Review Article Genetic Divergence, Implication of Diversity ...2. Genetic Divergence in Silkworm...

Review ArticleGenetic Divergence Implication of Diversity and Conservationof Silkworm Bombyx mori

Bharat Bhusan Bindroo and Shunmugam Manthira Moorthy

Central Sericultural Research and Training Institute Srirampura Mysore Karnataka 570 008 India

Correspondence should be addressed to ShunmugamManthira Moorthy moorthysm68gmailcom

Received 29 December 2013 Accepted 22 April 2014 Published 13 May 2014

Academic Editor Alexandre Sebbenn

Copyright copy 2014 B B Bindroo and S Manthira Moorthy This is an open access article distributed under the Creative CommonsAttribution License which permits unrestricted use distribution and reproduction in any medium provided the original work isproperly cited

Genetic diversity is critical to success in any crop breeding and it provides information about the quantumof genetic divergence andserves a platform for specific breeding objectives It is one of the three forms of biodiversity recognized by theWorld ConservationUnion (IUCN) as deserving conservation Silkworm Bombyx mori an economically important insect reported to be domesticatedover 5000 years ago by human tomeet his requirements Genetic diversity is a particular concern because greater genetic uniformityin silkworm can increase vulnerability to pests and diseases Hencemaintenance of genetic diversity is a fundamental component inlong-termmanagement strategies for genetic improvement of silkwormwhich is cultivated bymillions of people around the worldsfor its lusture silk In this paper genetic diversity studies carried out in silkworm using divergent methods (quantitative traits andbiochemical and molecular markers) and present level of diversity and factors responsible for loss of diversity are discussed

1 Introduction

Sericulture is a unique field of agriculture because silkwormsare reared on an extensive scale in rearing houses andtheir silk cocoons are utilized as fine material for clothingLike agriculture sericulture also requires a continuous flowof productive silkworm breeds and host plant varieties tomeet the ever-changing demand of people involved in theindustry besides the consumer sector To meet all theserequirements the breeder needs very wide and inexhaustiblegenetic resources to meet the ever-changing demands fromvarious sectors Considering the great economic importanceof Bombyx mori silk producing countries such as ChinaJapan India Russia Korea Bulgaria and Iran have collectednumber of silkworm breeds suitable for a wide range of agro-climatic conditions More than 4000 strains are maintainedin the germplasm of B mori and 46 institutes are involvingsilkworm genetic resources maintenance which includesunivoltine bivoltine and polyvoltine strains These differentgenotypes display large differences in their qualitative andquantitative traits that ultimately control silk yield It was

estimated that silkworm genome consists of about 481015840 108 bpits genetic information volume is about one-sixth of humanbeing There are over 450 morphological physiological andbiochemical characters recorded at present among them 300(including multiallele) had been located on 27 groups of thetotal 28 chromosomes [1] Apart from a rich biodiversity ofgeographical races there are also a large number of mutantsfor a variety of characters present in B mori [2]

2 Genetic Divergence in Silkworm

Study on genetic diversity is critical to success in any cropbreeding and it provides information about the quantumof genetic divergence and serves as a platform for specificbreeding objectives [3] Genetic diversity is usually thoughtof as the amount of genetic variability among individualsof a variety or population of a species [4] It results fromthe many genetic differences between individuals and maybe manifest in differences in DNA sequence in biochem-ical characteristics (eg in protein structure or isoenzymeproperties) in physiological properties (eg abiotic stress

Hindawi Publishing CorporationInternational Journal of BiodiversityVolume 2014 Article ID 564850 15 pageshttpdxdoiorg1011552014564850

2 International Journal of Biodiversity

resistance or growth rate) or in morphological characters[5] Genetic diversity has been conventionally estimated onthe basis of different biometrical techniques (Metroglyph1198632divergence analysis and principal component analysis) suchas phenotypic diversity index (119867) or coefficient of parentageutilizing morphological economical and biochemical data[6ndash9]

The genetic diversity ofBmori is derived from hybridiza-tion of different geographical origins mainly the JapaneseChinese European and Indian strains which have distincttraits Among these four geographical strains silkworm oftemperate origin produces a higher quantity of good finerstronger silk fiber whereas the tropical strains are hardytolerant to pathogen load and resistant to diseases Howeverthe tropical strains produce low amounts of silk which iscoarser and weaker [10] To help the breeders in the processto identify the parents that nick better several methods ofdivergence analysis based on quantitative traits have beenproposed to suit various objectives As most of the desirablecharacters in silkworm are quantitative nature multivariatestatistical methods have been employed to measure thegenetic diversity among the stocks Among them1198632 analysisof Mahalanobis [10] using Tocherrsquos optimization method [11]occupies a unique place and an efficient method to gaugethe extent of diversity among genotypes which quantifythe difference among several quantitative traits It is beingused by most of the workers and has been found as anextremely useful tool for estimating genetic divergence inthe silkworm [12ndash16] Table 1 summarizes genetic divergencestudy carried out by several workers in silkworm Usingthis method silkworm genotypes were formed into differentclusters indicating presence of distinct divergence amongthe genotypes Though divergence reported to exist amonggenotypes and mixed trend of clustering observed Jolly et al[13] subjected forty-nine silkworm breeds for this analysisand reported that these breeds were found to form threedistinct clusters indicating the presence of distinct diversityamong the breeds Subba Rao et al [14] and Govindan et al[15] reported that breeds derived from the same parents wereincluded in different clusters showing variation among thebreeds derived from the same source On the other handthe breeds derived from the same source were included inthe same cluster showing close affinity between advancedsister lines [12 14 15] and those of differing genetic back-ground occupied in single cluster indicated uniformity inselection procedures [12] However genotypes of temperateand tropical origin formed into separate clusters indicatingenvironmental influence on the expression of characters[17]Though theoretically geographical diversity is importantfactor it is not the whole determining factor for geneticdivergence [18 19] All these studies were aimed to identifysuitable parents for breeding programme and recommendedto cross the genotypes from different clusters [20ndash22] foryield improvementThoughmany characters in silkworm aresubjected for divergence study characters namely fecunditylarval weight single cocoon weight cocoon shell weightand filament length only contributed about 97 to the totalgenetic divergence [12 13 16 23 24]

3 Diversity in Silkworm

Genetic diversity is most often characterized using data thatdepict variation in either discrete allelic states or continuouslydistributed (ie quantitative) characters which lead to differ-ent possible metrics of genetic diversity [25] Genetic diver-sity can be assessed among different accessionsindividualswithin the same species (intraspecific) among species (inter-specific) and between genus and families [26] It plays animportant role in any breeding either to exploit heterosis or togenerate productive recombinantsThe choice of parents is ofparamount importance in any kind of breeding programmehence the knowledge of genetic diversity and relatednessin the germplasm is a prerequisite for crop improvementprogrammes Genetic diversity is also an essential aspectin conservation biology because a fundamental concept ofnatural selection states that the rate of evolutionary changein a population is proportional to the amount of geneticdiversity present in it [27] Decreasing genetic diversityincreases the extinction risk of populations due to a declinein fitness Genetic diversity also has the potential to affecta wide range of population community and ecosystemprocesses both directly and indirectly However these effectsare contingent upon genetic diversity being related to themagnitude of variation in phenotypic traits [28]

In general cocoon colour and cocoon shape larvalmarking and quantitative traits have been used for differen-tiation of silkworm genotypes and based on that parents arebeing selected However recent advent of different moleculartechniques led breeders to estimate genetic diversity onthe basis of data generated by different molecular markerswhich provided a means of rapid analysis of germplasm andestimates of genetic diversity which were often found tocorroborate phenotypic data These molecular markers arebroadly categorized as biochemical and molecular markers

31 Biochemical Markers Application of isoenzymes andother molecular markers helps to estimate genetic diversitymuch more accurately than that of morphological traitsElectrophoresis identifies variation (alleles) at loci that codesfor enzymes (usually termed isozymes or allozymes) Oneadvantage of allozyme loci is that they are codominantand heterozygotes can be scored directly Understanding thegenetic constitution of an individual in the population ofraces and allelic variations through isozyme studies is knownto reflect the differential catalytic ability of allelic genes andtheir significant role in the adaptive strategy of the genotypes[29]

The diversity study carried out in silkworm throughprotein profiles enzymes and isozymes are summarizedin Table 2 Isozymes like esterase acid phosphatase alka-line phosphatase amylase phosphoglucomutase aspartateaminotransferase malate dehydrogenase glucose 6 phos-phate dehydrogenase and carbonic anhydrase have beenused by various authors to study diversity in silkwormgenotypes [30ndash43] Among the different isoenzymes ana-lyzed esterase was most preferred because of its diversesubstrate specificity and polymorphic expression followed byacid phosphatase [36 37 44] Eguchi et al [32] found four

International Journal of Biodiversity 3

Table 1 Genetic divergence study reported in silkworm

SLnumber

Referencenumber

Number ofgenotypes usedand clustersformed

Measures of geneticdiversity Conclusion

1 [89] 49 and 3 Mahalanobis (1936) andTocher (1956)

(1) Presence of distinct diversity (2) Breeds derived from thesame parents were included in different clusters (3) Breedsderived from the same source were included in the same cluster

2 [23] 32 and 7 Mahalanobis (1936) andTocher

Geographical diversity did not contribute much to geneticdiversity


(1) Enough diversity present (2) Suggested for making crossesbetween different clusters


Cluster III was the largest consisting of 34 strains The clustersare compared for various features influencing silk production


Breeds derived from the same ancestry were included indifferent clusters and those of different genetic backgroundoccupied a single cluster

6 [20] 25 and 6 Mahalanobis (1936) andTocher (1956) The genetically divergent parents were grouped into four classes

7 [18] 30 and 5Mahalonobisrsquo1198632 values(Wardrsquos minimumvariance)

Geographical diversity though important is not the determiningfactor for genetic divergence


Genotypes of temperate and tropical origin formed separateclusters


The intracluster distance ranged from 000 to 168937 implyingthe prevalence of substantial amount of intracluster diversity


There is no relation between geographical diversity and geneticdiversity


Breeds in the optimum distance obtained cluster can be used inthe conventional silkworm breeding programme to improve silkquality


Geographic diversity had no association with genetic diversity

13 [120] 51 and 2 UPGMAClusters of individuals exhibited high internal (within clusters)homogeneity and high external (between clusters)heterogeneity

14 [121] 16 and 3 Mahalanobis (1936)UPGMA

The strains of the same origin did not group togetherdemonstrating they can have different biological anddevelopment performance


Genetic distance and not the geographic diversity is to beconsidered while identifying parents for hybridizationprogramme


Silkworm genotypes originating from the same geographicalregions fell in one cluster


Silkworm genotypes originating from different geographicalregions fell in one cluster while those originating from a singleregion fell in different clusters

18 [123] 51 and 4 Hierarchical agglomerativeclustering UPGMA

Inclusion of genotypes of the same origin in different clustersclearly indicates the presence of considerable genetic diversityamong the populations

19 [19] 19 and 3The hierarchical clusteranalysis using Euclidiandistance

Cluster analysis and conformity with the variability in theperformance of the genotypes for different traits Geographicdiversity had no association with genetic diversity

20 [124] 4 and 2 UPGMAmethod (Sokaland Michener)

The optimum level of genetic distance is necessary to obtainheterosis


Table 2 Genetic divergence study reported in silkworm using enzymes protein and isozymes

SLnumber

Referencenumber

Number ofgenotypes and

clusters


1 [32] mdash Esterase used forpolymorphism Polymorphism noticed among genotypes

2 [125] mdash Protein profiles used forgenetic diversity

Divergence arises internally after a relatively long aminoterminal sequence which appears to be conserved A plausibleexplanation for the observed genetic variability is theoccurrence of relatively large unequal crossing-over exchangesin the repetitive domain of the fibroin gene


4 [127] Acid phosphatase used forpolymorphism Polymorphism observed among genotypes

5 [128] 20 Enzymes Rich genetic diversity among genotypes

6 [129] 10 Esterase used forpolymorphism Polymorphism noticed among genotypes

7 [36] 12 and 6 Nei and Li (1978) [130] andYeh et al (1999) [131] Rich genetic diversity among genotypes

8 [132] 8 and 2 Enzymes and UPGMA Genetic diversity noticed among genotypes

9 [133] Nei and Li (1978) The protein profile of different breeds has indicated thepolymorphism and genetic diversity among silkworm breeds

10 [37] 15 mdash Esterase exhibited polymorphism among the bivoltine breeds11 [134] 6 and 3 Protein Genetic differentiation among populations of different races

12 [44] 12 and 2 Nei and Li (1978) UPGMA

The mean value of FST (02224) calculated on the base of theestablished polymorphism showed that 2224 of the geneticvariability was observed between the different strains whichcorresponds to the level of the interstrain genetic differentiation

13 [40] 21 and 8 Nei and Li (1978) UPGMA Genetic variations were observed and they can be identified byrelating with their morphology and geographical origins

14 [135] Nei and Li (1978) UPGMA

Protein profiles studied and presence of rich genetic diversityamong germplasm stocks Different origin accessionsestablished a close relationship indicating close affinity inprotein pattern

15 [41] 15 Esterase used forpolymorphism Variation in esterase pattern was observed among genotypes

16 [43] 10 and 2Nei (1978) by UPGMAdendrogram (Sneath andSokal 1973)

A perusal of genetic diversity within and among strainsindicated that 3472 of the observed variation occurred amongstrains and the rest of the variation (6528) within strainsTheir rich genetic diversity needs to be exploited inconservation and breeding programme

17 [136] 4 (Swofford and Selander1981)

The lower degree of observed heterozygosity and the higherdegree of homozygotes proved the inbreeding effect

18 [42] 15 and 3 Nei (1978) and UPGMA

Japanese and Chinese strains could not be totally separated bythe isoenzyme system analysis The results indicate that in spiteof the genetic distance and differentiation among the lineagesthey cannot be separate just with the isozymes alleles The highFST value (06128) allows the conclusion that the lineages aredifferentiated

fundamental types of esterase and about 70 of the JapaneseChinese and European races investigated belong to A typeand 20 to 0 type while B type was found only in Chineseraces Yoshitake et al [45] analyzed polymorphism pattern ofesterase and acid phosphates in 300 strains of silkworm andconcluded that distribution of acid phosphatase and esterase

was similar in European and Japanese strains and therewas resemblance between Chinese and European strainsA higher degree of interstrain variability was reported onthe acid phosphatase [43 44] and esterase [36 37 41 42]Acid phosphatase is also found to be a suitable marker foranalyzing the inter- and intrastrain diversity and the strain


differentiation [44] Isozyme analysis in different silkwormgenotypes by different authors indicated rich genetic diversitybetween the genotypes and results were mainly used toseparate populations and strains in order to use them inselection programs

32 Molecular Markers Molecular diversity studies assessall levels of genetic structure and species specific complexcomponents [46] The detection and exploitation of natu-rally occurring DNA sequence polymorphisms have widepotential applications in animal and plant improvement pro-grammes as a means for varietal and parentage identificationfacilitate genetic diversity and relatedness estimations ingermplasm [47] The results obtained from different molec-ular markers may themselves be quite different from thoseobtained by using biochemical markers such as isozymes ormorphological characters The molecular markers namelyRAPD RFLP ISSR and SSR have been effectively utilized inanalyzing the genetic diversity and phylogenetic relatednessin the domesticated silkworm Bombyx mori [48ndash56] Detailsof diversity study carried out in silkworm through molecularmarkers are summarized in Table 3 RAPD based dendro-gram resulted in a clear separation of two groups one com-prising of diapausing and other comprising of nondiapausinggenotypes [49 57ndash60] Among the diapausing genotypesall the ldquoChinese typerdquo genotypes which spin oval cocoonsgrouped separately while the ldquoJapanese typerdquo genotypeswhich spin peanut shaped cocoons were found in anothergroup Further genotypes which share the same geographicalorigin were grouped in the same cluster [57 61] SSRand mtDNA markers analysis revealed considerable geneticdiversity among the nondiapausing silkworm genotypes thatwere developed in India China and Bangladesh [62] Thedendrogram constructed analysing RFLP markers revealedtwo distinct groups as Khorasan native (Iran) and Japanesecommercial lines The distinct clustering of these two setsof strains and lines reflects differences of the geographicalorigin and morphological qualitative and quantitative traitsassociated with them [54] Kim et al [63] made phyloge-netic analysis using the individual or the nine concatenatedintronic sequences which showed no clustering on the basisof known strain characteristic such as voltinism moultinismegg colour blood colour cocoon colour or cocoon shapeFurthermore the tree obtained by them using the nine con-catenated intronic sequences comprising 5897 bp includingindels resulted in a similar conclusion However Tunca et al[64] statedmoderately low level of diversity among genotypesstudied Supporting this argument recently Jagadeesh Kumar[65] reported the low level of genetic distance between thebreeds on the basis of gene frequency evidenced by the bootstrap values in the constructed dendrogram with the help ofmolecular markers

On the whole the diversity study conducted usingphenotypic characters and molecular markers had reportedadequate genetic variation between genotypes But thesedifferentiations mostly based on voltinism and geographical

origin indicating narrow genetic base between the availablegenotypes

4 Status of Genetic Diversity in Silkworm

Zhang et al [51] reported that genetic distances withinJapanese strains are closer than those of Chinese strainsand within a strain the individual polymorphism is signif-icantly higher in wild silkworm than those of domesticatedsilkworm According to Liu et al [66] at the species levelAntheraea pernyi and Bombyx mori showed high levels ofgenetic diversity whereas Samia cynthia ricini showed lowlevel of genetic diversity However at the strains levelAnther-aea pernyi had relatively the highest genetic diversity and Bmori had the lowest genetic diversity Analysis of molecularvariance (ANOVA) suggested that 60 and 72 of geneticvariation resided within strains in Antheraea pernyi andSamia cynthia ricini respectively whereas only 16 of geneticvariation occurredwithin strains inBmori Similarly geneticvariation was measured using the population size scaledmutation ratewhichwas significantly smaller in domesticatedstrains (0011) when compared to the wild strains (0013) ofB mori The rate of heterozygosity in domesticated strainswas reported to be two times lower than that in wild varieties(0003 and 0008 resp) Recently Yukuhiro et al [67]analyzed PCR amplified carbamoyl-phosphate synthetase 2aspartate transcarbamylase and dihydroorotase (CAD) genefragments from 146 Bombyx mori native strains and foundextremely low levels of DNA polymorphism CAD haplotypeanalysis of 42 samples of Japanese B mandarina revealedfour haplotypes No common haplotype was shared betweenthe two species and at least five base substitutions weredetected These results suggesting that low levels of geneflow between the two species Further extremely low level ofDNA polymorphism in B mori compared to its wild relativessuggested that the CAD gene itself or its tightly linkedregions are possible targets for silkworm domestication Thisinformation clearly indicates narrow level of genetic diversityin silkworm

5 Causes for Loss of Genetic Diversity

The existence of genetic variation within a population is cru-cial for its ability to evolve in response to novel environmentalchallenges Genetically variable populations are expected toevolve morphological physiological or behavioural mech-anisms to cope with the novel conditions [68] This sort-ing process not only results in populations that are betteradapted to their local environments but may also at leastin theory cause a reduction in the genetic variation Forcesthat affect genetic variation within populations are effectivepopulation size mutation genetic drift gene flow inbreedingdepression out breeding depression and natural selectionIn silkworm reduction in genetic diversity might be mainlydue to domestication breeding systems selection geneticdrift and inbreeding In maize too selection and drift dueto the domestication are the principal factors that influence


Table 3 Molecular diversity reported in silkworm

Slnumber

Referencenumber


Cluster

Measures ofgenetic diversity Conclusion

1 [48] 13 and 2 RAPD

Silkworm genotypes were clustered into two groups oneconsisting of six diapausing and the other of sevennondiapausing genotypes RAPD technique could be used as apowerful tool to generate genetic markers that are linked totraits of interest in the silkworm

2 [57] 13RAPD and banded

krait minorsatellite DNA

The RAPD based dendrogram resulted in a clear separation oftwo groups one comprising diapausing and the othernondiapausing genotypes The clustering pattern of RFLPobtained was comparable to the phenogram resulting fromRAPD analysis

3 [49] 5 and 3 RAPDSome of the DNA fragments were strain specific and somecould differentiate the multivoltine from the bivoltine strains orvice versa

4 [50] 13 and 2 SSR

Detailed analysis of silkworm strains with microsatellite locirevealed a number of alleles ranging from 3 to 17 withheterozygosity values of 066ndash090 Along with strain specificmicrosatellite markers diapause and nondiapausestrain-specific alleles were also identified

5 [137] 13 and 2 ISSR and RAPDThe highest diversity index was observed for ISSRPCR (0957)and the lowest for RAPDs (0744) Differentiated diapause andnondiapause strains

6 [138] 31 and 7 SSR

The average heterozygosity value for each SSR locus rangedfrom 0 to 060 and the highest one was 096 (Fl0516 in 4013)The mean polymorphism index content (PIC) was 066 (rangeof 012ndash089) SSR markers are an efficient tool for fingerprintingcultivars and conducting genetic-diversity studies in thesilkworm

7 [139] 20 and 6 RAPD Multivoltine Silkworm has more genetic diverse than bivoltine

8 [51] 12 SSRWithin a strain the individual polymorphism of wild silkwormwas significantly higher in abundance than those ofdomesticated silkworm

9 [140] 5 RAPD

The genetic distances between the clusters and within theclusters estimated 6 percent variability between the 4 races andNistari RAPDs are very efficient in the estimation of geneticdiversity in populations that are closely related and acclimatizedto local environmental conditions

10 [52] 29 and 4 CAP Considerable genetic diversity observed Grouped strainsroughly according to their geographical origin

11 [55] 96 SSRThe mean polymorphism index content was 071 (range of0299ndash0919) UPGMA cluster analysis of Neirsquos genetic distancegrouped silkworm strains based on their origin

12 [141] 6 and 2 AFLP

Higher degree of genetic similarity within Japanese commerciallines than the Iranian native strains The distinct clustering ofthese two sets of strains and lines reflects differences of thegeographical origin and morphological qualitative andquantitative traits associated with them

13 [61] 7 and 2 AFLP

The genetic similarity estimated within and among silkwormscould be explained by the pedigrees historical and geographicaldistribution of the strains effective population size inbreedingrate selection intensity and gene flow

14 [64] 6 RAPDThe genetic diversity in studying strains was moderately lowEstimates of gene diversity in populations were higher in total(Ht) as compared to those within population diversity (Hs)

15 [142] 20 and 6 ISSR

In selected mutant genetic stocks the average number ofobserved alleles was (17080 plusmn 04567) effective alleles(15194 plusmn 03950) and genetic diversity (Ht) (02901 plusmn 00415)ISSR is a valuable method for determining the geneticvariability among mutant silkworm strains


Table 3 Continued

Slnumber

Referencenumber


Cluster


16 [63] 25 and 3 Intronic sequencesThe degree of sequence divergence in some introns is veryvariable suggesting the potential of using intronic sequences forstrain identification

17 [58] 12 RAPD ISSR andRFLP-STS

RAPD generated 936 ISSR was 8462 and RFLP was 756polymorphism Ability to discriminate bivoltine andmultivoltine

18 [143] 3 and 2 RAPDThe diversity within the populations (Hs) was 01334 and themagnitude of differentiation among the populations (GST) was02968

19 [56] 14 ISSR

ISSR markers has generated 92 percent werepolymorphicdiapausing and non-diapausing silkworm stockscould be distinguished by specific marker

20 [144] 8 and 2 ISSR RAPD andisozymes Sufficient polymorphism and genetic diversity observed

21 [145] 30 and 2 ISSR

PCA analysis helped to visualize the two major clusters whichincluded the multivoltines and bivoltines separately Thegrouping of bivoltines in the PCA analysis clearly showed highersimilarity among bivoltines as compared to the multivoltines

22 [62] 13 and 2 SSR and mtDNAThe heterozygosity generated by the seven pairs of SSR primersvaried from 0098 to 0396 Considerable genetic diversity ispresent among the 13 silkworm genotypes

23 [146] 30 and 2 ISSRThe grouping of bivoltines in the PCA analysis clearly showedhigher similarity among bivoltines as compared to themultivoltines

24 [66]A Pernyi-3

S cynthia ricini-12Bmori-12

RAPD

At the species level A pernyi and B mori showed high levels ofgenetic diversity whereas S cynthia ricini showed low level ofgenetic diversity However at the strain level A pernyi hadrelatively the highest genetic diversity and B mori had thelowest genetic diversity

25 [147] 14 and 2 RAPD ISSR High polymorphisms (7091 and 7470) were revealed by ISSRand RAPD markers

26 [59] 4 and 2 RAPD

Multivoltine silkworm races are genetically more distant thanthe two bivoltine silkworm Genetic distances among themultivoltine and bivoltine silkworm were 052 and 027respectively

27 [60] 9 and 3 RAPDThe average genetic distance between the samples was 053 Theaverage genetic distance from analyzed samples proved to berelatively high

28 [148] 8 and 6 RAPDGenetic distances varied from 028889 (B752-C14) to 092437(A12-A13) with an average of 058497 Silkworms group a highgenetic diversity

29 [65] 5 and 2 ISSR Artificial selection during seven continuous generationsgenerally caused lesser genetic distance between the breeds

[149] 6 and 3 ISSR This marker could not discriminate same geographical racescorrectly

30 [150] 10 and 3 RAPDThe genotypes were grouped based on voltinism and bivoltinesare subgrouped based on silk productivity nature of silkwormbreeds

31 [151] 10 SSRSufficient polymorphism and genetic diversity observed Thegenotypes were grouped based on voltinism and subdividedbased on cocoon shape and cocoon colour


the amount and distribution of genetic variation in cropgenomes as compared to their wild progenitors inmaize [69]

51 Domestication Over the past 12 000 years humans havesampled selected cultivated travelled through and colo-nized new environments thus inducing a plethora of bottle-necks drifts and selection Plant breeders have acceleratedthe whole process by selecting preferred genotypes [46]In the broadest sense alteration and narrowing of cropgenetic diversity began with the first domestication of wildplantsanimals Domestication represents a relatively recentevolutionary event occurring over the past 13000 years afterthe Neolithic revolution [70 71] This process frequentlyleads to the improvement of economically important traitsand the diversification of morphological traits in domesti-cated species compared to their wild ancestors Silkwormdomestication which is a relatively recent evolutionary eventmay have generated a large number of alterations anddiversification in the structure of an evolutionarily conservedmorphogenetic gene [72] There is an assumption that theprocess of domestication and selection has resulted in drasticnarrowing of the genetic variation and homozygosity inmulberry silkworm which has been domesticated over 5000years ago Xia et al [73] compared the whole genomesequencing of 29 B mori strains and 11 Chinese B mandarinaindividuals by 150 billion short reads and concluded that Bmoriwas clearly genetically differentiated fromBmandarinaAt the same time based on the high level of conservation ofgenetic variability the authors estimated that a large numberofBmandarina individuals were used for domestication (iethe population bottleneck during silkworm domesticationmight not have been severe) Therefore gene flow limitedto B mori could have occurred for many genes duringsilkworm domestication Recently Yu et al [74] and Guoet al [75] reported decreased level of genetic variation inB mori genes or regions compared to those in ChineseB mandarina in the domestication targeted gene About407 or 492 of the genetic diversity of wild silkwormwas lost in domesticated silkworm [74] Study conductedwith B Mandarina and B mori by Guo et al [75] revealsthat diversity of B mori is significantly lower than that of Bmandarina Further gene DefA showed signature of artificialselection by all analysismethods andmight experience strongartificial selection in B mori during domestication resultingless diversity [75] However when analysing the carotenoidbinding protein (CBP) genes in B mori identified large copynumber variations and retrotransposon associated structuraldifferences in CBP from B mori which were absent from Bmandarina and concluded that domestication can generatesignificant diversity of gene copy number and structure overa relatively short evolutionary time

52 Breeding Too Causes Loss of Variability Breeding systemsand life history traits govern the transmission of genesbetween generations and have been long recognized asimpacting the genetic diversity and population genetic struc-ture [76ndash78] Breeding is a strong force in the reduction ofgenetic diversity [79] and views the introduction of modern

varieties as evidence of genetic erosion [80] Silkworm breed-ing by definition is the selection of superior genotypes andorphenotypes over a period of time During the last decadesdevelopment and increased focus on more efficient selectionprogrammes have accelerated genetic improvement in anumber of breeds As a result highly productive silkwormbreeds have replaced local ones across the world [81ndash84]Thisdevelopment has led to growing concerns about the erosion ofgenetic resources As the genetic diversity of low-productionbreeds is likely to contribute to current or future traits ofinterest [85 86] they are considered essential formaintainingfuture breeding options

Selection naturally results in a narrowing of the geneticbase of the genotype Even if the breeder has introduced alle-les from indigenous races to his target genotype heshe mustthen begin the process of ldquoweeding outrdquo the alleles that areundesirable This weeding out of undesirable alleles is onceagain narrowing the genetic base of the line Practically abreeder typically uses the best genotypes available and selectssuperior progeny The continual use of the best genotypesas parents naturally narrows the gene pool to only thosealleles that are available from the elite parents and thereforetends to decrease the genetic variation of the population [87]There is also a threat or loss of genetic diversity as a result ofreplacement of wild species by exotic high-yielding varietiesTypically population size is also a major source of loss ofgenetic diversity

53 Effects of Selection on Diversity Patterns of diversityin any populations are likely to be affected by selectionBalancing selection due to overdominance (heterozygoteadvantage) or to frequency dependent selection may main-tain variants in populations and environmental differencesmay select for different genotypes in different populations [3]Purifying selection however removes deleterious variantsthat arise by mutation such variants are expected to bepresent at frequencies lower than predicted for the neutralequilibrium Another form of directional selection occurswhen advantageousmutations rapidly reach high frequencieswhether they spread throughout a species to fixation orjust within a population undergoing adaptation to its localenvironment [88 89] Artificial selection has been widelyutilized in the breeding programmes concerning B moriwhich is of commercially important insect Neverthelessthe genetic diversity of silkworm is greatly reduced duringsystematically extensive selection for a few target traits Ingeneral selection of superior individuals results in geneticgain but also loss in genetic diversity and it is stronglydependent on selection method and selection intensity [90]Selection will have two important consequences (1) thegenetic average value will be changed thus conventionally itmeasured a gain and (2) there will be change in diversityand this will be measured by relative effective number offamilies It is a well-known fact that diversity is affectedby directional selection Directional or disruptive selectionwill ultimately fix one allele and thereby deplete geneticvariation It has been suggested that directional selection


decreases the level of developmental precision or develop-mental stability [91] because it may prevent the evolutionof canalisation and possibly favour those mechanisms thatincrease the phenotypic variation [92 93] showing thatsystematic selection of parentsrsquo results in reduced geneticvariation among their offspring After 4-5 generations withthe same selection intensity the reduction will stabilize Classexample of diversity changes through directional selectionand inbreeding in silkworm was reported by Pradeep etal [94] They have separated larval populations of Nistaristrain based on the shortest larval duration (SLD) and thelongest larval duration (LLD) and maintained for 4 moregenerations RAPD and ISSR primers generated polymorphicprofiles in LLD and SLD lines Distinct markers specificto LLD individuals were observed from the 3rd generationand indicated selection induced differentiation of allelicvariants for longer larval duration This finding implies thatselection combined with inbreeding could result in lines withdifferent genetic properties following separation from theoriginal parental populations According to Strunnikov [95]continuous selection and inbreeding could have induced ahomozygous state of the recessive gene for longer larval dura-tionwhere shorter larval duration is the dominant andfitnesscharacterThough it introduced diversity because of losing itsdominant or fitness characters chances of survival becomevulnerable Further as reported by Seidavi [96] the geneticperformance of selected population of silkworm for cocoonweight trait after the fourth generation shown increasedsensitivity towards environment resulting in poor survivaldue to selection based on productivity traits indicating effectof selection on diversity

54 Genetic Drift Genetic drift is the chance changes in allelefrequency that result from the random sampling of gametesfrom generation to generation in a finite population It has thesame expected effect on all loci in the genome [97] In a largepopulation on the average only a small chance change in theallele frequency will occur as the result of genetic drift Onthe other hand if the population size is small then the allelefrequency can undergo large fluctuations in different gener-ations in a seemingly unpredictable pattern and can result inchance fixation (going to a frequency of 10) or the loss (goingto a frequency of 00) of an allele A classic illustration of howfinite population size affects allele frequency was provided byBuri [98] He looked at the frequency of two alleles at thebrown locus that affects eye color inDrosophila melanogasterin randomly selected populations of size 16 However around107 number of populations had 0 to 32 bw75 genes in different(19) generations The total number of populations fixed forone of the two alleles increased at nearly a linear rate aftergeneration 4 and in generation 19 it is nearly equal for thetwo alleles with 30 populations fixed for bw and 28 fixed forbw75 In silkworm germplasm maintenance centers at everycycle only 40ndash60 cocoons are selected from each strainbreedfor the next generation from which around 20 layings areprepared and subsequently only 5-6 layings are brushed fornext generationThis size of population is small whichmay belead to change in allele frequency as explained by Buri [98]

55 Effects of Inbreeding Loss of genetic diversity amongpopulations occurs due to the synergetic effects of inbreedingand environmental stressors [99] The negative interactionbetween inbreeding and environmental stress reflects onpopulation growth rates and inbreeding and environmentaleffects may interact in their effects on population dynamicsInbreeding is characterized by an increase in homozygosityresulting in increased expression of recessive deleteriousalleles (partial dominance hypothesis) [100] andor reducedopportunity to express heterozygote superiority (overdom-inance hypothesis) [101] Selfing has direct genetic conse-quences including its effect on the intensity of inbreedingdepression [102] and the partitioning of genetic diversitywithin and among populations [103] A consequence ofinbreeding is that it makes it much more likely that anindividual is homozygous for a rare gene because it ismore likely that two related parents simultaneously possessa rare allele and transmit it to their inbred offspring thanthe two unrelated individuals independently transmit thesame rare allele to noninbred offspring Thus inbreedingseems to reduce fitness because it reveals harmful genes inhomozygotes [104]

Sericulture practicing countries maintain hundreds ofinbred lines of silkworm in germplasm centres for severaldecades by selection and inbreeding Sibling mating of theprogenies derived from a single brood is preferred for purestocks so that the original traits of the races are maintainedthrough generations Generally breeders try to maintain theoriginal characters of the racesbreeds through selection withcare to avoid inbreeding depression However the effects ofinbreeding can accumulate over many generations as thefrequency of slightly deleterious alleles can gradually increaseover time due to genetic drift [105 106] This is a particularconcern in small populations where natural selection canbe inefficient for alleles that have only slight effects onfitness [107] The rate at which genetic diversity is lost willdepend on the populationrsquos size and degree of isolationsmall isolated populations can lose genetic diversity withina few generations whereas large continuous populationsmay not lose significant amounts of diversity over thousandsof years [108] In small populations where genetic drift ismost rapid the fixation of common alleles will result in thereduction of genetic diversityThis phenomenon is applicablein silkworm as different silkworm strains are maintainedwith small population leading to genetic drift thereby maybe reducing genetic diversity Further Li et al [109] whenanalysing genetic diversity in B mandarina and B moriconcluded that the polymorphism level (120579120587) of mt sequenceamong Chinese wild population (620 times 10minus3 nucleotidedifferences per site) is more than six times that amongdomesticated varieties (114 times 10minus3) and pointing out thatthe relative larger reduction in polymorphism is most likelycaused by inbreeding or population bottlenecking

6 Broadening the Genetic Diversity

Continuous breeding and selection of silkworm breed foruniformity narrows genetic diversity One of the approaches


to broaden the genetic diversity is the use of recent advancesin molecular biology and biotechnology which allow thetransfer of specific genes from diverse sources to targetgenotypes In [110] through transgenic approach by geneaddition subtraction and pathway redirection the geneticconstituents of crops can be modified and broadened result-ing in new and improved traits Another approach of broad-ening the genetic diversity is by the use of exotic germplasm[111] It will create genotypes with a diverse range of desirablecharacteristics The genetic variation that breeders need tointroduce these characteristics is often available only throughthe exchange of genetic resourcesThis exchange is necessarybecause some areas of the world have richer resources ofgenetic diversity which will be useful in creation of variation

7 Germplasm Conservation

Conservation of genetic diversity is essential to the long-term survival of any species particularly in light of changingenvironmental conditions Reduced genetic diversity maynegatively impact the adaptive potential for a species Increas-ing population size and maximizing genetic diversity areamong the primary goals of conservation management [112]The silkworm germplasm maintenance centres generallyfollow brushing of ldquocomposite populationrdquo type of all strainsto avoid inbreeding depression as well as genetic erosionand maintain the gene pool as far as possible Compositelaying is defined as collection of a known number of eggsfrom a known number of individual laying sources thatrepresents the whole population Though composite layingsmethod can retain gene pool there is a concern regardingpopulations as even slight selection has a drastic effect ongenetic variability when the effective population size N islarge [113] In thismethod only 250ndash500 larvae are retained ina strain improper selection can lead to inbreeding depressionand natural selection can be inefficient for alleles that haveonly slight effects on fitness [107]

8 Strategies Required for Conservation ofSilkworm Genetic Resources

(1) The curator of the germplasm bank should care-fully verify the available genetic resources and avoidduplicates before collection and introduction of newmaterial

(2) Development of cost-effective viable and cost-economic conservation practices through modifica-tion or development of long-term preservation ofsilkworm genetic resources to reduce the number ofcrop cycles is required

(3) Conservation through modern methods includescryopreservation of sperm artificial inseminationand induction of synthetic diapause hormones to beexplored

(4) Genetic resources should be categorized as mostsensitive and sensitive based on their availability inone place or in more than one place respectively

(5) The most sensitive genetic resources should be con-served in more than one place by establishing backupstations under the control ofmain germplasm station

(6) Establishment of centers for preservation of endan-geredlocal species under in situ condition is required

(7) Use of silkworm genetic resources for nonsericulturaluse other than cocoon production needs importance

9 Conclusion

Though silkworm has been domesticated for hundreds ofgenerations based on available literature it is speculatedthat it has not experienced any major reduction of geneticdiversity due to phenotypic selection and breeding But thereis concern that bottlenecks may restrict breeding flexibilityand slow response to new opportunities pests pathogensand other practices in the future To broaden the gene pool ofsilkworm exotic elite strains were required to be introducedfrom various countries The genomes of introduced exoticgermplasm will broaden the gene pool thereby diversity canbe maintained The original genetic composition of geneticresources should be maintained by avoiding genetic drift andselection process Maintaining adequate population size canprevent the loss of genetic variability due to genetic drift[114] Study on effects of inbreeding on inbreeding coefficientsin silkworm populations is limited Hence understandingthe effects of inbreeding for various traits can be verycrucial points in themanagement of germplasmAs suggestedby Doreswamy and Subramanya Gopal [115] during stockmaintenance in germplasm centers rigid selection for morenumbers of generations is required to retain original charac-teristics of the inbred lines and also reduces the deleteriouseffects of inbreeding

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

References

[1] J Nagaraju and M R Goldsmith ldquoSilkworm genomicsmdashprogress and prospectsrdquo Current Science vol 83 no 4 pp 415ndash425 2002

[2] Y Banno T Shimada Z Kajiura and H Sezutsu ldquoThesilkwormmdashan attractive bioresource supplied by Japanrdquo Exper-imental Animals vol 59 no 2 pp 139ndash146 2010

[3] D Charlesworth and T R Meagher ldquoEffects of inbreeding onthe genetic diversity of populationsrdquo Philosophical Transactionsof the Royal Society B Biological Sciences vol 358 no 1434 pp1051ndash1070 2003

[4] W L Brown ldquoGenetic diversity and genetic vulnerabilitymdashanappraisalrdquo Economic Botany vol 37 no 1 pp 4ndash12 1983

[5] V R Rao and T Hodgkin ldquoGenetic diversity and conservationand utilization of plant genetic resourcesrdquo Plant Cell Tissue andOrgan Culture vol 68 no 1 pp 1ndash19 2002

[6] I A Matus and P M Hayes ldquoGenetic diversity in three groupsof barley germplasm assessed by simple sequence repeatsrdquoGenome vol 45 no 6 pp 1095ndash1106 2002


[7] S A Mohammadi and B M Prasanna ldquoAnalysis of geneticdiversity in crop plantsmdashsalient statistical tools and consider-ationsrdquo Crop Science vol 43 no 4 pp 1235ndash1248 2003

[8] AA JaradatM Shahid andAAl-Maskri ldquoGenetic diversity inthe Batini barley landrace from Oman II Response to salinitystressrdquo Crop Science vol 44 no 3 pp 997ndash1007 2004

[9] Z Ahmad S U Ajmal M Munir M Zubair and M SMasood ldquoGenetic diversity for morpho-genetic traits in barleygermplasmrdquo Pakistan Journal of Botany vol 40 no 3 pp 1217ndash1224 2008

[10] M R Goldsmith ldquoRecent progress in silkworm genetics andgenomicsrdquo inMolecular Biology and Genetics of the LepidopteraM R Goldsmith and F Marec Eds pp 25ndash48 CRC BocaRaton Fla USA 2009

[11] P C Mahalanobis ldquoOn the generalized distance in statisticsrdquoProceedings of National Academy of Sciences India vol 2 no 1pp 49ndash55 1936

[12] C R RaoAdvanced Statistical Methods in Biometrical ResearchJohn Wiley and Sons New York NY USA 1952

[13] M S Jolly R K Datta M K R Noamani et al ldquoStudieson genetic divergence in mulberry silkworm Bombyx mori LrdquoSericologia vol 29 no 4 pp 545ndash553 1989

[14] G Subba Rao S K Das and N K Das ldquoGenetic divergenceamong fifteen multivoltine genetic stocks of silkworm (Bombyxmori L)rdquo Indian Journal of Sericulture vol 30 no 1 pp 72ndash741991

[15] R Govindan S Rangaiah T K Narayana Swamy M C Deva-iah andR S Kulkarni ldquoGenetic divergence amongmultivoltinegenotypes of silkworm (Bombyx mori L)rdquo Environmental Ecol-ogy vol 14 no 4 pp 757ndash759 1996

[16] N B Pal S MMoorthyM Z Khan N K Das and KMandalldquoAnalysis of genetic diversity in some multivoltine silkwormgenotypes of Bombyx mori Lrdquo in Proceedings of the GoldenJubilee National Conference on Sericulture Innovations Beforeand Beyond Abstract p 68 CSRTI Mysore India January2010

[17] KMandal SMMoorthy S Sen N KDas andC R Sahu ldquoAnanalysis of genetic variation and diversity in bivoltine silkworm(Bombyx mori L) genotypesrdquo in Proceedings of the NationalSymposium onDeccan Biodiversity Co-Existance of Funal Speciesin Changing Landscapes Abstract p 38 Osmania UniversityHyderabad December 2010

[18] P Mukherjee S Mukherjee and P Kumaresan ldquoAn analysis ofgenetic divergence in Indian multivoltine silkworm (BombyxmoriL) germplasmrdquo Sericologia vol 39 no 3 pp 337ndash347 1999

[19] N B Pal and S M Moorthy ldquoAssessment of variability in larvaland cocoon traits in some genotypes of bivoltine silkwormBombyx mori Lrdquo International Journal of Research in BiologicalSciences vol 1 no 4 pp 59ndash65 2011

[20] S K Sen B P Nair S K Das et al ldquoRelationship betweenthe degree of heterosis and genetic divergence in the silkwormBombyx mori Lrdquo Sericologia vol 36 no 2 pp 215ndash225 1996

[21] P Kumaresan T S Mahadevamurthy K Thangavelu andR K Sinha ldquoFurther studies on the genetic divergence ofmultivoltine silkworm (Bombyx mori L) genotypes based oneconomic charactersrdquo Entomon vol 28 no 3 pp 193ndash198 2003

[22] B Mohan N Balachandran M Muthulakshmi et al ldquoStratifi-cation of silkworm (Bombyx mori L) germplasm for establish-ing core-set using characterisation datardquo International Journalof Tropical Agricultur vol 29 no 3-4 pp 325ndash329 2011

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[32] M Eguchi N Yoshitake and H Kai ldquoTypes and inheritance ofblood esterase in the silkworm Bombyx mori Lrdquo Japan Journalof Genetics vol 40 pp 15ndash19 1965

[33] M Eguchi and N Yoshitake ldquoInterrelation of non specificesterase among various tissues in the silkworm Bombyx moriLrdquo Japan Journal of Sericulture vol 36 pp 193ndash198 1967

[34] M Eguchi Y TakahamaM Ikeda and SHorii ldquoAnovel variantof acid phosphatase isozyme from hemolymph of the silkwormBombyx morirdquo Japan Journal of Genetics vol 63 no 2 pp 149ndash157 1988

[35] A Shabalina ldquoEsterase genetic polymorphism in haemolymphof larvae Bombyx morirdquo Comptes Rendus de lrsquoAcademie Bulgaredes Sciences vol 43 pp 105ndash110 1990

[36] P Somasundaram K Ashok kumar K Thangavelu P K Karand R K Sinha ldquoPreliminary study on isozyme variation insilkworm germplasm of Bombyx mori (L) and its implicationfor conservationrdquo Pertanika Journal of Tropical AgriculturalScience vol 27 no 2 pp 163ndash171 2004

[37] S M Moorthy S K Das P R T Rao S Raje Urs and ASarkar ldquoEvaluation and selection of potential parents based onselection indices and isozyme variability in silkworm Bombyxmori Lrdquo Internatioanl Journal of Industrial Entomology vol 14pp 1ndash7 2007

[38] T Staykova andDGrekov ldquoStage specificity and polymorphismof haemolymph esterases in races and hybrids of silkworm(Bombyx mori L) kept in Bulgariardquo in Proceedings of theInternational Workshop on Silk Handcrafts Cottage Industriesand Silk Enterprises Development in Africa Europe Central Asiaand the Near East amp 2nd Executive Meeting of Black Caspianseas and Central Asia Silk Association (BACSA) pp 667ndash674Bursa Turkey March 2006


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[41] B B Patnaik T Datta A K Saha and M K MajumdharldquoIsozymic variations in specific and nonspecific esterase andits thermostability in silkworm Bombyx mori Lrdquo Journal ofEnvironmental Biology vol 33 pp 837ndash842 2012

[42] L Ronqui M Aparecida and M C C Ruvolo TakasusukldquoGenetic analysis of isoenzymes polymorphisms in silkworm(Bombyx mori L) strainsrdquo Acta Scientiarum Biological Sciencesvol 35 no 2 pp 249ndash254 2013

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[44] T Staykova E N Ivanova P Zenov Y VaSileva D ArkovaPantaleeva and Z Petkov ldquoAcid phosphatase as a marker fordifferentiation of silkworm (Bombyx mori) strainsrdquo Biotechnol-ogy and Biotechnological Equipment vol 24 no 2 pp 379ndash3842010

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[51] L Zhang YHuang XMiaoMQian andC Lu ldquoMicrosatellitemarkers application on domesticated silkworm and wild silk-wormrdquo Insect Science vol 12 no 6 pp 413ndash419 2005

[52] J H Huang S H Jia Y Zhang et al ldquoThe polymorphism ofsilkworm Bombyx mori (L) amylase generdquo Scientia AgriculturaSinica vol 39 no 11 pp 2390ndash2394 2006

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[54] S B Dalirsefat and S ZMirhoseini ldquoAssessing genetic diversityin Iranian native silk-worm (Bombyxmori) strains and Japanesecommercial lines using AFLP markersrdquo Iran Journal of Biotech-nology vol 5 no 1 pp 54ndash63 2007

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[58] A K Awasthi P K Kar P P Srivastava et al ldquoMolecular evalu-ation of bivoltine polyvoltine and mutant silkworm (Bombyxmori L) with RAPD ISSR and RFLP-STS markersrdquo IndianJournal of Biotechnology vol 7 no 2 pp 188ndash194 2008

[59] E Talebi M Khademi and G Subramanya ldquoRAPD markersfor understanding of the genetic variability among the foursilkworm races and their hybridsrdquo Middle-East Journal ofScientific Research vol 7 no 5 pp 789ndash795 2011

[60] E M Furdui L A Marghita D Dezmirean I F Pop CCoroian and I Pasca ldquoGenetic phylogeny and diversity ofsome Romanian silkworms based on RAPD techniquerdquoAnimalScience and Biotechnologies vol 44 no 1 pp 204ndash208 2011

[61] S Z Mirhoseini S B Dalirsefat and M PourkheirandishldquoGenetic characterization of iranian native Bombyxmori strainsusing amplified fragment length polymorphismmarkersrdquo Jour-nal of Economic Entomology vol 100 no 3 pp 939ndash945 2007

[62] K Vijayan C V Nair and S Raje Urs ldquoAssessment of geneticdiversity in the tropical mulberry silkworm (Bombyx mori L)with mtDNA-SSCP and SSR markersrdquo Emirate Journal of FoodAgriculture vol 22 no 2 pp 71ndash83 2010

[63] K Y Kim E M Lee I H Lee et al ldquoIntronic sequences of thesilkworm strains of Bombyx mori (Lepidoptera Bombycidae)high variability and potential for strain identificationrdquoEuropeanJournal of Entomology vol 105 no 1 pp 73ndash80 2008

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[65] T S Jagadeesh Kumar ldquoMolecular dynamics of genomic DNAof silkworm breeds for screening under higher temperatureRegimes utilising ISSR-primersrdquo International Journal of Sci-ence Environment and Technology vol 2 no 2 pp 275ndash2852013

[66] Y Liu L Qin Y Li et al ldquoComparative genetic diversity andgenetic structure of three chinese silkworm species Bombyxmori L (Lepidoptera Bombycidae) Antheraea pernyi guerin-meneville and samia cynthia ricini donovan (LepidopteraSaturniidae)rdquo Neotropical Entomology vol 39 no 6 pp 967ndash976 2010

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[78] K E Holsinger ldquoReproductive systems and evolution in vascu-lar plantsrdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 97 no 13 pp 7037ndash7042 2000

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[89] X Vekemans and C Lefebvre ldquoOn the evolution of heavy-metal tolerant populations in Armeria maritima evidencefrom allozyme variation and reproductive barriersrdquo Journal ofEvolutionary Biology vol 10 no 2 pp 175ndash191 2001

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[92] M G Bulmer ldquoThe effect of selection on genetic variabilityrdquoAmerican Nature vol 105 pp 201ndash211 1971

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[94] A R Pradeep S N Chatterjee and C V Nair ldquoGeneticdifferentiation induced by selection in an inbred population ofthe silkwormBombyxmori revealed byRAPDand ISSRmarkersystemsrdquo Journal of Applied Genetics vol 46 no 3 pp 291ndash2982005

[95] V A Strunnikov Control over Reproduction Sex and Heterosisof the Silkworm Harwood Academic Publishers Luxembourg1995

[96] A Seidavi ldquoEstimation of genetic parameters and selectioneffect on genetic and phenotype trends in silkworm commercialpure linesrdquo Asian Journal of Animal and Veterinary Advancesvol 5 no 1 pp 1ndash12 2010

[97] S Wright EVolution and the Genetics of Populations Volume 1Genetic and Biometric Foundations University of Chicago PressChicago Ill USA 1968

[98] P Buri ldquoGene frequency in small populations of mutantDrosophilardquo Evolution vol 10 pp 367ndash402 1956

[99] R Bijlsma J Bundgaard and A C Boerema ldquoDoes inbreedingaffect the extinction risk of small populations Predictions fromDrosophilardquo Journal of Evolutionary Biology vol 13 no 3 pp502ndash514 2000

[100] D H Reed ldquoThe effects of population size on populationviability from mutation to environmental catastrophesrdquo inConservation Biology Evolution in Action S P Carroll and CW Fox Eds pp 16ndash35 Oxford University Press New York NYUSA 2008

[101] D Charlesworth and B Charlesworth ldquoQuantitative genetics inplants the effect of the brreeding system on genetic variabilityrdquoEvolution vol 49 no 5 pp 911ndash920 1995

[102] D Charlesworth B Charlesworth and C Strobeck ldquoSelectionfor recombination in self-fertilising speciesrdquo Genetics vol 93pp 237ndash244 1979

[103] J L Hamrick and M J Godt ldquoAllozyme diversity in plantspeciesrdquo in Plant Population Genetics Breeding and GeneticResources A H D Brown M T Clegg A L Kahler and BS Weir Eds pp 43ndash63 Sinauer Sunderland Mass USA 1990

[104] R C Lacy ldquoImpacts of inbreeding in natural and captivepopulations of vertebrates implications for conservationrdquo Per-spectives in Biology and Medicine vol 36 no 3 pp 480ndash4961993

[105] R Lande ldquoRisk of population extinction from fixation of newdeleterious mutationsrdquo Evolution vol 48 no 5 pp 1460ndash14691994


[106] M C Whitlock ldquoSelection load and inbreeding depression ina large metapopulationrdquo Genetics vol 160 no 3 pp 1191ndash12022002

[107] S Wright Evolution and Genetics of Populations Volume 3Experimental Results and Evolutionary Deductions Universityof Chicago Press Chicago Ill USA 1977

[108] K Zittlau Population genetic analyses of North American cari-bou (Rangifer tarandus) [PhD dissertation] Department ofBiological Sciences University of Alberta Edmonton Canada2004

[109] D Li Y Guo H Shao et al ldquoGenetic diversity molecularphylogeny and selection evidence of the silkworm mitochon-dria implicated by complete resequencing of 41 genomesrdquo BMCEvolutionary Biology vol 10 no 1 article 81 10 pages 2010

[110] M L Wang J A Mosjidis J B Morris Z B Chen NA Barkley and G A Pederson ldquoEvaluation of Lespedezagermplasm genetic diversity and its phylogenetic relationshipwith the genusKummerowiardquoConservationGenetics vol 10 no1 pp 79ndash85 2009

[111] M Goodman ldquoBroadening the genetic diversity in maizebreeding by use of Exotic germplasmrdquo in The Genetics andExploitation of Heterosis in Crops G Coors and S Pandey Edspp 130ndash149 ASA-CSSA-SSSA Madison Wis USA 1999

[112] R Frankham J D Ballou and D A Briscoe Introductionto Conservation Genetics Cambridge University Press Cam-bridge UK 2002

[113] W-H Li ldquoMaintenance of genetic variability under the jointeffect ofmutation selection and randomdriftrdquoGenetics vol 90no 2 pp 349ndash383 1978

[114] P S Guzman and K R Lamkey ldquoEffective population size andgenetic variability in the BS11 maize populationrdquo Crop Sciencevol 40 no 2 pp 338ndash342 2000

[115] J Doreswamy and S Gopal ldquoInbreeding effects on quantitativetraits in random mating and selected populations of the mul-berry silkworm Bombyx morirdquo Journal of Insect Science vol 12pp 1ndash6 2012

[116] B K Gupta V K Kharoo and M Verma ldquoGenetic divergencein bivoltine strains of silkworm (Bombyx mori L)rdquo Bioved vol3 no 2 pp 143ndash146 1992

[117] T K Narayanaswamy R Govindan S R Anantha Narayanaand S Ramesh ldquoGenetic divergence among some breeds ofsilkworm Bombyx mori Lrdquo Entomon vol 27 no 3 pp 319ndash3212002

[118] P Kumaresan P R Koundinya S A Hiremath and R KSinha ldquoAn analysis of genetic variation and divergence on silkfibre characteristics of multivoltine silkworm (Bombyx mori L)genotypesrdquo International Journal of Industrial Entomology vol14 no 1 pp 23ndash32 2007

[119] M FarooqM A Khan andMN Ahmad ldquoGenetic divergenceamong bivoltine genotypes of silkworm Bombyx mori Lrdquoin Proceedings of the National Workshop on Seri-BiodiversityConservation pp 94ndash98 Central Sericultural GermplasmResources Centre Hosur India March 2009

[120] M S Nezhad S Z Mirhosseini S Gharahveysi M Mavva-jpour and A R Seidavi ldquoAnalysis of genetic divergence forclassification of morphological and larval gain characteristicsof peanut cocoon silkworm (Bombyx mori L) germplasmrdquoAmerican-Eurasian Journal of Agriculture and EnvironmentalSciences vol 6 no 5 pp 600ndash608 2009

[121] D B Zanatta J P Bravo J F Barbosa R E F Munhoz andM A Fernandez ldquoEvaluation of economically important traits

from sixteen parental strains of the silkworm Bombyx mori L(Lepidoptera Bombycidae)rdquo Neotropical Entomology vol 38no 3 pp 327ndash331 2009

[122] A Maqbool and H U Dar ldquoGenetic divergence in some bivol-tine silkworm (Bombyx mori L) breedsrdquo 2010 httpdspacesuokeduinjspuihandle11100

[123] M Salehi Nezhad S Z Mirhosseini S Gharahveysi MMavvajpour A R Seidavi and M Naserani ldquoGenetic diversityand classification of 51 strains of silkworm Bombyx mori (Lepi-doptera Bombycidae) germplasm based on larval phenotypicdata using Wardrsquos and UPGMA methodsrdquo African Journal ofBiotechnology vol 9 no 39 pp 6594ndash6600 2010

[124] E Talebi and G Subramanya ldquoGenetic distance and heterosisthrough evaluation index in the silkworm Bombyx mori (L)rdquoAmerican Journal of Applied Sciences vol 6 no 12 pp 1981ndash19872009

[125] P M Lizardi ldquoGenetic polymorphism of silk fibroin studiedby two-dimensional translation pause fingerprintsrdquoCell vol 18no 2 pp 581ndash589 1979

[126] T Egorova ENaletova andYNasirillaev ldquoPolymorphic systemof silkworm haemolymph esterases as a criterion to makeprograms for parental specimens crossingrdquo Biochemistry ofInsects pp 54ndash62 1985 (Russian)

[127] M Eguchi Y TakahamaM Ikeda and SHorii ldquoAnovel variantof acid phosphatase isozyme from hemolymph of the silkwormBombyx mori Lrdquo Japanese Journal of Genetics vol 63 pp 149ndash157 1988

[128] S N Chatterjee and R K Datta ldquoHierarchical clustering of54 races and strains of the mulberry silkworm Bombyx moriL significance of biochemical parametersrdquo Theoretical andApplied Genetics vol 85 no 4 pp 394ndash402 1992

[129] S K Das P K Chinya S Patinaik S K Sen and G SubbaRao ldquoStudies on genetic variability of heamolymph esterasesin some genetic stocks of mulberry silkworm Bombyx mori LrdquoPerspectives in Cytology and Genetics vol 7 pp 421ndash425 1992

[130] M Nei andW H LI ldquoMathematical model for studying geneticvariation in terms of restriction endonucleasesrdquo Proceedings ofthe National Academy of Sciences of the United States of Americavol 76 no 10 pp 5269ndash5527 1978

[131] F C Yeh R Yang and T Boyle ldquoPopulation genetic analysisPOPGENE version 131 Microsoftwindow-based freeware forpopulation genetic analysisrdquoQuickUserrsquosGuide A Joint ProjectDeveloped by Centre for International Forestry Research andUniversity of Alberta Alberta Canada 1999

[132] K Etebari S Z Mirhoseini and L Matindoost ldquoA study oninterspecific biodiversity of eight groups of silkworm (Bombyxmori) by biochemical markersrdquo Insect Science vol 12 no 2 pp87ndash94 2005

[133] V Kumar S K Ashwath and S B Dandin ldquoHeamolymphprotein variability among the silkworm (Bombyx mori) breedsand assessment of their genetic relationshiprdquo in Proceedings ofthe Asia Pacific Congress of Sericulture and Insect Biotechnology(APSERI rsquo06) Abstract p 54 Sangju Republic of KoreaOctober 2006

[134] S Bakkappa and G Subramanya ldquoElectrophoretic haemo-lymph protein pattern in a few bivoltine races of the silkwormBombyx morirdquoThe Bioscan vol 5 no 4 pp 541ndash544 2010

[135] J Anuradha S Somasundaram SVishnupriya andAManjulaldquoStorage protein-2 as a dependable biochemical index forscreening germplasm stocks of the silkworm Bombyxmori (L)rdquoAlbanian Journal of Agriculture Science vol 11 pp 141ndash148 2012


[136] T Staykova ldquoInter-and intra-population genetic variabilityof introduced silkworm (Bombyx mori L) strains raised inBulgariardquo Journal of Biosciences and Biotechnology vol 2 no1 pp 73ndash77 2003

[137] J Nagaraju K D Reddy GMNagaraja and B N SethuramanldquoComparison of multilocus RFLPs and PCR-based markersystems for genetic analysis of the silkworm Bombyx morirdquoHeredity vol 86 no 5 pp 588ndash597 2001

[138] M Li L Shen A Xu et al ldquoGenetic diversity among silkworm(Bombyx mori L Lep Bombycidae) germplasms revealed bymicrosatellitesrdquo Genome vol 48 no 5 pp 802ndash810 2005

[139] P P Srivastava K Vijayan A K Awasthi P K Kar KThangavelu and B Saratchandra ldquoGenetic analysis of silk-worms (Bombyx mori) through RAPDmarkersrdquo Indian Journalof Biotechnology vol 4 no 3 pp 389ndash395 2005

[140] B C K Murthy B M Prakash and H P Puttaraju ldquoFin-gerprinting of non-diapausing silkworm Bombyx mori usingrandom arbitrary primersrdquo Cytologia vol 71 no 4 pp 331ndash3352006

[141] S B Dalirsefat and S ZMirhoseini ldquoAssessing genetic diversityin Iranian native silkworm (Bombyx mori L) strains andJapanese commercial lines using AFLP markersrdquo Iranian Jour-nal of Biotechnology vol 5 no 1 pp 25ndash33 2007

[142] D Velu K M Ponnuvel M Muthulakshmi R K Sinha andS M H Qadri ldquoAnalysis of genetic relationship in mutantsilkworm strains of Bombyx mori using inter simple sequencerepeat (ISSR) markersrdquo Journal of Genetics and Genomics vol35 no 5 pp 291ndash297 2008

[143] D Eroglu and S Cakir Arica ldquoMolecular genetic analysisof three Turkish local silkworm breeds (Bursa Beyazı Alacaand Hatay Sarısı) by RAPD-PCR methodrdquo Journal of AppliedBiological Sciences vol 3 no 2 pp 17ndash20 2009

[144] K Ashok Kumar P Somasundaram K M Ponnuvel G KSrinivasa Babu S M H Qadri and C K Kamble ldquoIdentifica-tion of genetic variations among silkworm races ofBombyxmori(L) through bio-molecular toolsrdquo Indian Journal of Sericulturevol 48 no 2 pp 116ndash125 2009

[145] K Sanjeeva reddy C A Mahalingam K A Murugesh andS Mohankumar ldquoExploring the genetic variability in Bombyxmori L with molecular markerrdquo Karnataka Journal of Agricul-tural Sciences vol 22 pp 479ndash483 2009

[146] K A Murugesh S Mohankumar and C A MahalingamldquoMolecular marker analysis on genetic variation in domesti-cated silkwormrdquo Trends in Biosciences vol 3 no 2 pp 102ndash1052010

[147] P P Srivastava K Vijayan P K Kar and B SaratchandraldquoDiversity and marker association in tropical silkworm breedsof Bombyx mori (Lepidoptera Bombycidae)rdquo InternationalJournal of Tropical Insect Science vol 31 no 3 pp 182ndash191 2011

[148] B Vlaic L A Marghitas A Vlaic and P Raica ldquoAnalysis ofgenetic diversity of mulberry silkworm (Bombyx mori L) usingRAPDmolecular markersrdquoAnimal Science and Biotechnologiesvol 69 no 1-2 pp 292ndash296 2012

[149] R Radjabi A Sarafrazi A Tarang K Kamali and S TirgarildquoIntraspecific biodiversity of Iranian local races of silkwormBombyx mori by ISSR (Inter-Simple Sequence Repeat) molec-ular markerrdquo World Journal of Zoology vol 7 no 1 pp 17ndash222012

[150] S MMoorthy N Chandrakanth S K Ashwath V Kumar andB B Bindroo ldquoGenetic diversity analysis usingRAPDmarker insome silkworm breeds of Bombyx mori Lrdquo Annals of BiologicalResearch vol 4 no 12 pp 82ndash88 2013

[151] N Chandrakanth S M Moorthy P Anusha et al ldquoEvaluationof genetic diversity in silkworm (Bombyx mori L) strains usingmicrosatellite markersrdquo International Journal of Biotechnologyand Allied Fields vol 2 no 3 pp 73ndash93 2014

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


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International Journal of

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Zoology


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

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Evolutionary BiologyInternational Journal of



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Genetics Research International


Advances in

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Hindawi Publishing Corporationhttpwwwhindawicom

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Volume 2014

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Enzyme Research



Microbiology


resistance or growth rate) or in morphological characters[5] Genetic diversity has been conventionally estimated onthe basis of different biometrical techniques (Metroglyph1198632divergence analysis and principal component analysis) suchas phenotypic diversity index (119867) or coefficient of parentageutilizing morphological economical and biochemical data[6ndash9]

The genetic diversity ofBmori is derived from hybridiza-tion of different geographical origins mainly the JapaneseChinese European and Indian strains which have distincttraits Among these four geographical strains silkworm oftemperate origin produces a higher quantity of good finerstronger silk fiber whereas the tropical strains are hardytolerant to pathogen load and resistant to diseases Howeverthe tropical strains produce low amounts of silk which iscoarser and weaker [10] To help the breeders in the processto identify the parents that nick better several methods ofdivergence analysis based on quantitative traits have beenproposed to suit various objectives As most of the desirablecharacters in silkworm are quantitative nature multivariatestatistical methods have been employed to measure thegenetic diversity among the stocks Among them1198632 analysisof Mahalanobis [10] using Tocherrsquos optimization method [11]occupies a unique place and an efficient method to gaugethe extent of diversity among genotypes which quantifythe difference among several quantitative traits It is beingused by most of the workers and has been found as anextremely useful tool for estimating genetic divergence inthe silkworm [12ndash16] Table 1 summarizes genetic divergencestudy carried out by several workers in silkworm Usingthis method silkworm genotypes were formed into differentclusters indicating presence of distinct divergence amongthe genotypes Though divergence reported to exist amonggenotypes and mixed trend of clustering observed Jolly et al[13] subjected forty-nine silkworm breeds for this analysisand reported that these breeds were found to form threedistinct clusters indicating the presence of distinct diversityamong the breeds Subba Rao et al [14] and Govindan et al[15] reported that breeds derived from the same parents wereincluded in different clusters showing variation among thebreeds derived from the same source On the other handthe breeds derived from the same source were included inthe same cluster showing close affinity between advancedsister lines [12 14 15] and those of differing genetic back-ground occupied in single cluster indicated uniformity inselection procedures [12] However genotypes of temperateand tropical origin formed into separate clusters indicatingenvironmental influence on the expression of characters[17]Though theoretically geographical diversity is importantfactor it is not the whole determining factor for geneticdivergence [18 19] All these studies were aimed to identifysuitable parents for breeding programme and recommendedto cross the genotypes from different clusters [20ndash22] foryield improvementThoughmany characters in silkworm aresubjected for divergence study characters namely fecunditylarval weight single cocoon weight cocoon shell weightand filament length only contributed about 97 to the totalgenetic divergence [12 13 16 23 24]

3 Diversity in Silkworm

Genetic diversity is most often characterized using data thatdepict variation in either discrete allelic states or continuouslydistributed (ie quantitative) characters which lead to differ-ent possible metrics of genetic diversity [25] Genetic diver-sity can be assessed among different accessionsindividualswithin the same species (intraspecific) among species (inter-specific) and between genus and families [26] It plays animportant role in any breeding either to exploit heterosis or togenerate productive recombinantsThe choice of parents is ofparamount importance in any kind of breeding programmehence the knowledge of genetic diversity and relatednessin the germplasm is a prerequisite for crop improvementprogrammes Genetic diversity is also an essential aspectin conservation biology because a fundamental concept ofnatural selection states that the rate of evolutionary changein a population is proportional to the amount of geneticdiversity present in it [27] Decreasing genetic diversityincreases the extinction risk of populations due to a declinein fitness Genetic diversity also has the potential to affecta wide range of population community and ecosystemprocesses both directly and indirectly However these effectsare contingent upon genetic diversity being related to themagnitude of variation in phenotypic traits [28]

In general cocoon colour and cocoon shape larvalmarking and quantitative traits have been used for differen-tiation of silkworm genotypes and based on that parents arebeing selected However recent advent of different moleculartechniques led breeders to estimate genetic diversity onthe basis of data generated by different molecular markerswhich provided a means of rapid analysis of germplasm andestimates of genetic diversity which were often found tocorroborate phenotypic data These molecular markers arebroadly categorized as biochemical and molecular markers

31 Biochemical Markers Application of isoenzymes andother molecular markers helps to estimate genetic diversitymuch more accurately than that of morphological traitsElectrophoresis identifies variation (alleles) at loci that codesfor enzymes (usually termed isozymes or allozymes) Oneadvantage of allozyme loci is that they are codominantand heterozygotes can be scored directly Understanding thegenetic constitution of an individual in the population ofraces and allelic variations through isozyme studies is knownto reflect the differential catalytic ability of allelic genes andtheir significant role in the adaptive strategy of the genotypes[29]

The diversity study carried out in silkworm throughprotein profiles enzymes and isozymes are summarizedin Table 2 Isozymes like esterase acid phosphatase alka-line phosphatase amylase phosphoglucomutase aspartateaminotransferase malate dehydrogenase glucose 6 phos-phate dehydrogenase and carbonic anhydrase have beenused by various authors to study diversity in silkwormgenotypes [30ndash43] Among the different isoenzymes ana-lyzed esterase was most preferred because of its diversesubstrate specificity and polymorphic expression followed byacid phosphatase [36 37 44] Eguchi et al [32] found four



SLnumber

Referencenumber











































SLnumber

Referencenumber


clusters























18 [42] 15 and 3 Nei (1978) and UPGMA















Slnumber

Referencenumber


Cluster


1 [48] 13 and 2 RAPD






4 [50] 13 and 2 SSR



6 [138] 31 and 7 SSR




9 [140] 5 RAPD




12 [141] 6 and 2 AFLP


13 [61] 7 and 2 AFLP



15 [142] 20 and 6 ISSR



Table 3 Continued

Slnumber

Referencenumber


Cluster






19 [56] 14 ISSR



21 [145] 30 and 2 ISSR




24 [66]A Pernyi-3


RAPD



26 [59] 4 and 2 RAPD


































9 Conclusion




References





































































































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



SLnumber

Referencenumber











































SLnumber

Referencenumber


clusters























18 [42] 15 and 3 Nei (1978) and UPGMA















Slnumber

Referencenumber


Cluster


1 [48] 13 and 2 RAPD






4 [50] 13 and 2 SSR



6 [138] 31 and 7 SSR




9 [140] 5 RAPD




12 [141] 6 and 2 AFLP


13 [61] 7 and 2 AFLP



15 [142] 20 and 6 ISSR



Table 3 Continued

Slnumber

Referencenumber


Cluster






19 [56] 14 ISSR



21 [145] 30 and 2 ISSR




24 [66]A Pernyi-3


RAPD



26 [59] 4 and 2 RAPD


































9 Conclusion




References





































































































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



SLnumber

Referencenumber


clusters























18 [42] 15 and 3 Nei (1978) and UPGMA















Slnumber

Referencenumber


Cluster


1 [48] 13 and 2 RAPD






4 [50] 13 and 2 SSR



6 [138] 31 and 7 SSR




9 [140] 5 RAPD




12 [141] 6 and 2 AFLP


13 [61] 7 and 2 AFLP



15 [142] 20 and 6 ISSR



Table 3 Continued

Slnumber

Referencenumber


Cluster






19 [56] 14 ISSR



21 [145] 30 and 2 ISSR




24 [66]A Pernyi-3


RAPD



26 [59] 4 and 2 RAPD


































9 Conclusion




References





































































































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology












Slnumber

Referencenumber


Cluster


1 [48] 13 and 2 RAPD






4 [50] 13 and 2 SSR



6 [138] 31 and 7 SSR




9 [140] 5 RAPD




12 [141] 6 and 2 AFLP


13 [61] 7 and 2 AFLP



15 [142] 20 and 6 ISSR



Table 3 Continued

Slnumber

Referencenumber


Cluster






19 [56] 14 ISSR



21 [145] 30 and 2 ISSR




24 [66]A Pernyi-3


RAPD



26 [59] 4 and 2 RAPD


































9 Conclusion




References





































































































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


Table 3 Continued

Slnumber

Referencenumber


Cluster






19 [56] 14 ISSR



21 [145] 30 and 2 ISSR




24 [66]A Pernyi-3


RAPD



26 [59] 4 and 2 RAPD


































9 Conclusion




References





































































































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



























9 Conclusion




References





































































































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology































































































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

Review Article Genetic Divergence, Implication of Diversity ...2. Genetic Divergence in Silkworm...

Documents

Transcript of Review Article Genetic Divergence, Implication of Diversity ...2. Genetic Divergence in Silkworm...