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13UDC 635.6
Research
REPRODUCTIVE AND VIABILITY CHARACTERISTICS OF
F1, F2 GENERATIONS DURING THE IMPROVEMENT OF THE MULBERRY SILKWORM BREED BALANCED ON NONALLELIC EMBRYONIC Z-LETHAL
Bakhtiyar Nasirillaev
doctor of agricultural sciences, Murodkhuja Abdikodirov
Ph.D. student.
Annotation. Reproductive and viability indicators, among the economic traits of the mulberry silkworm, are important. This article reveals the results of research on the improvement of the unique C-8 ngl breed of Bombyx Mori L. mulberry silkworm balanced on embryonic Z-lethal and sex-limited at egg stage by transmitting high-tech traits of fine silk L-28 simple genetic line. In particular, in the F1 generation of mulberry silkworm, a reciprocal cross with a simple L-28 genetic line was performed. In the F2 generation, a complex combination in the form of С-8 ngl x L-28) x <^(L-28 x С-8 ngl) was obtained. In these studies, a comparative analysis of the parental components obtained in each generation, as well as indicators of reproductive and embryonic viability in F1 F2generations were implemented. According to the results of the analysis, despite the imbalance of embryonic lehal in F1 generation compared to the C-8 ngl breed taken as a control, a slight decrease in economic traits o-frred due to separation. In the F2 generation, however, it was found that its level had risen to the level of parental components, even higher. In the population of this genotype, it is possib e to create an almost new breed by conducting analytical selection from generation to generation.
Keywords: silkworm, egg, worm, breed, genetic 'ine embryonic Z-lethal, backcross breeding, sex determination.
Аннотация. Среди экономических характеристик тутового шелкопряда важны показатели репродуктивности и жизнеспособности. В этой статье раскрываются результаты исследований по улучшению уникальной породы С-8нгл тутового шелкопряда Bombyx Mori L., сбалансированной по эмбриональному Z-летальному типу и ограниченной по полу на стадии яйца, путем передачи высокотехнологичных признаков простой генетической линии Л-28. В частности, в поколении F1 тутового шелкопряда было проведено реципрокное скрещивание с простой генетической системой Л-28. В поколении F2 была получена сложная комбинация в виде С-8 нглхЛ-28) x <^(Л-28хС-8 нгл). В этих исследованиях был проведен сравнительный анализ родительских компонентов, полученных в каждом поколении, а также показателей репродуктивной и эмбриональной жизнеспособности в поколениях F1, F2. Согласно результатам анализа, несмотря на дисбаланс эмбриональной летальности в поколении F1 по сравнению с породой С-8нгл, взятой в качестве контроля, из-за разделения произошло небольшое снижение экономических признаков. Однако в поколении F2 было обнаружено, что его уровень поднялся до уровня родительских компонентов, даже выше. В популяции этого генотипа можно создать практически новую породу, проводя аналитический отбор из поколения в поколение.
Ключевые слова: шелкопряд, яйцо, червь, порода, генетическая линия, эмбриональная Z-летальность, обратное скрещивание, определение пола.
Аннотация. Тут ипак куртининг хужалик белгилари орасида репродуктив ва хаёт-чанлик курсаткичлари мухим ахамиятга эга. Мазкур макола Bombyx Mori L. тут ипак куртини эмбрионал Z-леталлари буйича мувозанатланган ва жинси тухумлик даврида нишонланган ноёб С-8нгл зотини юкори технологик хусусиятга эга, ингичка ипакли оддий Л-28 генетик тизим белгиларини утказиш оркали такомиллаштириш буйича олиб борилган тадкикот натижаларига багишланган. Жумладан, тут ипак куртининг F1 авлодида ингичка ипакли
оддий Л-28 генетик тизими билан реципрок чатиштирилди. F2 авлодда С-8 нглхЛ-28) х $(Л-28хС-8нгл) шаклидаги мураккаб комбинация олинган. Мазкур тадкикотларда цар бирида ота-оналик сифатида олинган компонентлар цамда F1, F2 авлодларда репродуктив ва эмбрионал цаётчанлик курсаткичлари киёсий тацлил килинди. Тацлил натижаларига кура, назорат сифатида олинган С-8нгл зотига нисбатан F1 авлодда эмбрионал леталлар мувозанати бузилишига карамасдан, хужалик белгилари бироз пасайганлиги ажралиш цисобига содир булди. F2 авлодда эса ота-оналик компонентлари даражасига, цаттоки, ундан цам юкорига кутарилганлиги аникланди. Шу олинган генотип популяциясида авлодма-авлод аналитик селекция ишларини олиб бориб, деярли янги зот яратиш имкони мавжуд.
Таянч сузлар: тут ипак курти, тухум, курт, зот, генетик тизма, эмбрионал Z-летал, бекросс чатиштириш, жинсни нишонлаш.
Today, mulberry silkworms are reared in more than 20 countries around the world and 840860 thousand tons of raw cocoons are produced. "The cocoon yield per box of worms in the world is 80.0-85.0 kg in the China, 78.0-80.0 kg in India, and the degree of uniformity of cocoon caliber is 90.0% in the leading countries" [1].
Nowadays, research is being conducted around the world to develop a scientific basis for increasing the viability and cocoon productivity of F1 hybrid generations, as well as the creation of high-heterosis industrial hybrids. In this regard, obtaining high-quality silk fiber that meets the requirements of the silk industry and obtaining purebred hybrid eggs with the hybridity of about 100%, as well as the further intensification of the genetics and practical selection of quantitative characteristics of the mulberry silkworm is of great scientific importance [2]. Taking into account all these abovementioned, it is possible to grow hightech cocoons using sex-regulation methods of the mulberry silkworm.
Sex-marking and regulation of sex ratio in silkworms is one of the first achieved biological objects. B.L.Astaurov and H.Hosimoto were the first in this direction in the 30s of the last century to conduct thorough fundamental research [3], [4]. Figure 1. Scheme of genetic sexing using a balanced lethal (BL) strain suggested by Strunnikov [1975]. A segment of the Z chromosome including wild-
type alleles of genes sl-1 and sl-2 (sl-sex lethal) has been translocated to the W resulting in a T(W; Z) chromosome. The strain is propagated with males heterozygous for lethal alleles (red, yellow) of the 2 genes in trans, and only this type of male offspring is viable.
BL Strain
sl-1 8 § sl-2 si-1 1
X
s J
z z z ■
T(W;Z)
z z z z z W zw
Males to be irradiated Lethal females
Since the 1960s, a group of scientists in Uzbekistan, led by V.A.Strunnikov, have managed to obtain a unique breed with lethal genes lx and l2 in mutual balance with the participation of the same sex-limited breeds (Fig.1) [5].
In 1996, the Zhejiang Agricultural Academy
and the Russian Academy of Sciences jointly researched unique and sex-regulated silkworm lines with balanced lethal genes created based on complex genetic schemes [6], [7]. In recent years, some Japanese scientists have conducted research to improve methods of obtaining the lines sex-limited larval markings, balanced on embryonic lethal [8], [9].
Today, the Uzbek Silk Research Institute is researching the preservation and monitoring of translocation in the genotype of the breeds balanced on Z-lethal genes, as well as on the creation of new hybrid combinations. In particular, Nasirillaev B.U., Umarov Sh. R., Jumaniyozov M.Sh., Khalilova M.F. [10], Achilov Sh. I., Umarov Sh.R. [11], Nasirillaev B.U. and Abdikodirov M.A. [12], [13], [14] are conducting genetic research to increase the technological indicators of the lines sex-limited on the color of the eggs by backcross breeding, to maintain a high level of cocoon productivity and to create new hybrid combinations and to recommend them in production.
The main goal of our study is to improve the background of the main economic traits of the unique C-8 ngl breed of mulberry silkworm balanced on embryonic Z-lethal by transferring to its genotype technological traits of fine fiber L-28 line by maintaining the lethal balance.
Materials and methods
The experiments were conducted by scientists from the Uzbek Silk Research Institute under the Committee for the Development of Sericulture and Wool Industry of the Republic of Uzbekistan and the Department of Genetics of the National University of Uzbekistan. In the experiment, the mulberry silkworm Bombyx mori L. sex-limited at the egg stage, C-8 ngl breed balanced on embryonic Z-lethal, sex-limited trw2w2 x L-67, as well as simple non sex-limited L-28 line were used. The genetic scheme of improvement of this C-8 ngl breed with the L-28 line with high
genetic characteristics is shown in Figure 2.
The worms of breed and lines selected for the experiment were reared at a young instar at a temperature of 26-27 °C and 70-75% relative humidity, and adult worms at a temperature of 2425 °C and relative humidity of 65-70% in a special cocoonery of the Uzbek Silk Research Institute. 1000 kg of mulberry leaves were used for 1 box of worms. The worms were fed with mulberry leaves of Uzbek breeding varieties Jarariq 4, Jarariq 5, and Jarariq 6. The sequence of determination of reproductive indicators in the study was carried out in the following order:
The prepared egg-layings - the breeds (lines) used as parents and the egg-layings with their participation were analyzed individually During the analys's, the eggs in each egg-laying were taken and their number was determined using the individual counting method. From these analyzed eggs, asexual and dried-up eggs were isolated, and physiological defect indicators were determined. Eggs in the egg-laying were individually weighed on a torsion scale.
To determine egg hatchability in the experimental material, the eggs of each breed (line) and hybrid were incubated during the spring worm feeding season. During the incubation period, the egg samples were taken by 100 eggs from egg-layings of each breed and F1 hybrid in 4 replications. Only in the whitening stage of the embryonic period of the C-8 ngl breed, 200 pieces were obtained due to the death of more than 50% of males and females under the influence of lethal genes.
The number of unhatched eggs was determined on the third day after mass hatching of the eggs. The hatchability rate was calculated by taking the ratio of the number of hatched worms and 100 total number of worms in the sample.
Research results and their discussion
Reproductive traits are the signs of egg fertility of silkworms. Such traits include the
number of normal eggs in the egg-laying, the weight of the normal eggs in the egg-laying, the weight of one egg, and the percentage of dried up eggs or asexual eggs in the egg-laying (physiological defect percentage). The number of normal eggs in the egg-laying represents the fertility of the breed (line) and the hybrid, and the fertility usually depends on the breed of the silkworm, i.e., the large cocoon breeds produce large cocoons (2.0-2.5 g). Naturally, large moths emerge from them and lay 800-1000 eggs. Medium-sized cocoon-producing breeds (1.75-2.0 g) give medium-sized moths. They lay about 600-800 eggs. Experiments have shown that small-sized cocoon-producing breeds relatively make small cocoons (1.2-1.6 g), from which small-sized moths emerge and lay 300-550 eggs.
Since our research aimed to improve the unique C-8 ngl breed by maintaining embryonic nonallelic Z-lethal balance, we identified exactly the leading reproductive traits in the background of lethal genes in this part of the experiment.
Before the spring silkworm rearing season of 2020-2021, the egg-layings obtained to determine the reproductive traits of selection breeds (lines) and F1 generation with their participation prepared in the 2019-2020 season were analyzed in the January-February months of this year (Table 1).
Table-1
Reproductive indicators in C-8 ngl breed and the F1 generation obtained during the improvement of C-8
ngl breed
Breeds and hybrids Number of stocks analyzed, pcs Number of eggs in stock, ±Sx, pcs Weight of eggs in stock, ±Sx, mg Physiological defect, ±Sx %
C-8 ngl (control) 229 723±0,11 452±5,75 1,75±0,33
L-28 (improver) 205 534±0,41 378±0,12 0,53±0,01
9L-28 x SC-8 gl F1 10 591±13,75 299±16,62 2,5±0,32
9C-8 ngl x SL-28 F1 14 700±7,98 408±9,38 2,2±0,31
From the table above, it can be seen that the indicators of F1 generation obtained by reciprocal crossbreeding were slightly lower than the reproductive indicators of the C-8 ngl breed and the L-28 line taken as a parental breed. In particular, the number of eggs in the egg-laying showed a high value at the C-8 ngl breed (723 eggs). Only in the combination of 9C-8 ngl x SL-28, the number of eggs (700 pieces) in the egg-laying did not differ from the C-8 ngl breed. The reliability of the difference is Pd = 0.862-0.999. A similar trend was observed in the weight of eggs in the egg-laying, which were 452 mg and 408 mg, respectively. It was detected in the L-28 line (0.53%) obtained as an improver on the physiological defect.
The F1 generation obtained from reciprocal crossbreeding formed a combination of 9 (C-8 ngl x L-28) x S (L-28 x C-8 ngl) by the mutual cross and F2 generation was obtained from it. The females (genotype has w2 gene) of tr24w2w2 x L-67 line of the paternal S (L-28 x C-8 ngl) were used for analytical cross, i.e to determine the presence of I and l2 genes in the male generation. Reproductive traits were also analyzed in the resulting F2 generation (Table 2).
Table-2
Reproductive traits in C-8 ngl breed and the F2 generation obtained in the improvement of C-8 ngl breed
Number of stocks analyzed, pcs Number of eggs in stock, ±Sx, pcs Weight of eggs in stock, ±Sx, mg Physiological defect, ±Sx %
С-8 ngl (control)
95 700±5,90 417±0,75 1,81±0,78
Improving 9(С-8 nglxL-28) x ^(L-28xC-8 ngl) F2
33 673±14,59 383±8,93 2,2±0,30
Crossbreeding under analysis9(tr24w2w2xL-67) x ^(L-28xC-8 ngl)
18 658±24,71 411±14,92 1,3±0,20
Based on the given table, we can say that 9 (C-8 ngl x L-28) x ^ (L-28 x C-8 ngl) prepared for improvement didn't differ from control breed C-8 ngl by the number of eggs (673 pieces), the weight of the eggs (383 mg) in the egg-laying (700 pieces and 417 mg, respectively). The reliability of the difference is Pd = 0.7860.999. The weight of the eggs (411 mg) and physiological defect (1.3%) in the 9 (tr24w2w2x L-67) x ^ (L-28xC-8 ngl) egg-laying created as a result of the analytical cross were at the level of the control C-8 ngl breed.
Embryonic and postembryonic viability traits of silkworm N^eds and hybrids are important in their evaluation on an industrial scale or in laboratoi F conditions Because, iflarge- cocoon silkworms have the required technological and productivity indicators, and if some lethal and sublethal genes are involved in their genotype, their eggs can hatch and make healthycocooning,get stable at different stages of ontogeny until the next life cycle.
Considering all these, worm hatching was studied in the background of lethal genes of parental and F1 and F2 generation. The results obtained are reflected in Tables 3 and 4.
Table-3
Embryonic viability in C-8 ngl breed and the F1 generation obtained in the improvement of C-8
ngl breed
Breeds and hybrids Number of returns (n), pcs Sex ratio, % Egg hatching, % Relatively on control, %
C-8 ngl (control) 3 509:50^ 89,3±1,45 100
L-28 (improver) 3 509:50^ 93,0±1,52 104,1
$L-28x^C-8 ngl F, 3 100^ 94,6±1,1 105,9
$C-8 nglx^L-28 F, 3 509:50^ 94,6±2,4 105,9
From the table above, it can be seen that the egg hatching in the F1 generation obtained by reciprocal crossbreeding of the C-8 ngl breed balanced on embryonic lethal with L-28 line on was at the level of the parental breeds. In particular, in the combinations 9L-28 x ^C-8 ngl and 9C-8 ngl x ^L-28 the same indicator - 94.6% was observed, 105.9% compared to the control breed C-8 ngl (reliability of the difference Pd = 0.907-0.999).
We can see that in the F1 generation obtained by this reciprocal crossbreeding, there is a growing trend in egg hatching relative to the parent breeds.
According to the improvement scheme of the obtained F1 generation, a crossbreeding in the form
9 (C-8 ngl x L-28) x 3 (L-28 x C-8 ngl) was performed to obtain the F2 generation.
In the spring worm rearing season of 2021, the embryonic viability was determined in the F2 generation obtained in the C-8 ngl balanced on embryonic lethal and in the improvement of C-8 ngl (Table 4).
Table-4
Embryonic viability in C-8 ngl and the F2 generation obtained during the improvement of C-8 ngl breed
Egg color and lethal Number of returns (n), pcs Sex ratio, % Egg hatching, % Relatively on control, %
С-8 ngl (control)
Gray 99 3 509 89,3 100
Light yellow 33 3 503 89,7 100
Improving 9(С-8 nglxL-28) x 3(L-28xC-8 ngl) F2
Gray -¿,99+33 3 679:333 78,8 88,4
Gray -,99+33 3 679:333 74,2 82,9
Light yellow -,33 3 1003 88,5 99,1
Light yellow -,33 3 1003 & 93,5 104,5
Crossbreeding under analysis9(tr24w2w/I-67) x 3(L-28xC-8 ngl)
Gray -, 99+33 3 679:333 70,1 78,3
Gray -,99+33 3 '/9:333 66,4 74,2
Light yellow - ,33 3 1003 88,8 99,2
Light yellow -,33 3 "003 93,5 104,5
As shown in the table above, egg hatching is a'most s able in the unique C-8 ngl breed due to the balance of lethal. Only the hatchability of gray-colo ed eggs of female worms was 89.3%, and the pale yellow eggs hatchability of male worms was 89.7%.
In the main 9 (C-8 ngl x L-28) x 3 (L-28 x C-8 ngl) combination of the F2 generation, egg hatching was noted to be high in females with gray eggs - l1 78.8 %, and in males with pale yellow eggs l2 93.5% due to the presence of one of the double lethal. This is 88.4 and 104.5% higher than the control breed (reliability of the difference Pd = 0.9 28-0.999.).
To clarify which lethal is taken by the main combination of the F2 generation, the indicators in 9 (tr24 w2w2 x L-67) x 3 (L-28 x C-8 ngl) combination created using a line that keeps translocation in the genotype in analytical crossbreeding, the upper limit was gray l1 70.1% in females, while in males pale yellow I 93.5%. This is 78.3 and 104.5% respectively higher than the control breed (reliability of the difference Pd = 0.891-0.999.).
In the genetic method we recommend, larval markings on the sexes by egg color allow the identification of lethal genes at an early stage of embryonic development. As it turns out, the highest quality, thin silk is obtained from male cocoons. The breeds balanced on embryonic lethal genes are used to create male industrial hybrids. Therefore, when obtaining high-quality silk, special attention is always paid to the genotype of the new breed and hybrids. It should be emphasized that no improvements have been made since the creation of the unique C-8 ngl breed. The task has been set to bring the silk of this breed to a high level of quality in the future. In this regard, of course, it is expedient to make effective use of methods of improvement. This method can be used not only in Uzbekistan but also by scientists from leading silkworm breeding countries. The advantages and ease of use of this method are that it is performed with the involvement of arbitrary breeds (lines), in a short period lethal genes are balanced and
high-tech features are collected in a single genotype. Given the above, newly created breeds and hybrids can be recommended in genetic, breeding research.
Within the framework of the improvement of the C-8 ngl breed balanced on embryonic lethal genes, theoretically expected results have been achieved practically in every generation.
From the obtained results of the study, it can be concluded as follows:
1. Imbalance of the double lethal in the unique C-8 ngl breed, embryonic viability was not significantly reduced as a result of this or that lethal acquisition. However, in the next stages of improvement work, it is advisable to increase the intensity of selection-breeding work and strictly control the lethal balance.
2. The high indicators of viability and satisfactory cocoon productivity of this new system, which is taken as an analog for the C-8 ngl breed, allow it to be recommended to silkworm egg processing enterprises.
3. Based on the weight and physiological defect indicators of the eggs of the egg-laying, along with the L-28 line taken as an improver the combination $ (tr24w2w2 x L-67) x $ (L-28 x C-8 ngl) created as a result of analytical crossbreeding was proved to be acceptable as a second improver for future generations.
4. The results obtained show that no adverse effects of embryonic Z-lethal genes on the reproductive traits of the mulberry silkworm were observed.
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Conclusion
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