Biology Bulletin, Vol. 27, No. 6, 2000, pp. 571–578. Translated from Izvestiya Akademii Nauk, Seriya Biologicheskaya, No. 6, 2000, pp. 679–687. Original Russian Text Copyright © 2000 by Strunnikov, Strunnikova.
GENETICS
The Nature of Heterosis and Methods of Enhancing and Fixing It in a Series of Generations without Hybridization V. A. Strunnikov and L. V. Strunnikova Kol’tsov Institute of Developmental Biology, Russian Academy of Sciences, ul. Vavilova 26, Moscow, 117808 Russia Received May 5, 2000
Abstract—The long-standing studies into the problem of heterosis on the silkworm and other objects are reviewed. Silkworms are divided by sex for hybridization using the genetic marking of the eggs by sex, an improved method of ameiotic parthenogenesis providing all-female progeny, or the method of obtaining allmale progeny as a result of the death of eggs with female embryos under the influence of two non-allele embryonic lethals balances in the Z-chromosome. The experimental data suggest that heterosis is determined by the heterozygosity of lethals and semilethals, rather than depending on the heterozygosity of all gene types. The number of modifier genes that control viability plays a big role in the vigor of heterosis. An effective method is proposed to enhance the vigor of heterosis through the selection of one or two parents of a hybrid for viability, both of which received a strong semilethal. In hybrids, this semilethal is suppressed by the normal allele, and then a more unbalanced complex of modifier genes initiates a heterosis two to three times more vigorous than in standard hybrids. A method is developed to fix heterosis through the backcrosses of females of the high-heterosis hybrids to the absolutely homozygous males of androgenetic origin obtained from these females. These crosses preserve the main genotype of the hybrid and eliminate lethals and semilethals, and, as a result, heterosis is not damped during standard intrahybrid reproduction.
INTRODUCTION Heterosis, the property of F1 hybrids to exceed the best of parents in certain features, was discovered about two hundred years ago. However, it was first used in practical breeding of silkworms only in the beginning of the XX century and, later, hybrids of rural plants were obtained. Its nature was not fully disclosed. A well known geneticist F. Hatt wrote that heterosis is one of the greatest mysteries of biology. Similar views were aired by many other well known biologists. Undoubtedly, a deep knowledge of the causes of heterosis and a development of the methods of its control would greatly help it to be used more efficiently in agriculture. The present authors began their studies into the problem in question after developing methods of artificially reproducing silkworms, such as ameiotic and meiotic parthenogenesis, gynogenesis, mono- and bipaternal androgenesis, polyploidization, depolyploidization, etc. Various methods were used according to the aims of investigation and, hence, their description is given in the corresponding chapters. Fundamental studies were carried mostly on the silkworm and then checked on other objects: Drosophila, barley and pea. MATERIALS, METHODS, AND RESULTS Hybridization of silkworm. The silkworm butterflies mate just after emerging from the cocoon and, hence, produce purebred eggs. Tens of technical methods of
separation of the females from males before natural intrabreed mating proved to be labor-consuming and far from precise. Like in some farm plants, the problem was solved by genetic methods. Astaurov (1968, 1978) induced ameiotic parthenogenesis, providing allfemale progeny by heating unfertilized eggs. However, the method was not successful, since the constancy of clone genotypes did not allow their improvement through selection. Strunnikov (1978) developed the method of selection of parthenoclones, after Terskaya and Strunnikov (1975) had discovered artificial meiotic parthenogenesis. As a result, the yield of parthenogenetic larvae and their viability reached 90–95%, and parthenoclones were used as initial parents for commercial hybridization. Another method was improved as well. Strunnikov and Maresin (1980) described an interesting phenomenon: when reproduced by meiotic parthenogenesis, the eggs of tetraploid females develop on the basis of a diploid pronucleus extruded during meiosis. Unlike the genetic patterns of free distribution of all chromosomes during meiosis, the sex W-chromosome finds itself alone, with few exceptions, in the pronucleus for some unknown reasons and, hence, the diploid progeny consists almost completely of females. Although the technique of polyploidization and depolyploidization is not complicated, this method of breeding has not yet been brought to perfection. Serebrovskii (1971, published posthumously) came up with the idea of genetically marking insects through
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Fig. 2. Scheme of production of genotypic variant II in group B through backcrosses. Dark symbols of sex designate individuals of parthenogenetic origin and light symbols, of normal origin. AP, ameiotic parthenogenesis; MP, meiotic parthenogenesis.
the translocation of a dominant gene that controls a very noticeable morphological feature onto the unpaired W-chromosome. Autosomes should carry recessive lethals of this gene, and the females express the dominant feature, while the males, the recessive one. Independently from Serebrovskii, Tazima (1941) marked the silkworm female larvae by a pattern on the integument, while the males were white. Strunnikov and Gulyamova (1969) marked the silkworm breeds at the embryonic stage by the same method. As a result, the eggs with female embryos have the normal dark coloration, while those with male embryos are white.
The photoelectric machines sort the eggs in two categories, females and males, at a very high rate. This method is used in sericulture. Strunnikov (1969) balanced two non-allele Z-lethals expressed at the egg stage by translocating a fragment of Z-chromosome onto the W-chromosome. The progeny of bilethal males obtained as a result of crosses with normal females consists almost totally of males (99.45%). The same result was obtained by Marec (1990) on Ephestia kuhniella when he used our method. This method attracted the attention of entomologists, since the release in nature of males that give allmale progeny upon crossing with the wild females of pest insects may be used for an effective control of pest insects. The described methods of separating the eggs and larvae by sex are sufficient for successful hybridization of the silkworm. Nature of heterosis. According to the received views, the main causes of heterosis in F1 hybrids are: suppression of the effect of harmful recessive genes, favorable combination of non-allele, fully dominant genes inherited separately from both parents (hypothesis of dominance), and favorable action of all types of alleles in the heterozygous state (hypothesis of superdominance). Our studies revealed the fourth factor of heterosis: the inheritance of a coordinated compensation complex of favorable genes that arise as a result of selection against the background of adverse genetic and ecological factors. In order to understand the essence of genetic events leading to heterosis, we determined the specific roles of all these factors in heterosis. Two groups of genotypic variants of the ameiotic parthenoclone PC-29 produced by a hybrid female with vigorous heterosis were obtained. The genotypic variants were obtained for five different artificial methods of reproduction, which can be realized jointly so far only in the silkworm. Both groups of PC-29 variants were characterized by their formation only from the PC-29 genes, without foreign genetic material. During the formation of variants in both groups, a similar decrease in heterozygosity is achieved by variants, starting from the first variant taken as 100% and terminating by a practically zero, last variant. The difference is that in the first group, all variants retain the frequency of harmful genes characteristic for the first variant. In the second group, they were mostly removed. The formation of all genotypic variants has already been described in detail (Strunnikov, 1994, 1995) and we will only recall here the data most important for our topic. The clone PC-29 served as a reliable control for both groups of variants, since all of its individuals were genetically identical and their genotype was fully BIOLOGY BULLETIN
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repeated in subsequent parthenogenetic generations. The indices of its features, including heterozygosity, were taken as 100%. The second genotypic PC-29 variant was obtained by transforming this clone into a tetraploid and subsequently returning it into a diploid. All pairs of alleles underwent the predictable changes: Aa Aaaa 1AA : 4Aa : 1aa. It can be seen that the heterozygosity of depolyploidized progeny decreased to 66.7%. The third variant was obtained as a result of crossing / PC-29 × ? F1(/ PC-29 × ? homozygous derived from PC-29). This type of crossing brought the lethal and semilethal genes back to the third variant, which is essential for the correct execution of an experiment with the first group of genotypic variants. The fourth variant was obtained through meiotic parthenogenesis, providing for an all-male progeny homozygous for all alleles. Thus, the heterozygosity of this variant was brought to zero. The indices of the genotypic PC-29 variants of this first group (Fig. 1a) represent a classical example of the total yield of cocoons, which determined the mean cocoon mass and survival rate from the genotypic variant heterozygosity. Similar conclusions have been repeatedly drawn in literature. Seemingly, these concepts are also confirmed by our data. But the genetic variants of the second group (Fig. 1b) radically changed our ideas about the causes of heterosis. In this group, as was already noted, recessive lethals were fully eliminated in variants 2–5, while semilethals were mostly transferred to the heterozygous state. In other words, this group had no active harmful genes. The first (control) variant was, as in the first group, the parthenoclone PC-29. The second variant represented the summarized indices of four backcross generations obtained according to the scheme presented in Fig. 2. The heterozygosity of all genes in each of these generations was preserved at a level of 50%, while the frequency of harmful genes decreased to 6.2% compared to the control. The third, fourth, and fifth variants were the first, second, and third inbred generations, respectively. Eight backcross families were reared for their production, and sisters were mated to brothers in each family. The inbred clutches obtained from eight families were intermixed. The second and then third inbred generations were obtained from the first in the same way. As a result, the heterozygosity of the first, second, and third inbred generations was 25, 12.5, and 6.25%, respectively, with reference to PC-29. If heterosis directly and fully depends on heterozygosity in the first group (group A), in the second group (group B) the yield of coons and, hence, a high level of heterosis, is not only preserved at the control level, but BIOLOGY BULLETIN
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even exceeds it, despite a sharply decreased heterozygosity, especially in the inbred generations. This suggests that there is no positive correlation between the levels of heterosis and heterozygosity. Hence, the results given for the first group, like the results of many investigations which seem to confirm the parallelism between the heterozygosity of all genes and heterosis, are in fact illusive. The heterozygosity of only recessive lethals and semilethals and their elimination from the genotype positively influence viability, rather the total heterozygosity. All this markedly reduces the significance of the hypothesis of “superdominance.” However, the dependence of viability on the heterozygosity of individual rare alleles described by some researchers appears to be real, although not widespread. In group B, there is no influence of semilethals on viability in the backcross and inbred generations, since they were preliminarily eliminated or transferred into the heterozygous state, distinctly showing the significant role of favorable homozygous genes integrated in compensation gene complexes (CGC) in heterosis. These complexes not only cup off the effects of weak semilethals that remained in the genotype, but also stimulate a vigorous development. The observed sharp shift of the level of viability, even after an insignificant change in the ratio of positive to negative genes, suggests that the degree of expression of each category of these genes does not correspond to a simple arithmetic sum of their effects, similar to the polymeric inheritance of quantitative traits. This effect increases in a geometric progression, with the denominator so far unknown. When the ratio of harmful to positive genes changes, not only as a
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Fig. 5. Changes in viability of semilethal lines (I–IV) and complete parthenogenesis (VI) as a result of selection for viability and parthenogenesis, respectively: (1) silkworm, recessive mutation w4/w4; (2) Drosophila, dominant mutant line +/L-90; (3) Drosophila, dominant mutant line +/Rev‚; (4) pea, recessive mutant line of chlorophyll deficiency; (5) barley, recessive mutation lys/lys; (6) silkworm, parthenoclones during selection.
result of elimination and suppression of the former but also as a result of accumulation of the latter, the viability should also increase in a geometric progression, but with a still greater denominator. This viewpoint makes the expression of viability and heterosis more understandable and shows that it is incorrect to calculate the force of heterosis by elementary or arithmetic addition of the effects of dominant genes. The significant role of CGCs in heterosis is additionally confirmed by the experiments in which small,
fully parthenogenetic hardly viable males, obtained from PC-29 by meiotic parthenogenesis and mated with the Yaponskaya 115 breed females, produced hybrids with a wield of 7%, on the average (p = 0.001), higher than the yield of hybrids obtained as a result of crossing large heterozygous PC-29 females of ameiotic origin to the Yaponskaya 115 males. Selection for combinative ability. The results of the above described experiments have already shown the role of the ratio of semilethal to favorable genes in the formation of combinative ability. However, the methods used in these experiments were not sufficiently effective for selection to combinative ability. We developed a more elaborate method of selection for combinative ability, which is realized by the selection of parental forms for viability against the background of depressing action of homozygous semilethals. Their action was suppressed by a CGC consisting of modifiers that arose during selection. Such a selection, as will be shown below, leads to a markedly strengthened heterosis of hybrids from selected parents in all studies. The first studies were carried out on the silkworm (Strunnikov, 1977, 1983). The breed derived for commercial use was divided in two lines: A1 and A2. They had identical genotypes, except line A1 contained semilethal gene w4, which decreases the survival rate to 18%. Since the eggs of the hybrids obtained in crosses of this line with breed B were precisely divided by sex (eggs with male embryos were light-brown, while those with females embryos were dark), we decided to increase the viability of line A1. As a result of an 8-year selection of the best families and, then, individuals for viability and phenotypic features, the survival rate increased from 18 to 72% (Fig. 3). Naturally, all of those individuals whose genotype did not contain enough genes for the formation of an effective CGC died or were so depressed that were eliminated by natural selection. During selection, line A1 and line A2, which had no semilethal gene, were crossed to the standard breed B. The hybrids were studied for five years in a network of state strain testing stations. In the beginning of selection, when the survival rate of mutant lines A1 was low, the yield of cocoons of the hybrid A1 × B was even less than that of A2 × B, but then it gradually increased and exceeded that of A2 × B by 18% (on average, p = 0.001) within the last three generations for 18 repetitions (Fig. 4). This was due to the increased cocoon mass and higher survival rate. A high heterosis degree of the hybrid A1 × B becomes even more impressive if we take into account that the mean A1 cocoon mass is 18% less than that of A2 cocoon. It is essential that our model of the heterotic genotype formation was fulfilled on line A1, which differed from line A2 in the beginning of selection by the presence of only one semilethal gene w4 in the homozygous BIOLOGY BULLETIN
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state. This allows a distinct evaluation of the role of this gene and selection for viability in the combinative ability formation. At the same time, the method does not include inbreeding, since otherwise the possibility of the appearance of heterosis prone forms related to this type of reproduction could not be excluded. Similar studies were also carried out on Drosophila, barley, and pea. The viability of genotypes containing homozygous semilethals increased as a result of selection (Fig. 5). It can be seen that all curves coincide in their general features: after several generations of selection, the initially low survival rate of mutant lines and silkworm parthenoclone increased and almost reached the level of normal selection lines and populations and, in some cases (Drosophila), exceeded it. We carried out 41 experiments with the testing of silkworm hybrids obtained as a result of crosses with the line carrying a semilethal in the homozygous state and selected for viability. In all of these experiments, the survival rate of the tested hybrids exceeded that of the control hybrids by 17.2 to 27.4%. Navolotskii (1989) increased the viability of the barley line homozygous for the recessive semilethal gene lys from 20 to 86% through selection. Following our experimental design, he crosses the lines with the tester. The grain mass of the hybrid derived from the selected line was 37% more than in the control hybrid derived from crosses with the nonselected line. Gostimskii et al. (1992) tried to obtain the pea lines with a high combinative ability using our method. In all tests (Fig. 6), hybrids obtained from crosses with the selected line homozygous for the recessive gene of chlorophyll deficiency (7s) gave a much higher harvest than the hybrids from crosses with the same varieties of the nonselected initial line (7ns). In another experiment, a commercial pea variety and selected line 7s were crossed to the same variety. The hybrid from crosses with line 7s gave a higher (by 60%, on average) harvest under unfavorable ecological conditions than the control hybrid. Acting on our advice, L.Z. Kaidanov analyzed his experimental materials for the combinative ability formation. Kaidanov et al. (1979) selected the males of an inbred Drosophila line for low (LA) and high mating activity (HA). Selection for the low activity led to a sharply decreased number of flies in the family and a low egg yield, while that for the high activity led to the opposite results. Surprisingly, the progeny of LA × HA crosses proved to be more (by 20%) abundant than that of line HA (p = 0.01). Also, in the progeny of LA × HA crosses, each female laid more (by 40%) eggs than the HA female (Kaidanov and Subbotin, 1991). Finally, the number of descendants in a family of (LA × HA)F1 was six-fold that in line HA (Taglina, 1991). These seemingly on the surface paradoxical phenomena can be explained from the viewpoint of our BIOLOGY BULLETIN
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Fig. 6. Harvest of pea seeds from a plot of F1 hybrids derived from crosses of the initial variety (7ns) and selected (7s) with two commercial varieties Viola (V) and Kapital (K) (Rybtsov, 1992).
hypothesis of the combinative ability formation. Selection for low mating activity simultaneously leads to the accumulation of recessive semilethal and dominant subvital genes in the homozygous state. The data of Kaidanov et al. (1979) confirmed this hypothesis. Since the viability of line LA is very low, the genotypically weak embryos and individuals will, naturally, die. Those individuals will survive, which, in addition to a lesser amount of harmful genes, contain a sufficient amount of genes that control viability. In hybrid LA × HA, the dominant genes of line HA suppress the effects of the harmful genes of line LA, and then the CGC, no more balanced by the harmful genes, stimulates a vigorous heterosis. All these data favor the hypothesis of combinative ability formation as a result of CGC activity and a high efficiency of the methods of selection for heterosis based on this hypothesis. Fixation of heterosis in subsequent generations without hybridization. Preservation of heterosis in a series of successive generations is indeed a global task. First, this will allow growing F1 hybrids known in practice without annual, technically complicated, hybridization. Second, we will be able to obtain hybrids of plants previously resistant to hybridization. And, third, it will be possible to use the individual hybrids that gave unique yields in the laboratory, but could not be reproduced in bulk by the standard methods because of the irreparable loss of vigorous heterosis that was inherent only in the first generation and insignificantly abundant. Cloning animals solves, in principle, the problem of preserving the hybrid heterosis in subsequent generations of a clone. However, only single descendants have so far been obtained that repeat the parental genotype. Although cloning silkworms is possible, it is impractical because only females are reproduced and they are less productive than males (Strunnikov, 1998). Difficulties of the development of efficient methods of heterosis fixation were accounted, above all, by insufficient studies if its nature. Therefore it is not sur-
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Fig. 8. Frequency of harmful genes in the heterozygous state (1), cocoon mass (2), and viability (3) in the initial hybrid (I) and transformed hybrid after four successive backcrosses with homozygous males (II) and in three subsequent inbred generations (III–V). Each genetic variant was reared simultaneously with the control parthenogenetic hybrid, whose indices were taken as 100%. In all genetic lines, heterosis is higher than in the initial hybrid, thus suggesting the fixation of heterosis.
prising that the idea of heterosis preservation in a series of successive generation without hybridization was derived from the results of studies in the nature of heterosis. Our conclusion that the heterozygosity of lethals and semilethals, rather than of all genes (hypothesis of “superdominance”), played an essential role in solving this problem. Hence, it follows that the suppression of heterosis in subsequent bisexual generations of hybrids is due to the transition of some harmful recessive genes in the homozygous state, which is inevitable for this
way of reproduction. Theoretically, the possibility of heterosis suppression due to the segregation of heterozygous alleles cannot be excluded but, as was experimentally shown, it is not significant. For example, we carried out 14 successive backcrosses of absolutely homozygous males obtained by meiotic parthenogenesis from PC-29 with females of the same clone. In the first seven backcrosses, viability, the main index of heterosis, was preserved at the level of initial hybrid, while in the following seven backcrosses, markedly exceeded it (Fig. 7). In two other experiments, from the first backcross generation and to the fourth, heterosis was markedly higher than in the initial parthenogenetic hybrid. These data unambiguously suggest that small rearrangements of normal heterozygous alleles in the course of backcrosses do not adversely affect the vigor of heterosis in the hybrids with fixed heterosis. The high viability of backcross generations, starting from the first, is of great interest because these generations are derived, in essence, as a result of self-fertilization, which usually reduces the viability. The matter is that backcrosses, unlike simple bisexual reproduction, do not lead to the appearance of harmful genes in the homozygous state in the offspring genotype. This is explained by simple genetic calculations. Let us reiterate that PC-29, like any highly heterotic hybrid, should be heterozygous for harmful genes. If a lethal gene comes to the male haploid pronucleus during the meiotic parthenogenesis of the clone in the zygotic nucleus formed from the duplication of the pronucleus, this gene passes to the homozygous state, which kills the embryo at a certain developmental stage. If the pronucleus has no harmful genes, but, on the contrary, accumulates favorable genes, the male (ZZ) embryos of such eggs are capable of development to imago. These cases are very rare: no more than 0.5% of individual survive. It is quite understandable that backcrosses of the males devoid of harmful genes in the homozygous or heterozygous state produce the progenies that also do not have lethals in the homozygous state. This and the presence of many modifier genes allows not only fixation, but also the enhancement of heterosis in all subsequent backcross generations. Thus, we obtained in principle the fixation of heterosis in subsequent hybrid generations without hybridization. But this method has no practical significance, since its effect is expressed only in the backcross generation; which is even more difficult to obtain in sericulture than F1 hybrids. Hence, the problem of fixating a heterosis can be solved by producing hybrids free of lethals and semilethals. The main genotype of the initial hybrid should not only be preserved, but even improved. This task is also solved by the method of backcrosses. The number of harmful genes in each new backcross generation is halved, as compared to the preceding generation. BIOLOGY BULLETIN
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In one of our experimental studies, we produced four backcross generations. They retained the heterozygosity of adaptively neutral and favorable genes at a level of 50%, while the frequency of harmful genes decreased from 50% to 25, 12.5, and 6.25% in the first, second, third, and fourth backcross generations, respectively. Three inbred crosses were then carried out, As a result, the heterozygosity was reduced to 25, 12.5, and 6.25%, and the frequency of recessive harmful genes, to 3.1, 1.5, and 0.7%, respectively. Despite a significant increase in homozygosity and, hence, contrary to the hypothesis of “superdominance,” the viability and cocoon mass in three successive inbred generations not only did not decrease, but were even higher than in the initial parthenogenetic hybrid (Fig. 8), while in the inbred generations of a standard breed, these indices usually markedly decrease. These result suggest that it is possible in principle to fix heterosis in the subsequent generation of hybrid lines that are fully free of lethals and semilethals and, on the contrary, are saturated with modifier genes. However, these studies only opened a way to fixate heterosis, but this method had no practical importance, since absolutely homozygous males obtained through meiotic parthenogenesis are extremely rare and their viability is very low. In the offspring obtained from female parthenogenetic clones by meiotic parthenogenesis, which were previously reproduced by ameiotic parthenogenesis, the rate of survival does not exceed 0.5%. In commercial silkworm breeds and hybrids with usual sexual reproduction, it is more often than not impossible to obtain viable parthenogenetic males. This obstacle has recently been overcome: we found that fully homozygous males can easily be produced in all silkworm breeds and hybrids by monospermic androgenesis. The monospermic origin of the androgenetic offspring is proved in our experiments by segregating androgenetic males in the ratio: 50% dominants : 50% recessives, if the paternal signal alleles were heterozygous. Although the viability of androgenetic offspring was low at all developmental stages, it was higher than in the offspring obtained through meiotic parthenogenesis (Strunnikov et al., 1991). This allowed us to propose an improved method of heterosis fixation based on five or six backcrosses with androgenetic males. The selected hybrid line is then transferred to standard reproduction. This scheme is now accessible to average breeders. Undoubtedly, although mass reproduction of hybrids with fixed heterosis will be close, they will not be inbred. This markedly reduces the frequency of newly arisen harmful genes in the homozygous state and, thus, will be beneficial. Commercially breeding hybrids with a fixed heterosis may be used in principle in all farm plants if it is possible to produce androgenetic fully homozygous individuals by different methods, such as transforming BIOLOGY BULLETIN
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haploid pollen in a cell with diploid nuclei, which then gives rise to viable and fertile plants. The production of hybrids with fixed heterosis may be of great economic advantage, if an outstanding (by heterotic properties) hybrid obtained from the parents, selected for viability against the background of active semilethals, will be taken as an initial hybrid. The authors have already received a patent for the above described method of heterosis fixation. REFERENCES Astaurov, B.L., Tsitogenetika razvitiya tutovogo shelkopryada I ee eksperimental’nyi kontrol’ (Developmental Cytogenetics of the Silkworm and Its Experimental Control), Moscow: Nauka, 1968. Astaurov, B.L., Selection for Capacity to Thermal Parthenogenesis and Production of Improved Silkworm Parthenoclones, Genetika, 1973, vol. 9, no. 9, pp. 93–106. Gostimskii, S.A., Ezhova, G.A., and Rybtsov, S.A., Possible Production of Heterotic Forms Based on Semilethal Chlorophyll Mutations in the Pea, S.-kh. Biol., 1992, no. 1, pp. 64–72. Hatt, F., Genetika zhivotnykh (Animal Genetics), Moscow: Kolos, 1969. Kaidanov, L.Z. and Subbotin, A.M., A Study of Combinative Ability in Drosophila melanogaster Inbred Lines, Tsitol. Genet., 1984, vol. 18, no. 6, pp. 429–432. Kaidanov, L.Z., Genova, G.K., and Gorbunova, V.N., Identification of Mutations Accumulated in Chromosomes 2 and Affecting Viability of Line LA, Issled. Genet., 1979, no. 8, pp. 54–62. Marec, F., Genetic Control of Pest Lepidoptera: Induction of Sex-Linked Recessive Lethal Mutation in Ephestia kuhniella (Pyralidae), Acta Entomol. Bohemoslov., 1990, vol. 87, pp. 445–458. Navolotskii, V.D., Selection of Spring Barley for the Conditions of Insufficient Water Content, Cand. Sci. (Bio.) Dissertation, Leningrad, 1989. Serebrovskii, A.S., Teoreticheskie osnovaniya translokatsionnogo metoda bor’by s vrednymi nasekomymi (Theoretical Foundations of the Translocation Method of Insect Pest Control), Moscow: Nauka, 1971. Strunnikov, V.A., Production of Male Progeny in the Silkworm, Dokl. Akad. Nauk SSSR, 1969, vol. 188, no. 5, pp. 1155–1158. Strunnikov, V.A., A Method of Production of Parthenogenetic Clones in the Silkworm, Byull. Izobr., 1977, no. 43. Strunnikov, V.A., Prospective Use of Balanced Sex-Linked Lethals for the Insect Pest Control, Genetika, 1978, vol. 14, no. 11, pp. 2002–2005. Strunnikov, V.A., A New Hypothesis of Heterosis: Its Scientific and Practical Significance, Vestn. S.-kh. Nauki, 1983, no. 1, pp. 34–40.
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Strunnikov, V.A., Priroda geterozisa I novye metody ego povysheniya (Nature of Heterosis and New Methods of Its Enhancement), Moscow: Nauka, 1994. Strunnikov, V.A., Control over Reproduction, Sex, and Heterosis of the Silkworm, New York: Harwood Academic, 1995. Strunnikov, V.A., Cloning of Animals: Theory and Practice, Priroda, 1998, no. 7, pp. 3–9. Strunnikov, V.A. and Gulyamova, L.M., Artificial Sex Regulation in the Silkworm. 1. Production of Sex-Linked Breeds, Genetika, 1969, vol. 5, no. 6, pp. 52–69. Strunnikov, V.A. and Maresin, V.M., Specific Features of Activation of the Silkworm Eggs, Dokl. Akad. Nauk SSSR, 1980, vol. 251, no. 3, pp. 720–724.
Strunnikov, V.A., Gubanov, E.A., and Pronyaeva, M.V., Imprinting in the Silkworm, Dokl. Akad. Nauk SSSR, 1991, vol. 317, no. 4, pp. 996–1000. Taglina, O.V., Studies of Structural-Functional Features of Polytene Chromosomes in Drosophila melanogaster with Reference to the Effect of Heterosis and Differences in Adaptively Significant Traits, Cand. Sci. (Biol.) Dissertation, Khar’kov, 1991. Tazima, Y., A Simple Method of Sex Discrimination by Means of Larval Markings in B. mori, J. Seric. Sci. Jpn., 1941, vol. 12, pp. 184–188. Terskaya, E.R. and Strunnikov, V.A., Artificial Meiotic Parthenogenesis in the Silkworm, Genetika, 1975, vol. 11, no. 3, pp. 54–67.
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