利用PCR从其他小麦族物种中分离并鉴定了该元件家族的成员。种间显著的序列差异有助于通过在不同严谨条件下的杂交后洗涤来鉴定单个植物基因组、染色体和染色体片段。Sec-1基因座的物理图谱在黑麦1R染色体的卫星区域得到了优化，并且鉴定出了大麦1H染色体上的同线基因座。（iii）通过高分辨率电子显微镜对rDNA序列进行物理作图：开发了一种利用高分辨率场发射扫描电子显微镜和背散射电子探测器进行原位杂交和信号检测的方法。胶体金颗粒定位在类似于30 nm纤维的染色体结构上。一个rDNA探针定位在大麦染色体的次缢痕和高度致密的相邻区域。
