Sequential four-molecule analysis experiments on Chlamydomonas reinhardtii

Summary

Neurosporacrassa belongs to the Ascomycetes class of fungi and is a good material for sequential tetramolecular analysis. The mycelium of Neurosporacrassa is haploid (n=7), and each mycelial cell contains dozens of nuclei. There were two kinds of conidia formed by the apical breakage of the mycelium: small conidia containing one nucleus and large conidia containing several nuclei. Conidium germination into mycelium, can be generated again conidia, and so on, forming the asexual reproduction process of rough vein cell bacteria. Source: Laboratory Course in Genetics

Operation method

basic program

Principle

Strains of P. vulgaris have two different mating types, denoted by A, a, or mt+, mt-. The mating types are controlled by a pair of alleles and conform to Mendel's law of segregation. The cells of different strains can reproduce sexually after joining. In the process of sexual reproduction, the mycelium of different splice types contact each other, the two nuclei are paired, but not fused, forming binucleosomes. With the development of binucleosomal mycelium, many elongated cystic sporangia, i.e., ascospores, develop in the ascospores' shells. The immature ascospores contain diploid conidia formed after fusion. After the formation of conidia will soon be in the development of cysts in meiosis, the formation of four spores, and then a mitosis, the formation of eight haploid cystospores, and the whole cyst shell will become mature cystospores (Figure 21-1) Rough vein cell bacterial cystospores is a haploid, that is, they are the product of meiosis, from which the mycelium is also a haploid germinated growth. Therefore, traits determined by a pair of alleles can be seen to segregate in the offspring of a cross. In Verticillium rougheri, the product of one meiotic division is contained in a single ascospore, so it is easy to visualize the segregation of a pair of alleles in the tetrad produced by one meiotic division from the traits of ascospores in a single ascospore. Moreover, the eight ascospores were sequentially arranged in a narrow and elongated ascospores, which made it possible to perform mitosis mapping and to detect geneconversion. If the two parent strains have a certain genetic trait difference, then each cyst formed by hybridization, there must be four ascospores belonging to one type and four ascospores belonging to another type, with a separation ratio of 1:1, and the ascospores are arranged in a certain order. If this pair of alleles is related to the color or shape of the ascospores, then the different arrangements of the ascospores can be directly observed under the microscope (Figure 21-2).

Materials and Instruments

Wild-type strain of Verrucomicrobium rougheri: Lys + Splice type A Conidia are pink in color Verrucomicrobium rougheri lysine-deficient strain: Lys - Splice type a Conidia are white in color.
Basic medium Supplementary medium Complete medium Hybridization medium 3% Lysol
Microscope Constant temperature incubator Tweezers Slides Test tubes and Petri dishes

Move

1. Hybridization: Inoculate the parental strains on the same hybridization medium. First connect the defective type, then connect the wild type, and inoculate the conidia or mycelium of the two parental strains on the hybridization medium at one time. Then put a sterilized folded filter paper on the medium and label the test tubes with the parents, date of hybridization and the name of the experimenter. 2.

2. Cultivation: The test tubes were put into a 25℃ incubator for cultivation. 5~7d later, we could see the appearance of many brown cysts, which gradually developed and matured, became bigger and blacker, and could be observed under a microscope in about 21d (3 weeks). It should be noted that lysine-deficient ascospores mature later, and when the wild-type ascospores have already matured and turned black, the defective ascospores are still gray, so we can observe the different ascospores directly under the microscope. If the time of observation is not properly chosen, good results cannot be observed. If the observation time is too early, the spores are immature and all of them are gray; if it is too late and all of them are mature and all of them are black, we cannot distinguish the ascospores from each other. Therefore, it is best to take a few cysts daily when the fruit develops to a mature size and the cyst shell starts to turn black, and then place them in the refrigerator at 4~5℃ at the right time to ensure that the observation can be made within 3~4 weeks. 3.

3. Pressure piece observation: put the strip of filter paper with cysts into 3% Lysol solution for 10min to kill the spores to prevent contamination of the laboratory. Take a slide, drop 1 to 2 drops of 3% Lysol solution, and then use the inoculation needle to pick out the cysts on the slide, cover with another slide, finger pressure piece, the cysts will break the fruit, placed in the microscope under low magnification observation, that is, a visible cysts will be scattered in the fruit of the cysts of 30 to 40 cysts. Observe the arrangement of ascospores. The reason for using a slide to cover the slide instead of a coverslip is that the ascospores are very hard and the coverslip is easy to break. This procedure does not require aseptic operation, but care should be taken to ensure that the conidia are not dispersed in such a way that the type of ascospores cannot be distinguished. Observed slides, used forceps and dissecting needles should be soaked in Lysol solution and then removed and washed to prevent contamination of the laboratory.

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