Taking the application of molecular marker technology in wheat stripe rust resistance breeding as an example.
molecular marker
In the field of agricultural basic and applied research, molecular marker technology has been applied to the research of agricultural germplasm resources and breeding, especially in the construction of molecular genetic map and the marking of target trait genes.
Compared with morphological markers, cell markers and biochemical markers, molecular markers have the following advantages: ① They can be detected in many tissues and growth stages of plants, and are not limited by time and space. ② The number is large, covering the whole genome. ③ Many markers are * * * dominant, which can distinguish whether genotypes are homozygous or not and provide complete genotypes. In the aspect of marking wheat leaf rust resistance genes, molecular markers can reveal the genetic mechanism of wheat rust resistance at a deeper level by looking for molecular markers closely linked with the rust resistance genes, which can not only specifically detect the target genes in breeding materials with different genetic backgrounds, but also screen multiple resistance genes at any growth stage at the same time, providing a faster, more stable and reliable method for understanding the resistance genes contained in resistant sources and resistant varieties.
At present, the molecular markers used to mark wheat leaf rust resistance genes mainly include the following:
2.2. Application of1RFLP technology in wheat leaf rust resistance gene marker
RFLP (Restriction Fragment Length Polymorphism), as a genetic analysis tool, began in 1974 and was applied to plants in 1980s. The basic principle is that the genomic DNA of a species produces a considerable number of DNA fragments of different sizes under the action of restriction endonucleases, and the fragments related to the labeled DNA are detected by using radioisotope-labeled DNA as probes, thus constructing a polymorphic map; It represents the difference in fragment length of genomic DNA after digestion with restriction enzymes. RFLP technology is widely used in the construction of wheat genetic map, the labeling and location of wheat target genes.
With regard to the RFLP marker of wheat leaf rust resistance gene, SCHACHERMAYR et al. [8] divided the 3.9Kb Hind fragment of Lrkl0 gene encoding receptor protein kinase into six sub-fragments with PST Ⅰ I, and used these sub-fragments as RFLP markers, and found the specific Lrk 10 fragment as a molecular marker closely linked to wheat leaf rust resistance gene Lr 10. Southern hybridization between this subfragment and single-gene lines with known disease resistance genes showed that the 3.9Kb hind Ⅲ Ⅲ fragment only existed in near-isogenic lines carrying leaf rust resistance gene Lr 10. In addition, the RFLP marker Krkl0-6 was transformed into STS marker STS lrk 10-6, and it was found that the 282bp fragment only existed in the varieties carrying Lr 10. F2 population segregation further proved that the 282bp fragment was closely linked with Lr 10. SCHACHERMAYR et al. [9] used near-isogenic lines to find RFLP and RAPD markers closely linked to leaf rust resistance gene Lr24. Of the 1 15 RFLP probes detected, 6 were closely linked to Lr24. Among 360 random RAPD primers, 1 1 primer was found, one of which was closely linked with Lr24. The RAPD products were cloned and sequenced, and transformed into more stable and reliable STS markers, which laid a good foundation for molecular marker-assisted breeding. FEUILLET et al. [10] used wheat near-isogenic line Lr 1/6*Thatcher and susceptible variety Frisal to separate F2 population, and located 16 in 37 RFLP in the fifth homologous group. In addition, it can reveal the polymorphism between Lr 1/6*Thatcher and Frisal, and I 1 RFLP probe can reveal the polymorphism between near-isogenic lines. The F2 population segregation analysis found that three of them were linked to the resistance gene, and a probe pTAG62 1 located on chromosome 5D was proved to be closely linked to Lr 1, and this RFLP marker was transformed into more.
AUTRIQUE[ 1 1] and others used four wheat near-isogenic lines with different disease resistance genes to select clones according to the position of disease resistance genes on their chromosomes. At the same time, other clones were screened from the RFLP linkage map of barley and the RFLP map of D genome. Searching for polymorphic molecular markers by hybridization. The results showed that eight molecular markers located on 7DL and 3DL chromosomes were isolated from the disease-resistant genes Lr 19 and Lr24***. Lr9 from Leymus chinensis is located on chromosome 6B. A clone XksuD27 was isolated from LR9 * *, and two RFLP markers were closely linked to Lr32, with genetic distances of (3.3±2.6)cM and (6.9±3.6)cM, respectively.
2.2 RAPD Marker of Wheat Leaf Rust Resistance Gene
NAIK et al [12] found a RAPD marker OPJ-O 1 from 80 random primers, which can reveal the polymorphism of donor parents and recurrent parents. The 387bp polymorphic product was cloned and sequenced, and it was designed as a more stable STS marker. Using BSA method to analyze F3 population, it was found that the specific product of 387bp only appeared in the resistant population, but not in the susceptible population. It was proved that RAPD markers OPJ-O 1 and STS markers were closely linked with Lr28. SIELDLER et al. [13] used 400 random primers and 14 DNA probes to screen the polymorphism of near-isogenic line Lr9, and conducted genetic linkage analysis through F2 population. The results showed that two RAPD markers and 1 RFLP markers could distinguish susceptible strains and were closely linked with Lr9. WILLIAM et al. [14] found three primers 0PG-05, 0P 1- 16 and OPR-03 from 400 random primers by RAPD technique, which can reveal the polymorphism in disease-resistant population and susceptible population. After cloning them into probes, Southern hybridization was performed on the recombinant population to determine the first two probes and lasting leaf resistance. SCHACHERMAYR et al [15] screened three RAPD markers linked to wheat leaf rust resistance gene Lr9 from 395 random primers, cloned and sequenced their specific products, and then transformed them into more stable STS markers. The detection of F2 and F3 isolates showed that three RAPD markers were 0PA-07, 0PJ-l3, OPR- 15 and respectively. Another RFLP marker, PSR546, is also closely linked to Lr9, which is linked to the above four markers.
The DNA markers are closely linked, and the genetic distance is (8 × 2.4)cM. The marker is located on the long arm 6BL of wheat chromosome. DEDRYVER et al. [16] used a wheat near-isogenic line containing Lr24. Among 125 random primers, only primer OP-H5 can be amplified in the resistant parent RL6064.
A specific band of 700bp was added, which was not found in the infected parent Thatcher. F2 population segregation proved that the marker was similar to Lr24 complete linkage. It is transformed into a stable and reliable SCAR marker, which provides a powerful tool for molecular marker-assisted breeding.
3 Other molecular markers
Although RFLP and RAPD are two common molecular markers, the polymorphism detected by RFLP in wheat is low, only 20%-38%. RAPD is a convenient and economical molecular marker, but its repeatability and stability are poor. Other molecular markers, such as SSR, ISSR and AFLP, are informative and have broad application prospects in the research of crop genetic resources, especially wheat genetic resources.
4 molecular marker-assisted selection
At present, molecular marker technology has been applied to breeding practice and has shown its unique advantages. Searching for molecular markers closely linked with important agronomic traits is the basis of molecular marker-assisted selection (also known as molecular breeding) and gene mapping cloning. Molecular-assisted selection (MAS) is a combination of biotechnology and traditional genetic breeding, which can reduce the linkage burden that is difficult to eliminate in the traditional backcross breeding process, and can also aggregate different leaf rust resistance genes into the same fine variety, thus achieving the same gene accumulation and obtaining lasting resistance. On the basis of obtaining stable molecular markers, the gene was isolated and cloned by chromosome walking and other methods.
Because molecular marker breeding technology is not mature and perfect at present, it can not be used as a breeding method alone. China has rich experience in traditional breeding, so we should pay attention to combining the advanced technology of molecular markers with the rich experience of breeders to make molecular marker-assisted selection play a greater role.