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:

• Those newly collected varieties should be separately raised during the first two generations when newly introduced, and could be raised in the same room of the existed stocks only after confirmed that they are pebrine disease free.
  
  
• The uniqueness of each variety is maintained only through systematic and scientific manipulation of the germplasm. Otherwise, it will degenerate to loose its original characteristics in genetic markers or in its performance of economic characters, or it will become lost because of drastic reduction or fertility to the extent that it is no longer able to produce viable offspring.
  
  
• Generally, rearing once a year in the congenial season for monovoltine and bvoltine races, two or three times for diapaused multivoltine, nonstop rearing for non-diapaused multivoltine.
  
  
• In order to prevent inbreeding depression and maintain the specific properties, the newly hatched larvae should be collected from at least 20 layings: cut one corner of eggs from each of the 20 layings, and stitch them together, brush 1500-2000 newly hatched larvae in the form of mixed rearing.
  
  
• Strictly maintain the purity of the variety in each stage of egg incubation, larvae rearing, mounting, cocoon collection, moth emergence, and egg production. In any case, if the abnormal character which is different from the original record, delete it, or separate if it could be a new mutation.
  
  
• Duplicate set of eggs should be produced so as to prevent the distinction of the stock in case of unsuccessful reproduction due to some unexpected circumstance.

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

3.2 Records of biological features and characteristics

• Race, voltinism, moltinism;
  
  
• Eggs: shape, color, shell color, coherence;
  
  
• Larvae: taxis, eating habit, marker, body shape, color; size;
  
  
• Cocoon: size, shape, color, wrinkle;
  
  
• Moth: size, color, mating habit, egg laying habits, laid egg number;
  
  
• Except the word records, photocopies of egg, larvae, cocoon, pupa moth should be maintained.

3.3 Investigation of economic characteristics

• Characteristics investigated during rearing: development uniformity, duration, survival rate of larvae, larva-pupa survival rate, rate of dead worm cocoons, cocoon weight, cocoon shell weight, cocoon shell ratio.
  
  
• Characteristics should be investigated by special inspection machine: filament weight, filament length and size, reelability, neatness, strength and elongation, cohesion, degumming loss percentage of cocoon shell, lousiness and etc..

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:

• To identify the tolerant ability to high temperature and humidity, larvae reared under the environment at the temperature 30-32°C, and RH over 90%;
  
  
• To identify the resistant ability to certain disease, inoculation of a specific bacteria or virus.
  
  
• To identify the tolerant ability to certain insecticide or fluoride pollution, by oral administration of miro-concentration of insecticide or fluoride.

3.5 Genetic analysis

• Gene behavior and gene location of the new mutation. There are high rate spontaneous mutation in silkworm, to identify and study its function, inheritance and location, enrich and modify the silkworm gene linkage map, are one of the important aspect of germplasm maintenance.
  
  
• Biochemical genetic research. To study isoenzyme pattern of hemolymph, skin, midgut and silk gland, protein type of blood and digestion juice in different development stage are also widely concerned in germpalsm investigation.
  
  
• Molecular linkage mapping by bio-technique such as RAPD, RELP, SADF, AFLP. This is the newly developed field and also widely recognized in silkworm germplasm research, will be the hot spot in the near future.

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 Gr^c01, by the system of ++/ch sm-2 × ch sm-2/ ch sm-2, E +/+ki × + ki/+ ki, + +/pGr^c01 × pGr^c01/pGr^c01. 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.