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3. Methodology used in maintenance and research of silkworm germplasm resource

3.1 Collection, assortment and identification

Investigation on germplasm varieties is one of the most important part in the maintenance of silkworm germplasm resource, only based on these basic research results that the germplasm could be effectively utilized in silkworm breeding and genetic research. Collection, assortment and identification are usually done in the early stage, and maintenance was strictly followed the specific rules in China, that is:

Investigations on the silkworm germplasm resources are mainly including following aspects:

3.2 Records of biological features and characteristics

3.3 Investigation of economic characteristics

3.4 Identification of resistant ability

Resistant ability is generally investigated under the artificially set environment condition to identify the specific property in different adverse conditions. For example:

3.5 Genetic analysis

Methodology of silkworm germplasm maintenance and research adopted by National Sericulture Institute of China Agriculture Science Academy refer to Annex 2.

3.5.1 Long term cold storage techniques for diapaused polyvoltine germplasm strains

The polyvoltine varieties have to be raised for many times a year in the germplasm maintenance, which not only created more works for the maintenance but also resulted in fast degeneration for more rearing generation in a year. The new cold storage technique had made it possible to rear diapaused polyvoltine varieties once a year. That is to rear the polyvoltine varieties in summer season, laid eggs (August 1-5) are preserved under 25°C for 35-40 days, then under 20°C during September to October, after that, the temperature gradually decreased to the natural temperature until 5°C, egg bath takes place in mid-December, and then be transferred into cold storage under 2.5 °C or 0°C, take out for intermediate temperature under 10°C-15°C for 7 days in late April, and put back to the cold storage under 2.5 °C until mid June before incubation starts. Silkworm will be reared again in summer season. Experiments had shown that there were no significance difference between the new method and normal rearing cycle in the rearing performance such as hatching rate, cocoon weight, cocoon shell weight, shell ratio, survival rate of larva-pupa, and voltinism character.

3.5.2 Maintenance of lethal genes

There are many lethal genes discovered in silkworm, and these genes require special techniques to be maintained in the germplasm resource. The small egg mutation sm, when the female moth is an homozygous sm, it lays 100% small eggs which are only half of the normal size and normal embryo cannot be developed and become dead eggs. Usually, to keep this gene, rear 5-10 layings of heterozygous sm separately, and select the layings with a ratio small egg: normal egg 1:1 as the seed layings, and 5-10 laying are reared separately again at the next generation. This will cost much more man power and material for maintenance, and moreover, very hard to be used for linkage analysis as marker gene. Now, take the dominant gene of larval zebra marking, which is in the same linkage as the marker, make the Ze +/+sm × + sm/+sm system to keep the sm gene, just use zebra marking × non-zebra marking, can produce the heterozygous sm eggs, therefore, only one batch of mixed rearing is required for the next generation. Here, the complete linkage of female silkworm was used, it is easy to distinguish that the zebra marking individual is heterozygous sm, to produce normal eggs, and non-zebra marking individual is homozygous sm, to produce small eggs. This principal can also be used in the maintenance of second small egg sm-2, kidney shape egg ki, shriveled egg Grc01, by the system of ++/ch sm-2 × ch sm-2/ ch sm-2, E +/+ki × + ki/+ ki, + +/pGrc01 × pGrc01/pGrc01. Other lethal genes like larval lethal gene l-nl, small non-molting larva nm-d, white dead egg al, stinky larva sku etc. can also make similar system to be maintained in a much easier method.

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