Since 1949, there is a great achievement not only in the collection, assortment and investigation of local varieties all over the China, but also introduced a lot of new silkworm varieties from Japan, former Soviet Union, and other countries to enrich the silkworm gene bank.
There are over 1000 strains or varieties of mulberry silkworm preserved in different institute in China at present, which including five aspects of the sources:
All these preserved resources could be divided into five different ecological types, that is:
There are 1000 varieties or strains (including mutations) preserved in Sericulture Research Institute of China Agricultural Science Academy, in Zhengjiang City of China. 300 breeding materials and 602 gene mutations are preserved in College of Sericulture, South-West Agricultural University, Chongqing City of China. And some other breeder's stocks maintained in different universities and provincial level institutes. The whole information is too much to be listed here, however, the overview of the gene mutations preserved in South-West University is listed in Annex 3. In 1987, the book "Record of Silkworm Varieties in China" was published. In this book, recorded 510 silkworm varieties from 26 preserved institutes and universities, which including 139 popularized varieties, 10 authorized improved varieties after national identification, 199 local varieties, 70 with special characteristics, 36 genetic material, 56 introduced variety from foreign countries. The description system used to record the characteristics of each variety was shown in Annex 4. Researches and achievements on silkworm germplasm resources are mainly on the following aspects.
Sericulture Research Institute of China Agricultural Science Academy was awarded for Second Prize of Scientific Advancement on Achievement of the research and application of silkworm germplasm resources in 1995. Their main achievements included following aspects:
· Finished the gene survey of silkworm germplasm in China
Systematical investigation on the gene bank of silkworm germplasm varieties was completed while keeping the target to discover and collect different types of gene mutations. In the national silkworm germplasm center (Zhengjiang), over 20 character had been investigated including morphological, physiological and biochemical characters of each variety such egg color, egg shape, chorion color, newly hatched larval color, body shape, cocoon color, cocoon shape, pupa color, pupa shape, moth color, moth shape, moth eye characters, globular cell of hemolymph, amylase of digestion juice, amylase of blood, albumin, of hemolymph, low molecular lipoprotein of pupa, eating habit of the whole preserved varieties(around 1000 accessions) in its germplasm center during 1985-1995. Meanwhile, 400 mutation individuals were discovered, among them 126 gene mutations were selected out and 50 were discovered as new mutation in the world. During the corresponding period, there was a total around 90 new mutations on mulberry silkworm discovered in the world, therefore, this institute alone had account for more than 50% of the total new discoveries. All these research discoveries had greatly enriched the silkworm germpalsm bank in China and supplied the materials for the genetic research and silkworm breeding.
· 83 gene mutations were analyzed
These 83 gene mutations were determined in loci, and its linkage group. Among them, 26 gene mutations are discovered and localized at first time in the world, such as Zhengjiang gray egg (GrZ, 2-6.9), Xu gray egg (GrXu, 2-6.9), second recessive chocolate color of newly hatched larvae(ch-2, 18.00), Zhengjiang narrow thorax (nb-Z, 19-12.0), Hainan flossy cocoon(Syc, 7-32.1), Jiang's striped marking(PJS, 2.00), black larva(blb, 9-22.9), second dark color of moth(mln2, 18-14.1), second face and tail marker (cts2, 16-4.6), Sheng's black marker (PBS, 2-0.0), oe translucent larva(oe, 10-?), densonucleosis-resistant gene (to Zhengjiang strain) (nsdZ, 15-?), non-lipis wing of moth (nlw, 13-?), second recessive gray egg (grc, 12-?), 013 translucent larvae(ox-4, 5-?),
· Determined the marker genes or representative genes for the total 28 linkage groups of silkworm Bombyx mori and modified the 18 linkage.
Marker genes for the 28 linkage groups were determined, its corresponding linkage retrieve system of the 28 linkage and detecting system for gene loci were established. The map of the 18th linkage group was modified based on the research result on genetic analysis of new gene mutation, the second recessive chocolate color of newly hatched larvae ch-2, by the application of the above established system. This modification was acknowledged and adopted quickly by the academic circles of the international sericulturists.
· Some valuable mutations were introduced into the improved varieties to breed practical varieties; a number of breeding materials and basic lines with specific characteristics were obtained through utilizing the mutation genes.
i. Normal color of silkworm moth is brown-rice color, it is very difficult to distinguish the Japanese or Chinese race for mating during the hybrid production. Research had approved that the new mutation mln2 had no negative effect on the economic characters, therefore, it was introduced into the improved variety "Fengyi" (Chinese race) to make the basic variety "Fenghei" with dark moth character. Its moth is deep dark color, and very easily be recognized from normal color moth, therefore, when mated with normal Japanese variety, it is so distinguish in color that any pure pair could be caught by eyes right away, and 100% hybridization can be completely ensured.
ii. The non-lepis wing mutation (nlw) was discovered in 1987, and localized in 13 linkage group. By introducing this character into improved variety, moth dust could be decreased by 95% and the production environmental condition could be greatly improved.
iii. Disease caused by parasitic maggot has been the one of major lost in cocoon production in the tropical areas. It was found that maggot fly seldom laid eggs on the black body larva with the black body gene mutation (blb). This gene mutation has also introduced into improved variety, thus could suuply basic material for this particular breeding purpose.
By utilizing the morphological characters to label the Densonucleosis-resistance gene (nsd-Z), a new method for breeding Densonucleosis-resistant silkworm varieties has developed. Linkage analysis had shown that the gene were closely linked with nsd-Z, desonucleosis-resistant varieties D01 (Chinese race) and D02 (Japanese race) were bred. By utilization of D01 and D02, it is easily to select DNV resistant individuals by the marker gene bl2 without DNV administration.
Polyphagous gene mutation was found both in Chinese race and Japanese race, and four strains with polyphagous gene were bred in Chinese race and Japanese race respectively. It is the first time to found the polyphagous gene in Chinese race in the world.
A gene bank that is the most complete one in China was established and corresponding database by FoxPro 2.5 completed. Some specific preserving techniques for lethal genes were developed. In order to optimize the structure of gene bank and decrease the risk of gene disappear, average 6 marker genes were carried by each preserved gene strain, and each gene mutation was carried by at two strains.
South-West University has been mainly focused on the research of egg color mutation, radiation induced gene mutation and molecular linkage map.
Since 80th, over 10 new gene mutations were discovered, and 220 marker genes were preserved, some valuable gene mutations were found, a total 602 gene mutations were maintained in this University. Dai F.-Y. et al. (2002) reported that A new mottled translucent oh, was discovered in the preserved system of silkworm in the Key Laboratory of Sericulture of the Ministry of Agriculture, it resulted from a recessive mutation in the 20th linage group, its gene locus is at 20-0.0 and ghe gene appears to be dominant over oh, this mutant is now tentatively designated with the gene symbol ohm and was found to have an epistatic effect over Chinese translucent (oc). Lu C. et al. (1999) found a citron-egg gene (ci) that belongs to the 20th linkage group for it linked with an hoarfrost translucent (oh 20-0.0) gene which is a marker gene of the 20th linkage group. Ci gene is located at the locus 20-21.9. Lu C. et al. (1991) found a spontaneous mutant of the new ellipsoid egg from Ng system. Its morphological appearance is similar to that of elp, but it is longer and thinner. Genetic analysis indicates new ellipsoid eff is controlled by a recessive gene that is independent of gene elp, named as elp-2.
Another spontaneous mutant or the chocolate purple has been discovered from the white-5 eggs system in 1990. Its morphological characters and hereditary mode are different from Ia, sch, cm, ch, and ch-2. The gene is marked with chp which is the allele of +ch and ch. Xiang Z.-H., (1997) studied the relationship between cocoon fluorescent color (FC) and varieties, sex, mounting environment and the inheritance pattern of FC, found that the pure yellow type was identified to be incompletely dominant to the pure purple type, cocoon fluorescent color is controlled by multiple genes, there are at least two pairs of genes (Yf-1 and Yf-2) that controlled the yellow color and at least two pairs of others (Vf-1 and Vf2) that controlled the purple color, the homozygous FC cocoons were less affected by environmental temperature and humidity, but the heterozygous FC cocoons were remarkably affected.
As above-mentioned, there is a total 26 units involved with silkworm germplasm resource maintenance or research. Except the above two major institutes, the relatively important ones are: Sericulture Research Institute of Zhejiang, Guangdong, Guangxi, Hubei, Hunan, Shuaanxi, Shangdong, Hebei, Xinjiang, Liaoning; Zhejaing University, Suzhou University, Southern Agricultural University, Huasheng Group of Sichuang Province, Qingzhou Egg Station, and some parent egg stations. There are over 100 silkworm varieties mainly in local varieties, special varieties and breeder's stock maintained in these institutes. Fox example, Sericulture Research Institute of Zhejaing Province had introduced the balanced lethal system from former Russia, and characteristic with the breeding of only-male rearing varieties and parthenogenesis. Department of Sericulture, Zhejiang University had maintained some special varieties such as flat-way spinning, nudity pupae, extra-fine or superthick filament size, varied cocoon color etc. Southern Agricultural University had maintained the tropical strains.
After a long period of continuous breeding research, several basal varieties with special characteristic were made. 651 and 652 (Rong J.-L., 1978) are the basal varieties with especially long filament length; EH1new (Zhang M.-Y. et al., 1991) with especially high cocoon shell weight, average 0.7g and shell ratio around 27%. "123" (Luo Y.-G., et al., 1993) with especially high cocoon shell ratio, TB with extremely high in cocoon shell ratio(Its average cocoon shell ratio by batch reached 30.58%, the highest of female individual reached 31.79%, and male 38.09%.); "125" (Luo Y.G., et al., 1997) with high raw silk rate(around 28 %); "SG" (HeY.-Y., et al., 1997) with dominant trimolter (especially fine filament size, 2.0d).
Moreover, the basal variety breeding of rich egg laying and superthick filament size was one of the national silkworm breeding program in the eighth five-year plan. The task of this program is to breed a variety which could produce more than 800 eggs per moth or filament size thicker than 4.0d and cocoon shell ratio exceeds 22%. Chen K.-P. et al. (1995) had reported that "He" with hyperlaying was discovered from the 400 preserved silkworm variety resources in 1986, and it was used as one the parent material to breed the basal variety HEK which lays 821 eggs per moth average, the most 983, cocoon shell ratio reached 22.08%, cocoon weight 2.208, shell weight 0.486g, larval duration 23-25d. Wu Y.-C., et al.(1996) also had reported that four rich egg laying varieties - GE, GCL, GLZ, GLBZ had been bred from 1991-1995 by classical breeding techniques. The highest one, GE reached 1017 eggs per moth on average, and the highest moth 1083 eggs. The new varieties with filament size 4.5d and shell ratio 25.45 also bred.
Lin C.Q et al. (1997) investigated on 251 preserved silkworm varieties for their cocoon shell production efficiency, the average (cocoon shell/food ingested) was found to be 10.11%, highest 12.19%, lowest 6.71%, bivoltine was higher, and followed by monovoltine, then polyvoltine. Among bivoltine, Chinese is the highest, followed by Japanese, then polyvoltine. It was positively correlated with cocoon shell rate, cocoon shell weight, average cocoon shell weight per day in 5th instar, no correlation with the 5th instar duration of larval duration, rate of dead worm cocoons, cocoon weight and digestibility.
Moreover, 200 silkworm varieties were investigated on their resistance to densonucleosis through oral administration of the pathogen. It was discovered that 28 of them had the resistance. Two resistant lines were selected and the densonucleosis resistant gene nsd-Z and eye spot gene bl liked lines were created, so that it is possible to breed densonucleosis resistant varieties with eye-spot as genetic marker instead of administration of the pathogen to silkworms. Chen K.P., et al.(1996) found significant difference among preserved silkworm varieties to NPV-sensitivity, the resistance is of incomplete dominance to susceptibility, it is controlled by over two pairs of genes.
Lin C.Q. et al. (1995) tested the cocoon filament size of 200 preserved silkworm varieties in spring rearing season of 1992. The average size of all varieties tested is 2.80 dtex., the thickest is 3.93, and finesse is 1.72. The size of different strains is in the sequence of Japanese>European>Chinese>tropical in geographical races, and monovoltine >bivoltine> multivoltine in voltinism classification. Filament size is negatively correlated with filament length, percentage of sericin in cocoon shell and neatness, but positively correlated with cocoon shell weight, pupa weight and filament weight.
Lin C.Q. et al. (1994) investigated 209 preserved silkworm varieties on their cocoon filament properties, results showed that the average filament length was 840m, longest 1273m, shortest 344m, average reelability is 79.4%, highest 100.0%, lowest 41.5%, average neatness was 93.9, highest 99.2, lowest 76.7.
It was also reported (Chen K.P. et al. 1988) that resistance to fluoride and NPV are significantly different among the preserved varieties, the resistance to fluoride in the varieties of Fengyi, 872, 54A, Fenyi × 54A, Huafeng × Xuesong, 871 × 872 are stronger than other varieties, the resistance to NPV in the varieties of 318, 416, Qiu3, 317 × 318, 415 × 416 and 871 × 872 are higher than the other tested varieties, there is no relationship between the resistance to fluoride and NPV, but it is possible to combine both of the character in the same hybrid such as 871 × 872 and 317 × 318 which showed high resistant ability to both fluoride pollution and NPV.
Furthermore, deguming loss percentage of cocoon shell in boiling water for 30 min was tested in 250 silkworm varieties preserved (Lin C.Q. et al. 1988), the highest and the lowest are 27.2% and 13.1% respectively, with an average 20.2%. As for voltinism concerned, monvoltine races is comparatively low and that of polyvoltine races is higher. it was positively correlated with sericin ratio of cocoon shell and cocoon shell ratio, but negative correlation with reelability. The average soluble sericin percentage of cocoon shell in tested races is 78.7% and the highest is 97.7%, lowest 50.3%. There is no significant difference between female and male cocoons. Significant difference (Wu D.X. et al. 1988) was found among different varieties to cold storage duration. No disadvantageous influence was found the two sex-linked genes of the balanced lethal line S-14 introduced form Russia on the viability and cocoon weight in the descendent generation (He K.R., et al. 1998).
Zhang Z.F., et al. (1999) reported that a new natural mutant: normal temperature lethal gene (ntl) was found from Chinese bivoltine native variety Yuhang 7. When the fifth instar larvae were reared the viability was 10.7% at the temperature of 23.3°C, and 90% at 29.4°C. The gene ntl is recessive, located at the 5th chromosome and very close to oc. Zhao Q.L. et al. (1997) discovered a mutant of translucent silkworm with medium transparency in the breeding variety Dong 3219, it causes a lethality of 21.16% during the first molting. This translucent character is controlled by a pair of recessive genes located on the fifth linkage group. Experiments showed it is different from other existed translucent mutant, and marked with ojc. Meng Z.Q. (1996) had discovered a new gene mutation, translucent of.
A series of experiments were carried out with silkworm eggs, larvae, cocoons and pupae of Bombyx mori L. onboard the 10th recoverable satellite of Russia in December of 1992(12 days flight), several mutations such crayfish pupa, exta crescents and trimolter were found in the recovered materials, whereas there was no relevant variation being found in the control group (Zhuang D.H., et al., 1995), photos reder to annex 6. And late, the Linkage inheritance of crayfish pupa (cf-h) mutant gene of Bombyx mori induced by space-flight was analyzed (Gui Z.Z., et al.1999), cf-h is a new mutant gene located in the 19th likage group.
Except the national sericulture research institute, there are over 20 provincial institutes and university dealt with silkworm breeding in China. New silkworm varieties bred since 1980 were listed in Annex 1, and the improvement of major economic character refers to Annex 7. It was considered that Chinese breeders had particular experience and techniques on the breeding of silkworm varieties adaptable to adverse rearing condition. A complete collection of thesis on silkworm breeding records, techniques and experiences will be published on October of 2002, in which 138 papers were included after 1949 in China. Besides, some silkworm varieties with special economic characters or for special purposes were bred. For example:
The International Silkworm Molecular Breeding Plan initiated in the beginning of 90s, and Silkworm Genome Plan started at present by cooperation with Japan and other foreign countries. During recent ten years, progress on the molecular linkage study had been rapidly advanced, especially in the construction of molecular linkage, isolation of important molecular markers, gene location of important economic characteristics. The major part of the silk gene and silkworm mitochondria genes had been successfully sequenced, and gene clone, QTLs and transgenic research are all on the way.
The construction of molecular linkage are mainly depending on the molecular marking techniques such as restriction fragment length polymorphism (RFLP), random amplified polymorphism DNA(RAPD), selective amplification DNA fragments (SADF) and amplified fragments length polymorphism (AFLP). (1999) had determined 7 RAPD marker were linked with the morphological gene Y, Nl1, Nd, Lc, Lca, od, and sch. He N.-J., et al. (2001) had reported that a molecular linkage map of silkworm was constructed successfully based on SADF and RAPD markers, refer to annex 8. Xiang Z. H. et al. It was expected that a high dense molecular linkage map could be establish soon after combining with morphological characters and isoezyme pattern.