WO2003087295A2 - Superior, novel, quality mulberry silkworm hybrids and a method thereof - Google Patents

Superior, novel, quality mulberry silkworm hybrids and a method thereof Download PDF

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WO2003087295A2
WO2003087295A2 PCT/IB2002/001349 IB0201349W WO03087295A2 WO 2003087295 A2 WO2003087295 A2 WO 2003087295A2 IB 0201349 W IB0201349 W IB 0201349W WO 03087295 A2 WO03087295 A2 WO 03087295A2
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hybrids
aps
ranging
cocoon
hybrid
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PCT/IB2002/001349
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French (fr)
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WO2003087295A3 (en
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Chandrashekharaiah
Chikati Guruprasad Rao
Vankadara Sivaprasad
Modumudi Ramesh Babu
Ajay Kumar Goel
Jonna Prasad
Javare Gowda Nagaraju
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Andhra Pradesh State Sericulture Research & Development Institute
Centre For Dna Fingerprinting And Diagnostics
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Priority to AU2002251435A priority Critical patent/AU2002251435A1/en
Priority to PCT/IB2002/001349 priority patent/WO2003087295A2/en
Publication of WO2003087295A2 publication Critical patent/WO2003087295A2/en
Publication of WO2003087295A3 publication Critical patent/WO2003087295A3/en

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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/033—Rearing or breeding invertebrates; New breeds of invertebrates
    • A01K67/04—Silkworms
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/033—Rearing or breeding invertebrates; New breeds of invertebrates

Definitions

  • the present invention relates to superior, novel Mulberry silkworm hybrids named Swarnandhra (APMi X APS 8 ), Kalpatharuvu (APS 9 X APS 8 ), and Hemavathy (APS 5 X APS 4 ), and a reliable and consistent method of producing the said hybrids.
  • Swarnandhra APMi X APS 8
  • Kalpatharuvu APS 9 X APS 8
  • Hemavathy APS 5 X APS 4
  • the breeding programme followed in the present invention includes integrated approaches of conventional selection and hybridization coupled with DNA marker technologies.
  • the main object of the present invention is to develop superior silkworm hybrids of high yield.
  • Another object of the present invention is develop silkworm hybrids producing international grade silk.
  • Yet another object of the present invention is to develop silkworm hybrids producing silk for all seasons.
  • Still another object of the present invention is to develop silkworm hybrids preferably well adapted for tropical regions.
  • Still another object of the present invention is to develop silkworm hybrids producing silk with high reelability.
  • Still another object of the present invention is to develop silkworm hybrids producing silk with low renditta.
  • Still another object of the present invention is to produce silkworm hybrids with resistance towards virus. Still object of the present invention is to develop silkworm hybrids with high survival rate.
  • Still another object of the present invention is to develop silkworm hybrids with reduced larval period.
  • Still another object of the present invention is to develop silkworm hybrids with desired heterozygosity
  • Further object of the present invention is to use DNA finge ⁇ rinting technology to evaluate the hybrid vigor of the silkworm hybrids.
  • Another object of the present invention is to use DNA finge ⁇ rinting to determine homozygosity of inbred lines.
  • the present invention relates to superior, novel Mulberry silkworm hybrids named Swarnandhra (APMi X APS 8 ), Kalpatharuvu (APS 9 X APS 8 ), and Hemavathy (APS 5 X APS 4 ), well adapted for all seasons and preferably adapted for tropical region, produced by crossing pureline inbreeds of Polyvoltine and Bivoltine and a reliable and consistent method of producing the hybrids, using steps comprising inbreeding, hybridizing, disease tolerance breeding, DNA finge ⁇ rinting, and selecting, wherein the said hybrids show high yield of international grade silk.
  • APMi X APS 8 Kalpatharuvu
  • APS 5 X APS 4 Hemavathy
  • the present invention relates to the superior novel Mulberry silkworm hybrid named Swarnandhra, well adapted for all seasons and preferably adapted for tropical region and the said hybrid is a cross of polyvoltine APMi derived from pureline inbreed of Madagascar hybrids and bivoltine APS 8 derived from pureline inbreed of Chinese hybrids, using steps comprising inbreeding, hybridization, disease tolerance breeding, DNA finge ⁇ rinting, and selection and is superior to conventional hybrids of the tropical regions mainly.
  • the said hybrid produces silk of superior quality and high yield.
  • silk has international grade of 2A-3A. In still another embodiment of the present invention, silk has reelability ranging between 80 and 90%.
  • silk has renditta ranging between 7.0 and 7.5 kg.
  • silk has floss percentage less than 5%.
  • silk has greenish yellow color.
  • said Swarnandhra hybrid produces larvae of superior quality.
  • larvae has survival rate ranging between 80 and 85%.
  • larval period is ranging between 22 and 23 days.
  • larvae is plain or without any visible markings.
  • Swarnandhra hybrid produces cocoon of good yield and superior quality.
  • cocoon has yield ranging between 50 and 55 per 100 dfls (kg).
  • single silk cocoon weight ranging between 1.60 and 1.70-g.
  • cocoon shell weight ranging between 0.290 and 0.320. In still another embodiment of the present invention, percentage shell ratio ranging between 18 and 19.
  • Fig 1 shows flow chart of methodology followed to develop silkworm hybrid Swarnandhra.
  • Fig 2 shows flow chart of methodology followed to develop and select pureline Chinese hybrids APS 5 and APS 9 .
  • Fig 3 shows flow chart of methodology followed to develop and select pureline Japanese hybrids APS 4 and APS 8 .
  • Fig. 4a Silkworm eggs laid (400-500 in number) by a single female moth of a polyvoltine- inbred line, APMi.
  • the eggs are pigmented by pigment accumulation in serosal cells and are non-diapause in nature.
  • the expression of pigments in the serosal cells in the non- diapause eggs is controlled by the gene, pnd located on chromosome 11.
  • Usually non- diapause eggs are non-pigmented in nature and APMi is exception to this
  • Fig 4b Silkworm eggs (450-550 in number) laid by a single female moth of bivoltine inbred line, APS 8 .
  • the eggs are pigmented by pigment accumulation in serosal cells and are diapause in nature.
  • Fig 4c The silkworm eggs laid by a single female moth of hybrid, APMi x APS 8 .
  • the eggs are pigmented similar to the maternal parent.
  • Fig.5a Fully-grown fifth instar silkworm larvae of polyvoltine inbred line, APMj. The larvae are devoid of any visible markings on body.
  • Fig.5b Fully-grown fifth instar silkworm larvae of bivoltine, APSg. The larvae are devoid of any visible markings on body.
  • Fig.5c Fully-grown fifth instar silkworm larvae of hybrid, APMi x APSg. The larvae are devoid of any visible markings on body.
  • Fig. ⁇ a Cocoons of APMi.
  • the cocoons are oval in shape and greenish yellow in color.
  • Fig.6b Cocoons of APSg.
  • the cocoons are white in color and peanut in shape.
  • APMi X APSg are greenish yellow in color similar to APMi , with prominent oval shape and mild peanut constriction appearance.
  • the greenish yellow cocoon color is dominant over white color in silkworm, Bombyx mori .
  • Fig.7a Silk fibre reeled from the cocoons of APMi.
  • Fig.7b Silk fibre reeled from the cocoons of APSg.
  • Fig.7c Silk fibre reeled from the cocoons of the hybrid, APMi x APSg.
  • the yarn colors reflect the cocoon color of he maternal parent in the hybrid.
  • FIG. 8 DNA finge ⁇ rinting profiles of APMi, APSg and APMi X APS 8 .
  • the finge ⁇ rints were generated using the Fluorescent Inter-Simple Sequence Repeat-anchored Polymerase Chain Reaction (FISSR-PCR).
  • FISSR-PCR Fluorescent Inter-Simple Sequence Repeat-anchored Polymerase Chain Reaction
  • the PCR products were resolved on GENSCAN (Perkin Elmer).
  • the PCR products specific to APMi and APSg are indicated by arrow and arrowheads, respectively.
  • M males (APSg)
  • F females (APMi)
  • Fi APMi x APSg hybrid (Swarnandhra).
  • Fig 9a Shows silkworm eggs laid (450-550 in number) by a single female moth of inbred line, APS 5 .
  • the eggs are pigmented by pigment accumulation in serosal cells and are diapause in nature.
  • Fig 9b shows silkworm eggs laid (450-550 in number) by a single female moth of inbred line APS 4 .
  • the eggs are pigmented by pigment accumulation in serosal cells and are diapause in nature.
  • Fig. 9c. shows silkworm eggs laid (450-550 in number) by a single female moth of inbred lines, APS .
  • the eggs are pigmented by pigment accumulation in serosal cells and are diapause in nature.
  • Fig 9d shows silkworm eggs laid (450-550 in number) by a single female moth of inbred lines APS 8 .
  • the eggs are pigmented by pigment accumulation in serosal cells and are diapause in nature.
  • Fig. 9e shows silkworm egg (450-600 in number) laid by single female moths of hybrid, Kalpatharuvu (APS 9 x APSg).
  • the eggs are pigmented similar to the parents and are diapause in nature.
  • Fig. 9f shows silkworm egg (450-600 in number) laid by single female moths of hybrid, Hemavathy (APS 5 x APS 4 ). The eggs are pigmented similar to the parents and are diapause in nature.
  • Fig.lOa. shows fully-grown fifth instar silkworm larvae of APS 5 .
  • Fig. 10b shows fully-grown fifth instar silkworm larvae of APS 4 .
  • Fig. 10c. shows fully-grown fifth instar silkworm larvae of APS 9 .
  • Fig. lOd. shows fully-grown fifth instar silkworm larvae of APSg.
  • Fig. lOe. shows fully-grown fifth instar silkworm larvae of the hybrid, Hemavathy (APS 5 x APS 4 ).
  • Fig. lOf shows fully-grown fifth instar silkworm larvae of the hybrid, Kalpatharuvu (APS x APSg).
  • Fig. 11a shows cocoons of APS 5 are oval in shape and white in color.
  • Fig. lib shows cocoons of APS 4 are peanut in shape and white in color.
  • Fig. lie shows cocoons of APS 9 are oval in shape and white in color.
  • Fig. lid shows cocoons of APSg are peanut in shape and white in color.
  • Fig. lie shows hybrid cocoons of Hemavathy (APS 5 x APS 4 ) are oval in shape with peanut appearance.
  • Fig. llf shows hybrid cocoons of Kalpatharuvu (APS 9 x APSg) are also oval in shape with peanut appearance.
  • Fig.l2a shows silk fibre reeled from the hybrids of Kalpatharuvu (APS 9 x APSg). The yarn color is also white.
  • Fig.l2b shows silk fibre reeled from the hybrids of Hemavathy (APS 5 x APS 4 ). The yarn color is also white.
  • Fig. 13 shows DNA finge ⁇ rinting profile of APS 4 , APS 5 , and hybrid APS 5 x APS 4 .
  • the finge ⁇ rints were generated using the Fluorescent Inter-Simple Sequence Repeat anchored Polymerase Chain Reaction (FISSR-PCR).
  • the PCR products were resolved on GENSCAN (Perkin Elmer).
  • the PCR products that are specific to the parental strains are indicated by arrows. M: males; F: females; Fi: hybrids.
  • Fig. 14 shows DNA finge ⁇ rinting profile of APS 9 , APS 8 and the hybrid, APS 9 x APSg.
  • the finge ⁇ rints were generated using the Fluorescent Inter-Simple Sequence Repeat anchored Polymerase Chain Reaction (FISSR-PCR).
  • the PCR products were resolved on GENSCAN (Perkin Elmer).
  • the PCR products that are specific to the parental strains are indicated by arrows. M: males; F: females; Fi: hybrids.
  • the percentage shell ratio of the Swarnandhra fall within the range of 18 and 19 and these have been clearly illustrated in Table 1 which is given below.
  • SWARNANDHRA (APMiXAPSg) AND THE TRADITIONAL HYBRID (PM X NB 4 D 2 )
  • Karnataka 1 total larval duration from the time of larval eclosion from the eggs upto the onset of secretion of silk.
  • filament length ranging between 700 and 800 m/cocoon.
  • cocoon has oval shape with mild peanut constriction appearance.
  • cocoon is greenish yellow color.
  • eggs are pigmented, and are non- diapause in appearance.
  • the polyvoltine inbred line APMi and the bivoltine-inbred line, APSg were found to produce the best hybrid combination. Further, combination recorded low floss% (1.30%). The analysis confirmed the superiority of APMi x APSg (Swarnandhra- please refer fig- 1). The superiority of APMi x APSg is also reconfirmed with multiple trait index method (Mano et. al, 1992).
  • a method to produce Mulberry silkworm hybrid named Swarnandhra, preferably well adapted for tropical regions and for all seasons of the year and the said hybrid is a cross of polyvoltine APMi , derived from pureline inbreed of Madagascar hybrids and bivoltine APS 8j derived from pureline inbreed of Chinese hybrids, and is superior to conventional hybrids of the tropical regions mainly and produces greenish yellow silk of international grade 2A - 3A, using steps comprising inbreeding, hybridization, disease tolerance breeding, DNA finge ⁇ rinting, and selection.
  • mass mating the progeny derived from the multiple random pair up to F 4 generation In still another embodiment of the present invention, mass mating the progeny derived from the multiple random pair up to F 4 generation.
  • cocoons from each of the cellular families based on several parameters comprising larval survival up to pupae stage, cocoon weight, silk content, silk filament length, cocoon shape, and floss percentage.
  • nine promising silkworm inbred lines including five polyvoltines and four bivoltines developed at this institute under breeding programme have been assessed at molecular level for their homozygosity within the strains and heterozygosity in between strains using five ISSR markers (Please refer Tables 3 and 4).
  • High molecular weight genomic DNA is extracted from the pupae and purified as per standard protocols (Nagaraju and Nagaraju, 1995; Sambrook et. al., 1982). DNA is amplified by PCR technique using five di and tri nucleotide repeat ISSR markers anchored with 5' degenerate nucelotides (Reddy et.al. 1999).
  • PCR reaction For 10 ul of PCR reaction, 50 ng of genomic DNA, IX PCR reaction buffer, 2.5 mM MgCl 2 , 25 urn each of dNTPs, and 0.5 units of Taq Gold DNA polymerase (Perkin Elmer) for dinucleotide primer and MBI Taq DNA polymerase for trinucelotide primers were used. Amplification is performed on MJ research PTC- 2000 thermal cycler and perkin Elmer Gene Amp PCR- 9600 system, With a programme of initial denaturation at 94°C for 3 min followed by 35 cycles Of 94°C for 30 sec, 50°C for 30 sec, 72°C for 1 min and final extension at 72°C for 10 min.
  • Taq Gold DNA polymerase Perkin Elmer
  • the initial denaturation at 94°C is for 10 min and annealing is at 42°C and the remaining programme is same as above.
  • the amplified DNA is separated on 2% agarose gels and generated DNA profiles are documented and analyzed for DNA band sharing levels within (homozygosity) and in between (heterozygosity) parental lines (Nei, 1978, Sharma et. al. 1999, Table 3 & 4).
  • DNA from the parents and the FI progeny is extracted, purified and amplified by PCR technique using six different ISSR markers (Table 4), (Kumar et.al. 2001).
  • the protocols and the PCR machines used were as described above except the addition of 5 ⁇ l of fluorescent dUTPs to the reaction mixture.
  • the amplified DNA is separated on 5% polyacrylamide gels using Perkin Elmer gene scan Machine and the generated FISSR-PCR DNA profiles of Swarnandhra (Fig. 5) are submitted for PCT filing. ⁇ lease refer Table - 6)
  • FISSR-PCR Fluorescent-inter-simple Sequence-repeat-polymerase chain reaction
  • bivoltine hybrids are selected from a group comprising APS 4 , APS 6 , APS 8 , and APSio.
  • polyvoltine hybrids are selected from a group comprising APMi, APM 2 , APM 3 , APM 4 , and APM 5 .
  • the line, APM] the female parent of "Swarnandhra"
  • GCA General Combining Ability
  • additive gene action is predominant in the line APMi as evidenced from variance (gca) > variance (sea) for majority of the characters (four).
  • the tester APSg combines well with majority of the lines and many characters.
  • APMi x APSg "Swarnandhra" the line APMi being good combiner recorded best mean performance.
  • Mid-Parent heterosis is an average heterosis averaged over two parents involved in the hybrid. It is the usual practice in silkworm to evaluate hybrids for mid-parental heterosis since the heterosis manifestation can be evaluated as less or more than the average performance of the parents.
  • the survival rate or in other words cocoon yield is a typical trait that manifests with high degree of heterosis over mid-parental value when hybrids are reared under adverse conditions. (Please read Table 8).
  • PR Pupation Rate
  • SR Cocoon shell ratio
  • the overall performance also confirmed the superiority of the hybrid APMj x APS 8 . This is further substantiated by multiple trait index analysis (Mano et.al. 1992) with maximum average evaluation Index
  • mulberry silkworm hybrid named Swarnandhra
  • a large F 2 populations contributed by the progeny from 8 - 10 pair matings of the F hybrid offspring (Fig- 4c) are reared and the mass mating of progeny derived from the multiple random pair is continued up to F4 generation.
  • the mass rearing without any selection is exercised to ensure desirable recombinants in the breeding population.
  • an outcross is made with Pure Mysore (Polyvoltine) in case of polyvoltine breeding plan, with NB 4 D 2 (bivoltine) in case of bivoltine breeding plan.
  • each breeding plan five cellular families (each cellular family is drawn from a single pair mating) are maintained.
  • 400 larvae are retained and reared up to cocooning in each of the cellular families.
  • the cocoons raised from each of the cellular families are selected, based on the larval survival upto pupal stage, single cocoon weight, silk content, silk filament length, cocoon shape and floss content.
  • the cellular families, which registered higher survival rate, were only selected for further Inbreeding.
  • inbreeding of the selected cellular families are continued up to Fg generation.
  • the Fg generation families are subjected to Bombyx mori densonucleosis virus 1, to isolate cellular families for BmDNNl resistance.
  • the inoculation dose of 10 BmDNNl dilution is used for the assay.
  • at Fio generation five females and five male moths from each of the lines are randomly picked and DNA is extracted by using standard protocol.
  • the DNA samples are subjected to finge ⁇ rinting using Fluorescent - Inter -Simple Sequence-Repeat - Polymerase Chain Reaction method (FISSR-PCR),( Fig- 8).
  • FISSR-PCR Fluorescent - Inter -Simple Sequence-Repeat - Polymerase Chain Reaction method
  • the inbred lines which show > 90% homozygosity based on DNA test are chosen for evaluation of hybrid vigor ( fig 8)
  • the isolated polyvoltine inbred lines (five lines) and the bivoltine-inbred lines (three lines) are crossed on a partial diallele pattern using polyvoltine as female parents and bivoltine lines as male parents.
  • the resultant hybrids (Fig- 4c) are reared in three replicates each retaining 400 larvae after third moult.
  • the hybrid performance is evaluated considering total larval duration, fifth instar larval duration, larval survival upto pupal stage, single cocoon weight, cocoon shell weight, silk filament length and floss content. (Fig - 5a, 5b, 5c)
  • the parents who gave rise to the highest hybrid vigor in the hybrids are selected.
  • the polyvoltine inbred line APMi and the bivoltine-inbred line, APSg are found to produce the best hybrid combination.
  • the excelled hybrid combination, APM, x APSg is further field tested with the farmers during different seasons of the year in different regions of Andhra Pradesh and Karnataka, to evaluate its silk cocoon productivity, yield consistency, silk cocoon yield attributes, and quality of silk.
  • Fig- 7a, 7b,and 7c (Please refer Tables 10 and 11) TABLE 10 - SEASONAL PERFORMANCE OF SWARNANDHRA ( APM, x APSg) AND TRADITIONAL HYBRID (PM x NB 4 D 2 ) WITH FARMERS OF A.P. & KARNATAKA
  • the new hybrid Swarnandhra has scored better over the ruling traditional hybrid in all the seasons of the year (Please refer Table- 3).
  • the grade of silk recovered from the new hybrid is classified as 2A -3 A, as compared to the ungraded silk from the ruling hybrid.
  • the newly developed hybrid ensure higher financial returns to the farmer to an extent of 30% to 40% over the returns from the ruling traditional hybrid.
  • inventors have successfully developed the elite silkworm hybrid, Swarnandhra and also are confident about higher economic returns to both silkworm rearers and silk reelers, and the silk obtained is graded 2A -3 A on the basis of international standard.
  • this is the first polyvoltine x bivoltine hybrid developed in India to meet the silk quality stipulated for international market.
  • the hybrids of these two inbred lines are raised and reared in different seasons of the year and different regions of Andhra Pradesh to evaluate the cocoon crop stability.
  • said hybrid is reared and recorded an average of 53.73 kg/100 dfls.
  • the cocoons can easily fetch Rs.30-50 more per kg of cocoons over the ruling hybrid Pure Mysore x NB 4 D2.
  • the new hybrid is named as 'Swarnandhra'.
  • Mulberry Silkworm hybrids named Kalpatharuvu and Hemavathy, preferably well adapted for tropical region and for all seasons of the year, are cross of bivoltines obtained from oval type Chinese and peanut type Japanese hybrids, using steps comprising inbreeding, hybridization, disease tolerance breeding, selection, and DNA finge ⁇ rinting, and are superior to conventional hybrids of the tropical regions mainly.
  • said Kalpatharuvu and Hemavathy hybrids produces silk of superior quality and high yield.
  • said silk is of international grade of
  • said silk has reelability ranging between 80 and 85%.
  • said silk has renditta ranging between 6.5 and 7.0 kg.
  • said silk is of white color.
  • larvae of superior quality with survival rate ranging between 80 and 85%.
  • said hybrid has larval period ranging between 22 and 23 days.
  • larvae is plain or without any visible marking.
  • cocoon of silkworm hybrids named Kalpatharuvu and Hemavathy has good yield and superior quality.
  • said cocoon yield ranging between 60 and 65 per 100 dfls (kg).
  • single silk cocoon weight ranging between 1.7 and 1.8g.
  • shell weight ranging between 0.350 and 0.390g.
  • percentage shell ratio ranging between 21 and 22.
  • filament length ranging between 750 and 800 m/cocoon.
  • oval shape with mild peanut constriction appearance in still another embodiment of the present invention, oval shape with mild peanut constriction appearance.
  • cocoon is white colored.
  • eggs of Kalpatharuvu and Hemavathy hybrids are pigmented and has diapause appearance.
  • crossing bivoltine strains of APS (oval) X APSg (peanut) and APS 5 (oval) X APS 4 (peanut) produces Kalpatharuvu and Hemavathy respectively.
  • pupation rate of Hemavathy is ranging between 92-96%.
  • pupation rate of Kalpatharuvu is ranging between 85-90%.
  • average evaluation Index of Hemavathy is ranging between 61-66%.
  • average evaluation Index of Kalpatharuvu is ranging between 65-70%.
  • the selected oval lines; APS 5 & APS 9 and two peanut lines; APS 4 , APSg lines are crossed in full diallele pattern.
  • the resultant 16 Fls and their reciprocals along with their parents hybrids are reared in three replicates each retaining 400 larvae after third moult.
  • the hybrids' performance is evaluated considering for six characters namely cocoon yield by weight, pupation rate, single cocoon weight, single shell weight, shell ratio and filament length.
  • the hybrid combinations APS 9 x APS 8 (Kalpatharuvu) and APS 5 x APS 4 (Hemavathy) showed promise.
  • the floss% in these hybrids is also found to less (APS 9 x APS 8 :0.972% & APS 5 x APS 4 : 1.056%). Further, multiple trait evaluation index (Mano et al, 1992) is calculated for the above commercially important characters and the superiority of APS 9 x APS 8 (Kalpatharuvu) and APS x APS (Hemavathy) is further confirmed. (Please refer Table 12)
  • the parent APS 5 the female parent of "Hemavathy” is showing positive General Combining Effects (GCA) effects for all the six characters followed by APS 9 , the female parent of "Kalpatharuvu", for 5 characters except Pupation Rate.
  • GCA General Combining Effects
  • the parent APS 4 the male of "Hemavathy” showed negative effects for all the characters so also APSg, the male parent of "Kalpatharuvu" except for Pupation Rate.
  • a met o to produce Mu erry Silkworm hybrids named Kalpatharuvu and Hemavathy, well adapted for tropical region mainly and for all seasons of the year, are cross of bivoltine, obtained from oval type Chinese and peanut type Japanese hybrids, using steps comprising hybridization, inbreeding, disease tolerance breeding, selection, and DNA finge ⁇ rinting, and are superior to conventional hybrids of the tropical regions mainly and produces white color silk of international grade 3A-4A.
  • cocoons raised from each of the cellular families on the basis of parameters comprising the larval survival upto pupation stage, single cocoon weight, silk content, silk filament length, cocoon shape, and floss content traits.
  • inbreeding the said cellular families up to F 8 generation inbreeding the said cellular families up to F 8 generation.
  • subjecting the F 8 generation families to Bombyx mori densonucleosis virus 1 (BmDNNl) and isolating cellular families resistant to BmDNNl.
  • BmDNNl Bombyx mori densonucleosis virus 1
  • ISSR - D ⁇ A markers for testing homozygosity are selected from a group comprising CGA(ATT)4, TA(CGA)4, (CA)7, T3(ATT)4, and CT(ATT)4.
  • FISSR Markers for testing heterozygosity are selected from a group comprising (CA)7, (TG)7, T(GA)9, T(GA)8, C(GA)7, and TA(CAG)4.
  • Taq polymerase is used in PCR.
  • homozygosity is tested on agarose gel of concentration ranging between 1-5%.
  • heterozygosity is tested on polyacrylamide gel with concentration ranging between 3-8%.
  • High molecular weight genomic DNA is extracted from the pupae and purified as per standard protocols (Nagaraju and Nagaraju, 1995; Sambrook et. al, 1982). DNA is amplified by PCR technique using five di and tri nucleotide repeat ISSR markers anchored with 5' degenerate nucelotides (Reddy et.al. 1999).
  • PCR reaction For 10 ul of PCR reaction, 50 ng of genomic DNA, IX PCR reaction buffer, 2.5 mM MgCl 2 , 25 urn each of dNTPs, and 0.5 units of Taq Gold DNA polymerase (Perkin Elmer) for dinucleotide primer and MBI Taq DNA polymerase for trinucelotide primers are used. Amplification is performed on MJ research PTC- 2000 thermal cycler and perkin Elmer Gene Amp PCR- 9600 system, With a programme of initial denaturation at 94°C for 3 min followed by 35 cycles Of 94°C for 30 se , 50°C for 30 se , 72°C for 1 min and final extension at 72°C for 10 min.
  • Taq Gold DNA polymerase Perkin Elmer
  • the initial denaturation at 94°C is for 10 min and annealing was at 42°C and the remaining programme is same as above.
  • the amplified DNA is separated on 2% agarose gels and generated DNA profiles are documented and analyzed for DNA band sharing levels within (homozygosity) and in between (heterozygosity) parental lines (Nei, 1978, Sharma et. al. 1999).
  • the protocols and the PCR machines used are as described above except the addition of 5 ⁇ l of fluorescent dUTPs to the reaction mixture.
  • the amplified DNA is separated on 5% polyacrylamide gels using Perkin Elmer gene scan Machine and the generated FISSR-PCR DNA profiles of Kalpatharuvu and Hemavathy hybrids (Fig. 5, please refer Table 17) are submitted for PCT filing.
  • evaluating the hybrid performance on the basis of total larval duration, fifth instar larval duration, larval survival upto pupal stage, single cocoon weight, cocoon shell weight, silk filament length, and floss content In still another embodiment of the present invention, selecting the parents giving rise to the highest hybrid vigor in the said hybrids.
  • the heterosis calculated for the Fls revealed significant to highly significant (P ⁇ 0.05 to P ⁇ 0.01) heterosis in the combination APS 5 X APS 4 (Hemavathy) for all the 6 characters and highly significant heterosis in APS X APSg (Kalpatharuvu) for the 5 characters out of 6 characters studied. (Please refer Table 19)
  • Evaluation Index (El) calculated on the basis of over all performance of the Fls also support the superiority of the hybrids APS 9 X APSg (Kalpatharuvu) and APS 5 X APS 4 (Hemavathy) through highest average indices (6 traits) of 67.12 and 63.15 respectively. (Please refer Table 20)
  • APS 9 x APSg and APSs x APS 4 which are named as "Kalpatharuvu' and ⁇ emavathy' respectively.
  • the exotic Chinese and Japanese hybrids are used as donor parental genetic material for breeding bivoltine inbred lines APS 4 , APS 5 , APSg and APS 9 .
  • the breeding is carried out by exercising selection for desired characters particularly for oval and peanut cocoon shape with uniform shape and size, hardiness of cocoon, higher pupation rate, shorter larval duration, higher single cocoon weight, higher cocoon shell weight, longer silk filament length, better reelability and overall enhancement in the quality of silk (as quantified by international silk grade).
  • outcross is made with another qualitatively superior bivoltine breed to inco ⁇ orate the beneficial characters to the newly bred inbred lines.
  • inbred lines are separated based on the presence/absence of markings on the larval body and shape of the cocoons (oval or peanut shaped).
  • inbred lines after the inbred lines attained some degree of uniformity for various quantitative traits, they are tested for homozygocity using different DNA markers.
  • the highly inbred and homozygous lines are subjected to hybridization studies in the breeding laboratory.
  • the excelled hybrid combinations, APS 9 x APSg and APS 5 x APS 4 are field tested during different seasons of the year in different regions of Andhra Pradesh to evaluate its silk cocoon productivity, yield consistency, silk cocoon yield attributes and quality of silk.
  • the new hybrids, Kalpatharuvu and Hemavathy yielded better crop results in all the seasons of the year.
  • the grade of silk recovered from new hybrids is tested and classified as 3A-4A.
  • the overall financial returns to the farmers can be about 30-40% higher than the traditional polyvoltine x bivoltine hybrids.
  • the new hybrids are also expected to perform better in the tropical states of Karnataka and Tamil Nadu, where there is paucity of productive bivoltine hybrids.
  • two breeding plans are initiated separately using the bivoltine silkworm hybrids obtained from China and Japan as donor genetic materials. Each breeding plan is aimed at isolating Chinese (oval) type and Japanese (peanut) type of cocoons. In both the breeding plans, breeding methods involving inbreeding, hybridization, disease tolerance breeding, DNA finge ⁇ rinting, and selection for the desired traits are followed.
  • Silkworm rearing is conducted under standard rearing conditions. Initially, a large F2 populations contributed by the progeny from 8-10 pair matings of the F, hybrid offsprings is reared. The mass mating of progeny derived from the multiple random pair is continued upto F 4 generation. The mass rearing without any culling is exercised to ensure desirable combinations in the breeding plan.
  • an outcross is made with elite oval for oval lines and peanut for peanut cocoon lines.
  • F 5 generation selection was resorted to larval markings and cocoon shape. After two generations of mass mating in each breeding plan, five cellular families (each cellular family is drawn from a single pair mating) are maintained.
  • 400 larvae are retained and reared upto cocooning in each of the cellular families.
  • the cocoons raised from each of the cellular families are selected based on the larval survival upto pupation stage. Single cocoon weight, silk content, silk filament length, cocoon shape and floss content traits are measured.
  • the cellular families which registered higher survival rate, are only selected for further inbreeding.
  • diapause eggs which is characteristic feature of bivoltines, are only selected.
  • each of the selected cellular families 25 males and 25 females are selected. The moths emerged from these cocoons are mated among themselves. Inbreeding of the selected cellular families is continued upto F ⁇ 0 generation.
  • the Fin generation families are also subjected to Bombyx mori densonucleosis virus 1 (BmDNNl) to isolate cellular families for BmDNNl resistance. The inoculation dose of 10 "2 BmDNNl is used for the assay.
  • five females and five male moths from each of the lines are randomly picked at Fin generation and DNA is extracted by using the standard protocol.
  • the DNA samples are subjected to finge ⁇ rinting using Fluorescent Inter-Simple Sequence Repeat anchored Polymerase Chain Reaction (FISSR- PCR) based analysis.
  • FISSR- PCR Fluorescent Inter-Simple Sequence Repeat anchored Polymerase Chain Reaction
  • the isolated oval cocoon (four lines) and peanut cocoon (four lines) lines are crossed on a full diallele pattern.
  • the resulted hybrids are reared in three replicates each retaining 400 larvae after third moult.
  • the hybrid performance is evaluated considering total larval duration, fifth instar larval duration, larval survival upto pupal stage, single cocoon weight, cocoon shell weight, silk filament length and floss content.
  • the parents which give rise to the highest hybrid vigor in the hybrids, are selected.
  • the inbred line APS 9 (oval) with APSg (peanut) and APS 5 (oval) with APS 4 (peanut) are found to produce the best hybrid combination.
  • these two promising hybrids are reared in different seasons of the year and different regions of Andhra Pradesh to evaluate the cocoon crop stability with the farmers.
  • the performance is tabulated in Table 21.
  • the said hybrids are reared and have recorded an average of 61.43kg/100 dfls.
  • the cocoons can easily fetch about Rs. 50-60 more per kg of cocoons over the ruling polyvoltine x bivoltine hybrid, Pure Mysore x NB 4 D 2 .
  • the new hybrids, APS 9 x APSg and APS 5 x APS 4 are named respectively as 'Kalpatharuvu' and ⁇ emavathy'.
  • the bivoltine hybrids obtained from China and Japan are utilized as donor parental genetic materials in the breeding programmes.
  • the inbred lines of oval and peanut shaped cocoons have been isolated through selection for desired metric traits and controlled progeny mating.
  • the isolated inbred lines, which carried desired characters for larval survival, silk cocoon weight, silk content and silk quality are bred upto Fin-Fn generations. These lines are stabilized for the target traits between Fin and Fn generations.
  • the DNA finge ⁇ rinting technology which is now well proven and globally used in crop improvement programme is used to estimate the zygocity status of the developed inbred lines.
  • These inbred lines are also screened simultaneously for Bombyx mori densonucleosis virus 1 (BmDNVl) resistance.
  • BmDNVl Bombyx mori densonucleosis virus 1
  • the highly homozygous inbred lines of oval cocoon shape are crossed systematically with similarly developed peanut shape inbred lines to analyse their hybrid vigor.
  • the silkworm hybrid combinations, which showed higher-level heterosis, are selected for further trials with farmers.
  • DNA finge ⁇ rinting is been used for the first time to test homozygosity and heterozygosity in silkworms for the first time.
  • DNA finge ⁇ rinting reflects not genetic constitution of the hybrids but also the stability of the same.
  • Introduction of DNA finge ⁇ rinting is a major step forward towards developing high quality silkworm hybrids.
  • Instant application is an initiative towards achieving close association between advancement in the field of biotechnology with molecular Biology in particular and sericulture. The instant Application has overcome a major hurdle as regards uncertainty involved with random crossing of parents with hit and trials to develop desired hybrid. Applicants have made a breakthrough in the field of sericulture. Advantages of the in present invention
  • the main advantage of the present invention is development of novel, superior silkworm hybrids.
  • Another main advantage of the present invention is development of a reliable and consistent method to develop superior silkworm hybrids.
  • Yet another advantage of the present invention is development of a silkworm hybrid producing international standard silk.
  • Still another advantage of the present invention is that silk produced by the novel hybrids is with high reelability.
  • Still another advantage of the present invention is that hybrids are resistant to viruses.
  • Still another advantage of the present invention is the use of DNA finge ⁇ rinting to develop silkworm hybrids of desired heterogeneity and traits.
  • Still another advantage of the present invention is the development of hybrids with reduced larval period.
  • Still another advantage of the present invention is the use of DNA finge ⁇ rinting to ascertain hybrid vigor in silkworms.
  • Still another advantage of the present invention is to develop hybrids with high survival rate.

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Abstract

The present invention relates to superior, novel Mulberry silkworm hybrids named Swarnandhra (APM1 X APS8), Kalpatharuvu (APS9 X APS8), and Hemavathy (APS5 X APS4), well adapted for all seasons and preferably adapted for tropical region, produced by crossing pureline inbreeds of Polyvoltine (APM) and Bivoltine (APS)and a reliable and consistent method of producing the hybrids, using steps comprising inbreeding, hybridizing, disease tolerance breeding, DNA fingerprinting, and selecting, wherein the said hybrids show high yield of international grade silk.

Description

SUPERIOR, NOVEL, QUALITY MULBERRY SILKWORM HYBRIDS AND A METHOD THEREOF
Technical Field
The present invention relates to superior, novel Mulberry silkworm hybrids named Swarnandhra (APMi X APS 8), Kalpatharuvu (APS 9 X APS 8), and Hemavathy (APS5 X APS4), and a reliable and consistent method of producing the said hybrids.
Background Art
At present silk cocoon production in the southern Indian states of Andhra Pradesh, Karnataka and Tamil Nadu, which fall under tropical India, is mainly contributed by the traditional hybrid of polyvoltine (Pure Mysore) x bivoltine breeds (NB4D2), which accounts for more than 96% of the total silk produced in these states. The traditional hybrid known for consistent performance under tropical conditions but the silk produced is of inferior quality and silk cocoon productivity is considerably low. As a result, silkworm rearers (sericulturists) and silk reelers get poor financial returns besides producing internationally ungraded silk. Non-availability of alternative productive polyvoltine x bivoltine hybrids has resulted in the over-exploitation of the existing traditional hybrid and hence, its performance with farmers is gradually declining.
Among several contributory factors, focused silkworm breeding effort directed towards silk yield contributory traits such as survival rate of silkworm larvae, weight of silk cocoon assume special importance in improving the silk productivity and silk quality. So the primary attempt that needs investment is to provide suitable breeds that can give consistent cocoon crops over a range of seasons and regions and record higher silk cocoon yield with the farmers.
This demand for better quality silk has created a supply demand gap of 7000 Mt, which is presently imported from China. To meet the changing demand pattern, the import may increase in the coming years. Besides, the silk exporting community prefers to import international grade silk for their products to meet the expectations of buyers abroad as the local silk do not meet the standard. It has been established that the bivoltine hybrids have the inherent character of producing superior quality international grade silk. Therefore it is essential to develop high silk yielding bivoltine strains to be used in tropical regions of India. This would herald a bivoltine era in tropical countries like India. The quality of silkworm races is by far the most important input of sericulture technology and much effort should be devoted to the breeding of better races to meet the requirements. So the urgent need in the present scenario is to produce suitable bivoltine hybrids that can give consistent cocoon crop over a range of seasons and regions and record higher productivity and better returns to the farmers.
Further, it is possible to assign heredity unambiguously between parents and progeny, by showing that 50% of the bands in any individual are derived from a particular parent. This is the basis of the technique known as DNA fingerprinting.
In this backdrop of emerging demand/trend, we have focused our attention to develop silkworm hybrids more suited to tropical conditions. These hybrids give increased silk cocoon yield and gradable raw silk production. The breeding programme followed in the present invention includes integrated approaches of conventional selection and hybridization coupled with DNA marker technologies.
Objects of the present invention
The main object of the present invention is to develop superior silkworm hybrids of high yield.
Another object of the present invention is develop silkworm hybrids producing international grade silk.
Yet another object of the present invention is to develop silkworm hybrids producing silk for all seasons.
Still another object of the present invention is to develop silkworm hybrids preferably well adapted for tropical regions.
Still another object of the present invention is to develop silkworm hybrids producing silk with high reelability.
Still another object of the present invention is to develop silkworm hybrids producing silk with low renditta.
Still another object of the present invention is to produce silkworm hybrids with resistance towards virus. Still object of the present invention is to develop silkworm hybrids with high survival rate.
Still another object of the present invention is to develop silkworm hybrids with reduced larval period.
Still another object of the present invention is to develop silkworm hybrids with desired heterozygosity
Further object of the present invention is to use DNA fingeφrinting technology to evaluate the hybrid vigor of the silkworm hybrids.
Another object of the present invention is to use DNA fingeφrinting to determine homozygosity of inbred lines.
Summary of the Invention
The present invention relates to superior, novel Mulberry silkworm hybrids named Swarnandhra (APMi X APS 8), Kalpatharuvu (APS9 X APS8), and Hemavathy (APS5 X APS4), well adapted for all seasons and preferably adapted for tropical region, produced by crossing pureline inbreeds of Polyvoltine and Bivoltine and a reliable and consistent method of producing the hybrids, using steps comprising inbreeding, hybridizing, disease tolerance breeding, DNA fingeφrinting, and selecting, wherein the said hybrids show high yield of international grade silk.
Detailed Description of the present invention Accordingly, the present invention relates to the superior novel Mulberry silkworm hybrid named Swarnandhra, well adapted for all seasons and preferably adapted for tropical region and the said hybrid is a cross of polyvoltine APMi derived from pureline inbreed of Madagascar hybrids and bivoltine APS8 derived from pureline inbreed of Chinese hybrids, using steps comprising inbreeding, hybridization, disease tolerance breeding, DNA fingeφrinting, and selection and is superior to conventional hybrids of the tropical regions mainly.
In an embodiment of the present invention, the said hybrid produces silk of superior quality and high yield.
In yet another embodiment of the present invention, silk has international grade of 2A-3A. In still another embodiment of the present invention, silk has reelability ranging between 80 and 90%.
In still another embodiment of the present invention, silk has renditta ranging between 7.0 and 7.5 kg.
In still another embodiment of the present invention, silk has floss percentage less than 5%.
In still another embodiment of the present invention, silk has greenish yellow color.
In still another embodiment of the present invention, said Swarnandhra hybrid produces larvae of superior quality.
In still another embodiment of the present invention, larvae has survival rate ranging between 80 and 85%.
In still another embodiment of the present invention, larval period is ranging between 22 and 23 days.
In still another embodiment of the present invention, larvae is plain or without any visible markings.
In still another embodiment of the present invention, Swarnandhra hybrid produces cocoon of good yield and superior quality.
In still another embodiment of the present invention, cocoon has yield ranging between 50 and 55 per 100 dfls (kg).
In still another embodiment of the present invention, single silk cocoon weight ranging between 1.60 and 1.70-g.
In still another embodiment of the present invention, cocoon shell weight ranging between 0.290 and 0.320. In still another embodiment of the present invention, percentage shell ratio ranging between 18 and 19.
Brief description of the accompanying drawings: Fig 1 shows flow chart of methodology followed to develop silkworm hybrid Swarnandhra.
Fig 2 shows flow chart of methodology followed to develop and select pureline Chinese hybrids APS5 and APS9.
Fig 3 shows flow chart of methodology followed to develop and select pureline Japanese hybrids APS4 and APS8.
Fig. 4a. Silkworm eggs laid (400-500 in number) by a single female moth of a polyvoltine- inbred line, APMi. The eggs are pigmented by pigment accumulation in serosal cells and are non-diapause in nature. The expression of pigments in the serosal cells in the non- diapause eggs is controlled by the gene, pnd located on chromosome 11. Usually non- diapause eggs are non-pigmented in nature and APMi is exception to this
Fig 4b. Silkworm eggs (450-550 in number) laid by a single female moth of bivoltine inbred line, APS8. The eggs are pigmented by pigment accumulation in serosal cells and are diapause in nature.
Fig 4c.The silkworm eggs laid by a single female moth of hybrid, APMi x APS8. The eggs are pigmented similar to the maternal parent.
Fig.5a. Fully-grown fifth instar silkworm larvae of polyvoltine inbred line, APMj. The larvae are devoid of any visible markings on body.
Fig.5b. Fully-grown fifth instar silkworm larvae of bivoltine, APSg. The larvae are devoid of any visible markings on body.
Fig.5c. Fully-grown fifth instar silkworm larvae of hybrid, APMi x APSg. The larvae are devoid of any visible markings on body.
Fig.όa. Cocoons of APMi. The cocoons are oval in shape and greenish yellow in color. Fig.6b. Cocoons of APSg. The cocoons are white in color and peanut in shape.
Fig.όc. Cocoons of the hybrid, APMi X APSg are greenish yellow in color similar to APMi, with prominent oval shape and mild peanut constriction appearance. The greenish yellow cocoon color is dominant over white color in silkworm, Bombyx mori .
Fig.7a. Silk fibre reeled from the cocoons of APMi.
Fig.7b. Silk fibre reeled from the cocoons of APSg.
Fig.7c. Silk fibre reeled from the cocoons of the hybrid, APMi x APSg. The yarn colors reflect the cocoon color of he maternal parent in the hybrid.
Fig. 8. DNA fingeφrinting profiles of APMi, APSg and APMi X APS8. The fingeφrints were generated using the Fluorescent Inter-Simple Sequence Repeat-anchored Polymerase Chain Reaction (FISSR-PCR). The PCR products were resolved on GENSCAN (Perkin Elmer). The PCR products specific to APMi and APSg are indicated by arrow and arrowheads, respectively. M: males (APSg), F: females (APMi); Fi: APMi x APSg hybrid (Swarnandhra).
Fig 9a. Shows silkworm eggs laid (450-550 in number) by a single female moth of inbred line, APS5. The eggs are pigmented by pigment accumulation in serosal cells and are diapause in nature.
Fig 9b shows silkworm eggs laid (450-550 in number) by a single female moth of inbred line APS4 . The eggs are pigmented by pigment accumulation in serosal cells and are diapause in nature.
Fig. 9c. shows silkworm eggs laid (450-550 in number) by a single female moth of inbred lines, APS . The eggs are pigmented by pigment accumulation in serosal cells and are diapause in nature.
Fig 9d shows silkworm eggs laid (450-550 in number) by a single female moth of inbred lines APS8. The eggs are pigmented by pigment accumulation in serosal cells and are diapause in nature. Fig. 9e shows silkworm egg (450-600 in number) laid by single female moths of hybrid, Kalpatharuvu (APS9 x APSg). The eggs are pigmented similar to the parents and are diapause in nature.
Fig. 9f shows silkworm egg (450-600 in number) laid by single female moths of hybrid, Hemavathy (APS5 x APS4). The eggs are pigmented similar to the parents and are diapause in nature. Fig.lOa. shows fully-grown fifth instar silkworm larvae of APS5.
Fig. 10b. shows fully-grown fifth instar silkworm larvae of APS4.
Fig. 10c. shows fully-grown fifth instar silkworm larvae of APS9.
Fig. lOd. shows fully-grown fifth instar silkworm larvae of APSg.
Fig. lOe. shows fully-grown fifth instar silkworm larvae of the hybrid, Hemavathy (APS5 x APS4).
Fig. lOf shows fully-grown fifth instar silkworm larvae of the hybrid, Kalpatharuvu (APS x APSg).
Fig. 11a shows cocoons of APS5 are oval in shape and white in color.
Fig. lib shows cocoons of APS4 are peanut in shape and white in color.
Fig. lie shows cocoons of APS9 are oval in shape and white in color.
Fig. lid shows cocoons of APSg are peanut in shape and white in color.
Fig. lie shows hybrid cocoons of Hemavathy (APS5 x APS4) are oval in shape with peanut appearance.
Fig. llf shows hybrid cocoons of Kalpatharuvu (APS9 x APSg) are also oval in shape with peanut appearance.
Fig.l2a shows silk fibre reeled from the hybrids of Kalpatharuvu (APS9 x APSg). The yarn color is also white.
Fig.l2b shows silk fibre reeled from the hybrids of Hemavathy (APS5 x APS4). The yarn color is also white. Fig. 13 shows DNA fingeφrinting profile of APS4, APS5, and hybrid APS5 x APS4. The fingeφrints were generated using the Fluorescent Inter-Simple Sequence Repeat anchored Polymerase Chain Reaction (FISSR-PCR). The PCR products were resolved on GENSCAN (Perkin Elmer). The PCR products that are specific to the parental strains are indicated by arrows. M: males; F: females; Fi: hybrids.
Fig. 14 shows DNA fingeφrinting profile of APS9, APS8 and the hybrid, APS9 x APSg. The fingeφrints were generated using the Fluorescent Inter-Simple Sequence Repeat anchored Polymerase Chain Reaction (FISSR-PCR). The PCR products were resolved on GENSCAN (Perkin Elmer). The PCR products that are specific to the parental strains are indicated by arrows. M: males; F: females; Fi: hybrids.
The percentage shell ratio of the Swarnandhra fall within the range of 18 and 19 and these have been clearly illustrated in Table 1 which is given below.
TABLE 1 - COMPARATIVE YIELD PROFILE OF NEW CROSS-BREED,
SWARNANDHRA (APMiXAPSg) AND THE TRADITIONAL HYBRID (PM X NB 4D2)
Figure imgf000009_0001
* based on 100070 disease free egg layings tested with 546 farmers of Andhra Pradesh and
Karnataka
** based on 129505 disease free egg layings tested with 543 farmers of Andhra Pradesh and
Karnataka 1 total larval duration from the time of larval eclosion from the eggs upto the onset of secretion of silk.
2 total weight of cocoons harvested from the total number of larvae used for rearing
3 total weight of cocoons harvested from the larvae raised from 100 disease free egg layings
4 the ratio of single cocoon shell weight to the weight of single silk cocoon
5 an average length of silk fibre recovered from a single silk cocoon
6 the recovery percentage of continuous silk fibre from cocoons
7 the number of kgs of cocoons required to produce 1 kg of raw silk
8 silk is graded based on international standards considering the quality, neatness and tenacity of silk fibre.
In still another embodiment of the present invention, filament length ranging between 700 and 800 m/cocoon.
In still another embodiment of the present invention, cocoon has oval shape with mild peanut constriction appearance.
In still another embodiment of the present invention, cocoon is greenish yellow color.
In still another embodiment of the present invention, eggs are pigmented, and are non- diapause in appearance.
In further embodiment of the present invention, five polyvoltine inbred lines viz., APM(, APM2, APM3, APM4 and APM5, which were developed at this Institute are subjected for DNA Fingeφrinting and established their homozygocity. (Please refer Table 2) Table 2 - General Combining Ability (GCA) of the parents
Figure imgf000010_0001
=> Female parent of "Swarnandhra" & ** => Male parent of "Swarnandhra'' In still another embodiment of the present invention, all these five inbred lines were crossed each with four testers namely, APS4, APSβ, APSg, & APSin - Peanut shape in Line x Tester design. The resultant 20 hybrids are reared in three replicates each retaining 400 larvae after third moult. Accordingly, all the 20 combinations are evaluated for their performance. The analysis is conducted for six commercially important characters viz., Cocoon yield by weight/ 10000 larvae, Pupation rate, Cocoon Weight, Cocoon shell weight, Cocoon shell Ratio and Filament Length. The polyvoltine inbred line APMi and the bivoltine-inbred line, APSg were found to produce the best hybrid combination. Further, combination recorded low floss% (1.30%). The analysis confirmed the superiority of APMi x APSg (Swarnandhra- please refer fig- 1). The superiority of APMi x APSg is also reconfirmed with multiple trait index method (Mano et. al, 1992).
In another embodiment of the present invention, a method to produce Mulberry silkworm hybrid named Swarnandhra, preferably well adapted for tropical regions and for all seasons of the year and the said hybrid is a cross of polyvoltine APMi, derived from pureline inbreed of Madagascar hybrids and bivoltine APS8j derived from pureline inbreed of Chinese hybrids, and is superior to conventional hybrids of the tropical regions mainly and produces greenish yellow silk of international grade 2A - 3A, using steps comprising inbreeding, hybridization, disease tolerance breeding, DNA fingeφrinting, and selection.
In yet another embodiment of the present invention, taking polyvoltine and bivoltine strains of Madagascar hybrids and Chinese hybrids respectively for separate breeding plan to generate pureline inbreeds.
In still another embodiment of the present invention, mating Fi hybrid offsprings and rearing the progeny.
In still another embodiment of the present invention, mass mating the progeny derived from the multiple random pair up to F4 generation.
In still another embodiment of the present invention, making outclass at F generation with pure Mysore (polyvoltine) and NB4D2 (bivoltine) in the case of polyvoltine and bivoltine breeding plans respectively. In still another embodiment of the present invention, mass mating up to F6 generation, and maintaining cellular families of said generation drawn from a single pair mating in each breeding plan. In still another embodiment of the present invention, retaining larvae from each of the cellular families and rearing the retained larvae up to cocooning.
In still another embodiment of the present invention, selecting cocoons from each of the cellular families based on several parameters comprising larval survival up to pupae stage, cocoon weight, silk content, silk filament length, cocoon shape, and floss percentage.
In still another embodiment of the present invention, selecting cellular families which register high survival rate for further inbreeding.
In still another embodiment of the present invention, selecting non-diapause egg laying from selected families.
In still another embodiment of the present invention, selecting males and females in each of the cellular families and mating the moths emerging from the cocoons of each breeding plan among themselves.
In still another embodiment of the present invention, inbreeding of the selected families up to F8 generation and exposing F8 generation cellular families to Bombyx mori densonucleosis virus.
In still another embodiment of the present invention, isolating F8 generation cellular families resistant to BmDNN 1.
In an embodiment of the present invention, nine promising silkworm inbred lines including five polyvoltines and four bivoltines developed at this institute under breeding programme have been assessed at molecular level for their homozygosity within the strains and heterozygosity in between strains using five ISSR markers (Please refer Tables 3 and 4).
Table 3: ISSR- DΝA markers used for the estimation of Homozygosity in the parental lines
Figure imgf000012_0001
Table 4. DNA band sharing percentage within the strains (Molecular marker homozygosity)
Figure imgf000013_0001
In another embodiment of the present invention, ten individuals (five males and five females) from each strain (9x10= 90) are analyzed for their homozygosity. High molecular weight genomic DNA is extracted from the pupae and purified as per standard protocols (Nagaraju and Nagaraju, 1995; Sambrook et. al., 1982). DNA is amplified by PCR technique using five di and tri nucleotide repeat ISSR markers anchored with 5' degenerate nucelotides (Reddy et.al. 1999). For 10 ul of PCR reaction, 50 ng of genomic DNA, IX PCR reaction buffer, 2.5 mM MgCl2, 25 urn each of dNTPs, and 0.5 units of Taq Gold DNA polymerase (Perkin Elmer) for dinucleotide primer and MBI Taq DNA polymerase for trinucelotide primers were used. Amplification is performed on MJ research PTC- 2000 thermal cycler and perkin Elmer Gene Amp PCR- 9600 system, With a programme of initial denaturation at 94°C for 3 min followed by 35 cycles Of 94°C for 30 sec, 50°C for 30 sec, 72°C for 1 min and final extension at 72°C for 10 min. For Gene Amp PCR machine, the initial denaturation at 94°C is for 10 min and annealing is at 42°C and the remaining programme is same as above. The amplified DNA is separated on 2% agarose gels and generated DNA profiles are documented and analyzed for DNA band sharing levels within (homozygosity) and in between (heterozygosity) parental lines (Nei, 1978, Sharma et. al. 1999, Table 3 & 4).
In still another embodiment of the present invention, for patenting the hybrid Swarnandhra (APM, X APS8), FISSR-PCR DNA profiling is carried out. (Please refer Table- 5)
Figure imgf000014_0001
In still another embodiment of the present invention, DNA from the parents and the FI progeny is extracted, purified and amplified by PCR technique using six different ISSR markers (Table 4), (Kumar et.al. 2001). The protocols and the PCR machines used were as described above except the addition of 5 μl of fluorescent dUTPs to the reaction mixture. The amplified DNA is separated on 5% polyacrylamide gels using Perkin Elmer gene scan Machine and the generated FISSR-PCR DNA profiles of Swarnandhra (Fig. 5) are submitted for PCT filing.φlease refer Table - 6)
Table 6: FISSR Markers used for DNA fingerprinting of Silkworm Hybrid "Swarnandhra"
Figure imgf000014_0002
In still another embodiment of the present invention, fingeφrinting the DNA samples using Fluorescent-inter-simple Sequence-repeat-polymerase chain reaction (FISSR-PCR) and selecting the inbred lines showing more than 90% homozygosity based on said DNA test.
In still another embodiment of the present invention, crossing the above selected lines on a partial diallele pattern using polyvoltine as female parents and bivoltine lines as male parents. In still another embodiment of the present invention, rearing the resultant hybrids and retaining larvae in each of the resultant hybrid after third moult.
In still another embodiment of the present invention, using DNA fingeφrinting technology to test the hybrid vigor and selecting the parents giving rise to maximum hybrid vigor.
In still another embodiment of the present invention, finding cross of pureline inbreed polyvoltine (APMi) and bivoltine (APS8) to have maximum hybrid vigor.
In still another embodiment of the present invention, wherein bivoltine hybrids are selected from a group comprising APS4, APS6, APS8, and APSio.
In still another embodiment of the present invention, wherein polyvoltine hybrids are selected from a group comprising APMi, APM2, APM3, APM4, and APM5.
In still another embodiment of the present invention, wherein bivoltine hybrids are used as tester.
In an embodiment of the present invention, as per the General Combining Ability effects, the line, APM] the female parent of "Swarnandhra", showed positive effects for all the six traits studied except SR %. On the other hand the line APM5 showed negative General Combining Ability (GCA) for all characters. Among the 4 testers APS4 followed by APSg showed better GCA. The tester APS6 showed negative GCA for all the six characters studied.
In still another embodiment of the present invention, combinations APM x APS , APM3 x APSg, APM5 x APSg and APMi x APS8 (except for SR%) showed positive Specific Combining Ability (SCA) effects for all the six traits. However, in the combination, APMi x APSg, APMi is found to be the good combiner. (Please refer Table 7) Table 7- Specific Combining Ability (SCA) of the hybrids
Figure imgf000016_0001
In still another embodiment of the present invention, in comparison to other lines, additive gene action is predominant in the line APMi as evidenced from variance (gca) > variance (sea) for majority of the characters (four).
In still another embodiment of the present invention, it is evident from the SCA analysis, the tester APSg combines well with majority of the lines and many characters. In the hybrid combination APMi x APSg "Swarnandhra" the line APMi being good combiner recorded best mean performance.
Further embodiment of the present invention, Mid-Parent heterosis is an average heterosis averaged over two parents involved in the hybrid. It is the usual practice in silkworm to evaluate hybrids for mid-parental heterosis since the heterosis manifestation can be evaluated as less or more than the average performance of the parents. The survival rate or in other words cocoon yield is a typical trait that manifests with high degree of heterosis over mid-parental value when hybrids are reared under adverse conditions. (Please read Table 8).
Figure imgf000017_0001
PR: Pupation Rate; SR: Cocoon shell ratio; ** significance at 1% level
In still another embodiment of the present invention, the overall performance also confirmed the superiority of the hybrid APMj x APS8. This is further substantiated by multiple trait index analysis (Mano et.al. 1992) with maximum average evaluation Index
(60.50).
In still another embodiment of the present invention, Evaluation Index (El) is calculated based on the following formula and the rank will be assigned on the basis of average evaluation index; Evaluation index (El) = (A-B)/ C X 10 + 50 Where, A= Mean of particular hybrid for a trait ; B= Overall mean of all hybrids including control for a trait ; C= standard Deviation over means for a trait; 10= standard unit & 50= Base index value. (Please refer Table 9)
Table 9 - Mean Performance & Evaluation Indices (El) (in descending order)
Figure imgf000018_0001
In an embodiment of the present invention, for the development of the mulberry silkworm hybrid named Swarnandhra, a large F2 populations contributed by the progeny from 8 - 10 pair matings of the F, hybrid offspring (Fig- 4c) are reared and the mass mating of progeny derived from the multiple random pair is continued up to F4 generation. In another embodiment of the present invention, the mass rearing without any selection is exercised to ensure desirable recombinants in the breeding population.
In yet another embodiment of the present invention, at F4 generation an outcross is made with Pure Mysore (Polyvoltine) in case of polyvoltine breeding plan, with NB4D2 (bivoltine) in case of bivoltine breeding plan.
In still another embodiment of the present invention, for the next two generations, mass mating is carried out.
In still another embodiment of the present invention, after two generations of mass mating, in each breeding plan five cellular families (each cellular family is drawn from a single pair mating) are maintained.
In still another embodiment of the present invention, 400 larvae are retained and reared up to cocooning in each of the cellular families.
In still another embodiment of the present invention, the cocoons raised from each of the cellular families are selected, based on the larval survival upto pupal stage, single cocoon weight, silk content, silk filament length, cocoon shape and floss content. The cellular families, which registered higher survival rate, were only selected for further Inbreeding.
In still another embodiment of the present invention, in polyvoltine breeding plan, only non-diapause egg layings (Fig - 4a) and in bivoltine breeding plan only diapause egg layings (Fig- 4b) are selected.
In still another embodiment of the present invention, in each of the selected cellular family 25 males and 25 females are selected and the moths emerging from these cocoons are mated among themselves.
In still another embodiment of the present invention, inbreeding of the selected cellular families are continued up to Fg generation.
In still another embodiment of the present invention, the Fg generation families are subjected to Bombyx mori densonucleosis virus 1, to isolate cellular families for BmDNNl resistance. The inoculation dose of 10 BmDNNl dilution is used for the assay. In still another embodiment of the present invention, at Fio generation, five females and five male moths from each of the lines are randomly picked and DNA is extracted by using standard protocol.
In still another embodiment of the present invention, the DNA samples are subjected to fingeφrinting using Fluorescent - Inter -Simple Sequence-Repeat - Polymerase Chain Reaction method (FISSR-PCR),( Fig- 8).
In still another embodiment of the present invention, the inbred lines which show > 90% homozygosity based on DNA test are chosen for evaluation of hybrid vigor ( fig 8)
In still another embodiment of the present invention, the isolated polyvoltine inbred lines (five lines) and the bivoltine-inbred lines (three lines) are crossed on a partial diallele pattern using polyvoltine as female parents and bivoltine lines as male parents. The resultant hybrids (Fig- 4c) are reared in three replicates each retaining 400 larvae after third moult.
In still another embodiment of the present invention, the hybrid performance is evaluated considering total larval duration, fifth instar larval duration, larval survival upto pupal stage, single cocoon weight, cocoon shell weight, silk filament length and floss content. (Fig - 5a, 5b, 5c)
In still another embodiment of the present invention, the parents who gave rise to the highest hybrid vigor in the hybrids are selected.
In still another embodiment of the present invention, the polyvoltine inbred line APMi and the bivoltine-inbred line, APSg are found to produce the best hybrid combination.
In still another embodiment of the present invention, the excelled hybrid combination, APM, x APSg is further field tested with the farmers during different seasons of the year in different regions of Andhra Pradesh and Karnataka, to evaluate its silk cocoon productivity, yield consistency, silk cocoon yield attributes, and quality of silk. (Fig- 7a, 7b,and 7c) (Please refer Tables 10 and 11) TABLE 10 - SEASONAL PERFORMANCE OF SWARNANDHRA ( APM, x APSg) AND TRADITIONAL HYBRID (PM x NB 4D2) WITH FARMERS OF A.P. & KARNATAKA
Figure imgf000021_0001
Table 11 - ST ATE- WISE PERFORMANCE OF SWARNANDHRA (APM , x APSg)
Figure imgf000021_0002
In still another embodiment of the present invention, the new hybrid, Swarnandhra has scored better over the ruling traditional hybrid in all the seasons of the year (Please refer Table- 3).
In still another embodiment of the present invention, the grade of silk recovered from the new hybrid is classified as 2A -3 A, as compared to the ungraded silk from the ruling hybrid. The newly developed hybrid ensure higher financial returns to the farmer to an extent of 30% to 40% over the returns from the ruling traditional hybrid. In still another embodiment of the present invention, inventors have successfully developed the elite silkworm hybrid, Swarnandhra and also are confident about higher economic returns to both silkworm rearers and silk reelers, and the silk obtained is graded 2A -3 A on the basis of international standard.
In still another embodiment of the present invention, this is the first polyvoltine x bivoltine hybrid developed in India to meet the silk quality stipulated for international market. In still another embodiment of the present invention, the hybrids of these two inbred lines are raised and reared in different seasons of the year and different regions of Andhra Pradesh to evaluate the cocoon crop stability. The check hybrid, Pure Mysore X NB4D2 included as control in all these studies. The performance is tabulated in Tables 9 & 10.
In still another embodiment of the present invention, said hybrid is reared and recorded an average of 53.73 kg/100 dfls. The cocoons can easily fetch Rs.30-50 more per kg of cocoons over the ruling hybrid Pure Mysore x NB4D2.
In still another embodiment of the present invention, the new hybrid is named as 'Swarnandhra'.
In further embodiment of the present invention, Mulberry Silkworm hybrids named Kalpatharuvu and Hemavathy, preferably well adapted for tropical region and for all seasons of the year, are cross of bivoltines obtained from oval type Chinese and peanut type Japanese hybrids, using steps comprising inbreeding, hybridization, disease tolerance breeding, selection, and DNA fingeφrinting, and are superior to conventional hybrids of the tropical regions mainly.
In another embodiment of the present invention, said Kalpatharuvu and Hemavathy hybrids produces silk of superior quality and high yield.
In yet another embodiment of the present invention, said silk is of international grade of
3A-4A.
In still another embodiment of the present invention, said silk has reelability ranging between 80 and 85%.
In still another embodiment of the present invention, said silk has renditta ranging between 6.5 and 7.0 kg.
In still another embodiment of the present invention, said silk is of white color.
In still another embodiment of the present invention, larvae of superior quality with survival rate ranging between 80 and 85%.
In still another embodiment of the present invention, said hybrid has larval period ranging between 22 and 23 days. In still another embodiment of the present invention, larvae is plain or without any visible marking.
In still another embodiment of the present invention, cocoon of silkworm hybrids named Kalpatharuvu and Hemavathy has good yield and superior quality.
In still another embodiment of the present invention, said cocoon yield ranging between 60 and 65 per 100 dfls (kg).
In still another embodiment of the present invention, single silk cocoon weight ranging between 1.7 and 1.8g.
In still another embodiment of the present invention, shell weight ranging between 0.350 and 0.390g.
In still another embodiment of the present invention, percentage shell ratio ranging between 21 and 22.
In still another embodiment of the present invention, filament length ranging between 750 and 800 m/cocoon.
In still another embodiment of the present invention, oval shape with mild peanut constriction appearance.
In still another embodiment of the present invention, cocoon is white colored.
In still another embodiment of the present invention, eggs of Kalpatharuvu and Hemavathy hybrids are pigmented and has diapause appearance.
In still another embodiment of the present invention, crossing bivoltine strains of APS (oval) X APSg (peanut) and APS5 (oval) X APS4 (peanut) produces Kalpatharuvu and Hemavathy respectively.
In still another embodiment of the present invention, pupation rate of Hemavathy is ranging between 92-96%.
In still another embodiment of the present invention, pupation rate of Kalpatharuvu is ranging between 85-90%. In still another embodiment of the present invention, average evaluation Index of Hemavathy is ranging between 61-66%.
In still another embodiment of the present invention, average evaluation Index of Kalpatharuvu is ranging between 65-70%.
In still another embodiment of the present invention, ultimately, based on the overall performance and uniformity in cocoon shape two oval lines viz., APS5 & APS9 and two peanut lines viz., APS4, APSg are selected (Fig 2 and 3)
In still another embodiment of the present invention, the selected oval lines; APS5 & APS9 and two peanut lines; APS4, APSg lines are crossed in full diallele pattern. The resultant 16 Fls and their reciprocals along with their parents hybrids are reared in three replicates each retaining 400 larvae after third moult. The hybrids' performance is evaluated considering for six characters namely cocoon yield by weight, pupation rate, single cocoon weight, single shell weight, shell ratio and filament length. The hybrid combinations APS9 x APS8 (Kalpatharuvu) and APS5 x APS4 (Hemavathy) showed promise. The floss% in these hybrids is also found to less (APS9 x APS8:0.972% & APS5 x APS4: 1.056%). Further, multiple trait evaluation index (Mano et al, 1992) is calculated for the above commercially important characters and the superiority of APS9 x APS8 (Kalpatharuvu) and APS x APS (Hemavathy) is further confirmed. (Please refer Table 12)
TABLE 12. COMPARATIVE YIELD PROFILE OF NEW BIVOLTINE HYBRIDS,
HEMAVATHY (APSS x APS4) & KALPATHARUVU (APS9 x APSg)
AND TRADITIONAL HYBRID (PM x NB4D2)
Figure imgf000024_0001
* based on 100070 disease free egg layings tested with 546 farmers of Andhra Pradesh and Karnataka ** based on 40425 disease free egg layings tested with 191 farmers of Andhra Pradesh
1 total larval duration from the time of larval eclosion from the eggs upto the onset of secretion of silk.
2 total weight of cocoons harvested from the total number of larvae used for rearing
3 total weight of cocoons harvested from the larvae raised from 100 disease free egg layings
4 the ratio of single cocoon shell weight to the weight of single silk cocoon
5 an average length of silk fiber recovered from a single silk cocoon
6 the recovery percentage of continuous silk fibre from cocoons
7 the number of kgs of cocoons required to produce 1 kg of raw silk
8 silk is graded based on international standards considering the quality, neatness and tenacity of silk fibre.
In still another embodiment of the present invention, among the 4 parents, the parent APS5, the female parent of "Hemavathy", is showing positive General Combining Effects (GCA) effects for all the six characters followed by APS9, the female parent of "Kalpatharuvu", for 5 characters except Pupation Rate. While the parent APS4, the male of "Hemavathy" showed negative effects for all the characters so also APSg, the male parent of "Kalpatharuvu" except for Pupation Rate. (Please refer Table 13)
Table 13 - General Combining Ability (GCA) of the parents
Figure imgf000025_0001
In an em o ment o t e present nvent on, a met o to produce Mu erry Silkworm hybrids named Kalpatharuvu and Hemavathy, well adapted for tropical region mainly and for all seasons of the year, are cross of bivoltine, obtained from oval type Chinese and peanut type Japanese hybrids, using steps comprising hybridization, inbreeding, disease tolerance breeding, selection, and DNA fingeφrinting, and are superior to conventional hybrids of the tropical regions mainly and produces white color silk of international grade 3A-4A.
In another embodiment of the present invention, taking the bivoltine strains of the Chinese and the Japanese hybrids and initiating two exclusive breeding plans to generate pureline inbreeds of high yield from both hybrids. In yet another embodiment of the present invention, mating Fi hybrid offsprings in each breeding plan and rearing the progeny.
In still another embodiment of the present invention, mass mating of progeny derived from the multiple random pair up to F4 generation and without any culling to ensure desirable combinations in the breeding plan.
In still another embodiment of the present invention, making an outclass at F generation with elite oval line SHOWA of Japanese origin for oval lines, and with indigenous peanut line NB4D2 for peanut cocoon lines.
In still another embodiment of the present invention, selecting F5 generation on the basis of desired larval markings and cocoon shape and mass mating selected multiple lines up to F6 generation.
In still another embodiment of the present invention, obtaining cellular families from above-mentioned separate single pair mating and thereby obtaining larvae from said cellular families.
In still another embodiment of the present invention, rearing said larvae up to cocooning in each of the cellular families.
In still another embodiment of the present invention, selecting cocoons raised from each of the cellular families on the basis of parameters comprising the larval survival upto pupation stage, single cocoon weight, silk content, silk filament length, cocoon shape, and floss content traits.
In still another embodiment of the present invention, selecting cellular families registering higher survival rate.
In still another embodiment of the present invention, selecting only diapause eggs for subsequent inbreeding and selecting males and females emerging from eggs of said cellular families.
In still another embodiment of the present invention, mating the moths emerging from the cocoons of the said families among themselves.
In still another embodiment of the present invention, inbreeding the said cellular families up to F8 generation. In still another embodiment of the present invention, subjecting the F8 generation families to Bombyx mori densonucleosis virus 1 (BmDNNl) and isolating cellular families resistant to BmDNNl.
In still another embodiment of the present invention, inbreeding the said resistant cellular families up to F]0 generation.
In still another embodiment of the present invention, picking up female and male moths from each of the lines at Fin generation at random.
In still another embodiment of the present invention, extracting DΝA from selected moths at Fio generation and subjecting isolated DΝA samples to fingeφrinting, using Fluorescent Inter-Simple Sequence Repeat anchored Polymerase Chain Reaction (FISSR-PCR) based analysis.
In still another embodiment of the present invention, ISSR - DΝA markers for testing homozygosity are selected from a group comprising CGA(ATT)4, TA(CGA)4, (CA)7, T3(ATT)4, and CT(ATT)4.
In still another embodiment of the present invention, FISSR Markers for testing heterozygosity are selected from a group comprising (CA)7, (TG)7, T(GA)9, T(GA)8, C(GA)7, and TA(CAG)4.
In still another embodiment of the present invention, Taq polymerase is used in PCR.
In still another embodiment of the present invention, homozygosity is tested on agarose gel of concentration ranging between 1-5%.
In still another embodiment of the present invention, heterozygosity is tested on polyacrylamide gel with concentration ranging between 3-8%.
In still another embodiment of the present invention, eight promising bivoltines silkworm inbred lines developed at this institute under breeding programme have been assessed at molecular level for their homozygosity within the strains and heterozygosity in between strains using five ISSR markers (Please refer Tables 14 and 15). Table 14 - ISSR- DNA markers used for the estimation of homozygosity in the parental lines
Figure imgf000028_0001
15
Table 15 - DNA band sharing percentage within the strains (Molecular marker homozygosity)
Figure imgf000028_0002
In still another embodiment of the present invention, ten individuals (five males and five females) from each strain (8x10= 80) are analyzed for their homozygosity. High molecular weight genomic DNA is extracted from the pupae and purified as per standard protocols (Nagaraju and Nagaraju, 1995; Sambrook et. al, 1982). DNA is amplified by PCR technique using five di and tri nucleotide repeat ISSR markers anchored with 5' degenerate nucelotides (Reddy et.al. 1999). For 10 ul of PCR reaction, 50 ng of genomic DNA, IX PCR reaction buffer, 2.5 mM MgCl2, 25 urn each of dNTPs, and 0.5 units of Taq Gold DNA polymerase (Perkin Elmer) for dinucleotide primer and MBI Taq DNA polymerase for trinucelotide primers are used. Amplification is performed on MJ research PTC- 2000 thermal cycler and perkin Elmer Gene Amp PCR- 9600 system, With a programme of initial denaturation at 94°C for 3 min followed by 35 cycles Of 94°C for 30 se , 50°C for 30 se , 72°C for 1 min and final extension at 72°C for 10 min. For Gene Amp PCR machine, the initial denaturation at 94°C is for 10 min and annealing was at 42°C and the remaining programme is same as above. The amplified DNA is separated on 2% agarose gels and generated DNA profiles are documented and analyzed for DNA band sharing levels within (homozygosity) and in between (heterozygosity) parental lines (Nei, 1978, Sharma et. al. 1999).
In still another embodiment of the present invention, for patenting the hybrids "Kalpatharuvu" (APS9 X APS8) and "Hemavathy" (APS5 X APS4), FISSR-PCR DNA profiling is carried out. DNA from the parents and the FI progeny is extracted, purified and amplified by PCR technique using six different ISSR markers (Please refer Table 16), (Kumar et.al. 2001).
Table 16 - Molecular marker heterozygosity measured in between 13 silkworm parental lines
Figure imgf000029_0001
In still another embodiment of the present invention, the protocols and the PCR machines used are as described above except the addition of 5 μl of fluorescent dUTPs to the reaction mixture. The amplified DNA is separated on 5% polyacrylamide gels using Perkin Elmer gene scan Machine and the generated FISSR-PCR DNA profiles of Kalpatharuvu and Hemavathy hybrids (Fig. 5, please refer Table 17) are submitted for PCT filing. Table 17 - FISSR Markers used for DNA Fingerprinting of two Silkworm Hybrids
Figure imgf000030_0001
In still another embodiment of the present invention, selecting the inbred lines showing >90% homozygosity based on DNA tests for evaluating hybrid vigor.
In still another embodiment of the present invention, isolating oval and peanut shaped cocoon lines showing said degree of homozygosity.
In still another embodiment of the present invention, crossing the isolated cocoons on a full diallele pattern.
In still another embodiment of the present invention, rearing the resulting hybrids and retaining larvae after third moult.
In still another embodiment of the present invention, evaluating the hybrid performance on the basis of total larval duration, fifth instar larval duration, larval survival upto pupal stage, single cocoon weight, cocoon shell weight, silk filament length, and floss content In still another embodiment of the present invention, selecting the parents giving rise to the highest hybrid vigor in the said hybrids.
In still another embodiment of the present invention, crossing bivoltine strains of APS (oval) X APS8 (peanut) and APS5 (oval) X APS4 (peanut) produces the superior quality and high yield hybrids named Kalpatharuvu and Hemavathy respectively. In still another embodiment of the present invention, among all the hybrid combinations (FI) studied APS5 X APS4 (Hemavathy), APS9 X APSg (Kalpatharuvu) showed positive Specific Combining Ability (SCA) for all the characters studied. These hybrids involving good X poor combiners indicate that additive x dominant gene interaction were important in determining the SCA effects. (Please refer Table 18). Table 18 - Specific Combining Ability (SCA) of the hybrids
Figure imgf000031_0001
* => "Hemavathy" & ** => "Kalpatharuvu"
In still another embodiment of the present invention, the heterosis calculated for the Fls revealed significant to highly significant (P < 0.05 to P < 0.01) heterosis in the combination APS5 X APS4 (Hemavathy) for all the 6 characters and highly significant heterosis in APS X APSg (Kalpatharuvu) for the 5 characters out of 6 characters studied. (Please refer Table 19)
Table 19 - Mid Parent (MP) Heterosis in Hybrids
Figure imgf000031_0002
* => P< 0.05 & ** => P≤ O.01
In still another embodiment of the present invention, Evaluation Index (El) calculated on the basis of over all performance of the Fls also support the superiority of the hybrids APS9 X APSg (Kalpatharuvu) and APS5 X APS4 (Hemavathy) through highest average indices (6 traits) of 67.12 and 63.15 respectively. (Please refer Table 20)
Figure imgf000032_0001
In an embodiment of the present invention, applicants have developed two new bivoltine hybrids, APS9 x APSg and APSs x APS4, which are named as "Kalpatharuvu' and Ηemavathy' respectively.
In still another embodiment of the present invention, the exotic Chinese and Japanese hybrids are used as donor parental genetic material for breeding bivoltine inbred lines APS4, APS5, APSg and APS9.
In still another embodiment of the present invention, the breeding is carried out by exercising selection for desired characters particularly for oval and peanut cocoon shape with uniform shape and size, hardiness of cocoon, higher pupation rate, shorter larval duration, higher single cocoon weight, higher cocoon shell weight, longer silk filament length, better reelability and overall enhancement in the quality of silk (as quantified by international silk grade). In still another embodiment of the present invention, during the course of inbreeding, outcross is made with another qualitatively superior bivoltine breed to incoφorate the beneficial characters to the newly bred inbred lines.
In still another embodiment of the present invention, during the process of inbreeding, different inbred lines are separated based on the presence/absence of markings on the larval body and shape of the cocoons (oval or peanut shaped).
In still another embodiment of the present invention, after the inbred lines attained some degree of uniformity for various quantitative traits, they are tested for homozygocity using different DNA markers. The highly inbred and homozygous lines are subjected to hybridization studies in the breeding laboratory.
In still another embodiment of the present invention, the excelled hybrid combinations, APS9 x APSg and APS5 x APS4 are field tested during different seasons of the year in different regions of Andhra Pradesh to evaluate its silk cocoon productivity, yield consistency, silk cocoon yield attributes and quality of silk.
In still another embodiment of the present invention, the new hybrids, Kalpatharuvu and Hemavathy yielded better crop results in all the seasons of the year. The grade of silk recovered from new hybrids is tested and classified as 3A-4A.
In still another embodiment of the present invention, the overall financial returns to the farmers can be about 30-40% higher than the traditional polyvoltine x bivoltine hybrids. Thus applicants have successfully developed elite silkworm hybrids, Kalpatharuvu and Hemavathy and demonstrated that the large-scale exploitation would lead to higher economic returns to both silkworm rearers and silk reelers.
In still another embodiment of the present invention, since the silk obtained is graded as 3A-4A, which is the quality stipulated for international market. The country can benefit from the export of internationally competitive quality silk. The rearing of these hybrids in large-scale will also help to meet the industrial needs for quality silk and curbs the importing of Chinese silk.
In still another embodiment of the present invention, the new hybrids are also expected to perform better in the tropical states of Karnataka and Tamil Nadu, where there is paucity of productive bivoltine hybrids. In still another embodiment of the present invention, two breeding plans are initiated separately using the bivoltine silkworm hybrids obtained from China and Japan as donor genetic materials. Each breeding plan is aimed at isolating Chinese (oval) type and Japanese (peanut) type of cocoons. In both the breeding plans, breeding methods involving inbreeding, hybridization, disease tolerance breeding, DNA fingeφrinting, and selection for the desired traits are followed.
In still another embodiment of the present invention, Silkworm rearing is conducted under standard rearing conditions. Initially, a large F2 populations contributed by the progeny from 8-10 pair matings of the F, hybrid offsprings is reared. The mass mating of progeny derived from the multiple random pair is continued upto F4 generation. The mass rearing without any culling is exercised to ensure desirable combinations in the breeding plan.
In still another embodiment of the present invention, at F4 generation an outcross is made with elite oval for oval lines and peanut for peanut cocoon lines. At F5 generation selection was resorted to larval markings and cocoon shape. After two generations of mass mating in each breeding plan, five cellular families (each cellular family is drawn from a single pair mating) are maintained.
In still another embodiment of the present invention, 400 larvae are retained and reared upto cocooning in each of the cellular families. The cocoons raised from each of the cellular families are selected based on the larval survival upto pupation stage. Single cocoon weight, silk content, silk filament length, cocoon shape and floss content traits are measured.
In still another embodiment of the present invention, the cellular families, which registered higher survival rate, are only selected for further inbreeding. In both the breeding plans only diapause eggs, which is characteristic feature of bivoltines, are only selected.
In still another embodiment of the present invention, in each of the selected cellular families 25 males and 25 females are selected. The moths emerged from these cocoons are mated among themselves. Inbreeding of the selected cellular families is continued upto Fι0 generation. The Fin generation families are also subjected to Bombyx mori densonucleosis virus 1 (BmDNNl) to isolate cellular families for BmDNNl resistance. The inoculation dose of 10"2 BmDNNl is used for the assay. In still another embodiment of the present invention, five females and five male moths from each of the lines are randomly picked at Fin generation and DNA is extracted by using the standard protocol. The DNA samples are subjected to fingeφrinting using Fluorescent Inter-Simple Sequence Repeat anchored Polymerase Chain Reaction (FISSR- PCR) based analysis. The inbred lines which showed >90% homozygocity based on DNA tests are chosen for evaluation of hybrid vigor.
In still another embodiment of the present invention, the isolated oval cocoon (four lines) and peanut cocoon (four lines) lines are crossed on a full diallele pattern. The resulted hybrids are reared in three replicates each retaining 400 larvae after third moult. The hybrid performance is evaluated considering total larval duration, fifth instar larval duration, larval survival upto pupal stage, single cocoon weight, cocoon shell weight, silk filament length and floss content.
In still another embodiment of the present invention, the parents, which give rise to the highest hybrid vigor in the hybrids, are selected. The inbred line APS9 (oval) with APSg (peanut) and APS5 (oval) with APS4 (peanut) are found to produce the best hybrid combination.
In still another embodiment of the present invention, these two promising hybrids, APS9 x APSg and APS5 x APS4, are reared in different seasons of the year and different regions of Andhra Pradesh to evaluate the cocoon crop stability with the farmers. The performance is tabulated in Table 21.
TABLE 21 - SEASONAL PERFORMANCE OF KALPATHARUVU (APS9 x APSg) AND HEMAVATHY (APSS x APS4 ) WITH FARMERS OF A.P.
Figure imgf000035_0001
In still another embodiment of the present invention, the said hybrids are reared and have recorded an average of 61.43kg/100 dfls. The cocoons can easily fetch about Rs. 50-60 more per kg of cocoons over the ruling polyvoltine x bivoltine hybrid, Pure Mysore x NB4D2. The new hybrids, APS9 x APSg and APS5 x APS4 are named respectively as 'Kalpatharuvu' and Ηemavathy'.
In still another embodiment of the present invention, the bivoltine hybrids obtained from China and Japan are utilized as donor parental genetic materials in the breeding programmes. The inbred lines of oval and peanut shaped cocoons have been isolated through selection for desired metric traits and controlled progeny mating. The isolated inbred lines, which carried desired characters for larval survival, silk cocoon weight, silk content and silk quality are bred upto Fin-Fn generations. These lines are stabilized for the target traits between Fin and Fn generations.
In still another embodiment of the present invention, the DNA fingeφrinting technology, which is now well proven and globally used in crop improvement programme is used to estimate the zygocity status of the developed inbred lines. Four inbred lines, APS5 and APS9 (oval), APS4 and APSg (peanut), which, showed higher homozygocity (>90%) are chosen for further studies. These inbred lines are also screened simultaneously for Bombyx mori densonucleosis virus 1 (BmDNVl) resistance. The highly homozygous inbred lines of oval cocoon shape are crossed systematically with similarly developed peanut shape inbred lines to analyse their hybrid vigor. The silkworm hybrid combinations, which showed higher-level heterosis, are selected for further trials with farmers.
In still another embodiment of the present invention, DNA fingeφrinting is been used for the first time to test homozygosity and heterozygosity in silkworms for the first time. DNA fingeφrinting reflects not genetic constitution of the hybrids but also the stability of the same. Introduction of DNA fingeφrinting is a major step forward towards developing high quality silkworm hybrids. Instant application is an initiative towards achieving close association between advancement in the field of biotechnology with molecular Biology in particular and sericulture. The instant Application has overcome a major hurdle as regards uncertainty involved with random crossing of parents with hit and trials to develop desired hybrid. Applicants have made a breakthrough in the field of sericulture. Advantages of the in present invention
1. The main advantage of the present invention is development of novel, superior silkworm hybrids.
2. Another main advantage of the present invention is development of a reliable and consistent method to develop superior silkworm hybrids.
3. Yet another advantage of the present invention is development of a silkworm hybrid producing international standard silk.
4. Still another advantage of the present invention is that silk produced by the novel hybrids is with high reelability.
5. Still another advantage of the present invention is that hybrids are resistant to viruses.
6. Still another advantage of the present invention is the use of DNA fingeφrinting to develop silkworm hybrids of desired heterogeneity and traits.
7. Still another advantage of the present invention is the development of hybrids with reduced larval period.
8. Still another advantage of the present invention is the use of DNA fingeφrinting to ascertain hybrid vigor in silkworms.
9. Still another advantage of the present invention is to develop hybrids with high survival rate.

Claims

Claims
1. A Mulberry silkworm hybrid named Swarnandhra, preferably well adapted for tropical region and for all seasons of the year and the said hybrid is a cross of polyvoltine APMi derived from pureline inbreed of Madagascar hybrids and bivoltine APS8 derived from pureline inbreed of Chinese hybrids, said hybrid being produced using steps comprising inbreeding, hybridizing, disease tolerance breeding, DNA fingeφrinting, and selecting, said hybrid is superior to conventional hybrids of the tropical regions and produces:
(a) silk of superior quality and high yield with:
(i) international grade of 2A-3 A,
(ii) reelability ranging between 80 and 90%,
(iii) renditta ranging between 7.0 and 7.5 kg,
(iv) floss percentage less than 5%, and (v) greenish yellow color;
(b) larvae of superior quality with:
(i) survival rate ranging between 80 and 85%, (ii) larval period ranging between 22 and 23 days, and (iii) plain or no visible markings;
(c) cocoon of good yield and superior quality with:
(i) yield ranging between 50 and 55 per 100 dfls (kg),
(ii) single silk cocoon weight ranging between 1.60 and 1.70 g, (iii) cocoon shell weight ranging between 0.290 and 0.320,
(iv) percentage shell ratio ranging between 18 and 19,
(v) filament length ranging between 700 and 800 m/cocoon,
(vi) oval shape with mild peanut constriction appearance, and
(vii) greenish yellow color; (d) eggs with :
(i) pigmentation, and
(ii) non-diapause appearance.
2. A hybrid as claimed in claim 1, wherein said hybrid has average evaluation index about 60.50.
3. A hybrid as claimed in claim 1, wherein pupation rate is ranging between 92-98%.
4. A reliable and consistent method to produce Mulberry silkworm hybrid named Swarnandhra, well adapted for all seasons and preferably adapted for tropical region and the said hybrid is a cross of polyvoltine APMi, derived from pureline inbreed of
Madagascar hybrids and bivoltine APS8; derived from pureline inbreed of Chinese hybrids, and is superior to conventional hybrids of the tropical regions mainly and produces greenish yellow silk of international grade 2A - 3 A, said method comprising,
(i) mass-mating polyvoltine and bivoltine strains of Madagascar hybrids and
Chinese hybrids respectively with separate breeding plan to generate pureline inbreeds up to F4 generation,
(ii) producing outclass at F generation with pure Mysore (polyvoltine) and NB4D2 (bivoltine) in polyvoltine and bivoltine breeding plans respectively,
(iii) mass mating outclass hybrid up to F6 generation,
(iv) rearing larvae up to cocooning,
(v) selecting cellular families with cocoons having several characteristics comprising high larval survival up to pupae stage, cocoon weight, high silk content, silk filament length, cocoon shape, and floss percentage,
(vi) selecting non-diapause egg laying from selected families,
(vii) inbreeding selected families up to F8 generation,
(vϋi) exposing F generation cellular families to Bombyx mori densonucleosis virus,
(ix) isolating F8 generation cellular families resistant to BmDNV 1,
(x) fingeφrinting DNA samples of selected families using Fluorescent-inter- simple Sequence-repeat-polymerase chain reaction (FISSR-PCR) with ISSR DNA markers, (xi) selecting inbred lines showing more than 90% homozygosity based on the DNA test,
(xii) crossing selected inbred lines on a half diallele pattern using polyvoltine as female parents and bivoltine lines as male parents,
(xiii) rearing resultant hybrids and retaining larvae after third moult,
(xiv) using the DNA fingeφrinting technology to test hybrid vigor of said larvae using Fluorescent-inter-simple Sequence-repeat-polymerase chain reaction (FISSR-PCR) with FISSR DNA markers, and
(xv) selecting the parents giving rise to maximum hybrid vigor with cross of pureline inbreed polyvoltine (APMi) and bivoltine (APSg) having maximum hybrid vigor.
5. A method as claimed in claim 4, wherein bivoltine hybrids are selected from a group comprising APS4, APS6, APSg, and APSio.
6. A method as claimed in claim 4, wherein polyvoltine hybrids are selected from a group comprising APMb APM2, APM3, APM4, and APM5.
7. A method as claimed in claim 4, wherein bivoltine hybrids are used as tester.
8. A method as claimed in claim 4, wherein ISSR DNA markers are selected from a group comprising CGA (ATT)4, TA (CGA)4, (CA)7, T3 (ATT)4, and CT (ATT)4.
9. A method as claimed in claim 4, wherein FISSR - DNA markers are selected from a group comprising (CA)7, (TG)7, T(GT)9, T(GA)8, C(GA)7, and TA(CAG)4.
10. A method as claimed in claim 4, wherein Taq polymerase is used in PCR.
11. A method as claimed in claim 4, wherein homozygosity is tested on agarose gel of concentration ranging between 1-5%.
12. A method as claimed in claim 4, wherein heterozygosity is tested on polyacrylamide gel with concentration ranging between 3-8%.
13. Mulberry Silkworm hybrids named Kalpatharuvu and Hemavathy, preferably well adapted for tropical region and for all seasons of the year, which are cross of bivoltine strains APS9 (oval) X APSg (peanut) and APS5 (oval) X APS4 (peanut) respectively, wherein the oval and peanut types are obtained from oval type Chinese and peanut type Japanese hybrids, said hybrids being produced by using steps comprising inbreeding, hybridization, disease tolerance breeding, selection, and DNA fingeφrinting, said hybrids are superior to conventional hybrids of the tropical regions mainly and produces:
(a) silk of superior quality and high yield with:
(i) international grade of 3 A-4A, (ii) reelability ranging between 80 and 85%, (iii) floss percentage ranging between 0.9-1.1 %,
(iv) renditta ranging between 6.5 and 7.0 kg, and (v) white color;
(b) larvae of superior quality with:
(i) survival rate ranging between 80 and 85%, (ii) larval period ranging between 22 and 23 days, and
(iii) plain or no visible marking;
(c) cocoon of good yield and superior quality with:
(i) yield ranging between 60 and 65 per 100 dfls( kg),
(ii) single silk cocoon weight ranging between 1.7 and 1.8g, (iii) shell weight ranging between 0.350 and 0.390g,
(iv) percentage shell ratio ranging between 21 and 22, (v) filament length ranging between 750 and 800 m cocoon, (vi) oval shape with mild peanut constriction appearance, and (vii) white color; (d) eggs with:
(i) pigmentation, and (ii) diapause appearance.
14. Hybrids as claimed in claim 13, wherein pupation rate of Hemavathy is ranging between 92-96%.
15. Hybrids as claimed in claim 13, wherein pupation rate of Kalpatharuvu is ranging between 85-90%.
16. Hybrids as claimed in claim 13, wherein average evaluation Index of Hemavathy is ranging between 61-66%.
17. Hybrids as claimed in claim 13, wherein average evaluation Index of Kalpatharuvu is ranging between 65-70%.
18. A reliable and consistent method to produce Mulberry Silkworm hybrids named Kalpatharuvu and Hemavathy, well adapted for all seasons and preferably adapted for tropical region, which are cross of bivoltines strains APS (oval) X APSg (peanut) and APS5 (oval) X APS4 (peanut) respectively, wherein the oval and peanut types are obtained from oval type Chinese and peanut type Japanese hybrids, and are superior to conventional hybrids of the tropical regions mainly and produces white color silk of international grade 3A-4A, and said method comprising,
(i) mass mating bivoltine strains of the Chinese and the Japanese hybrids and initiating two exclusive breeding plans to generate pureline inbreeds of high yield from both hybrids up to F4, (ii) Producing an outclass at F generation with elite oval line SHOW A of Japanese origin for oval cocoon lines and with indigenous peanut line NB4D2 for peanut cocoon lines, (iii) selecting F5 generation on the basis of desired larval markings and cocoon shape, (iv) mass mating selected multiple lines up to F6 generation to obtain larvae,
(v) rearing said larvae of each cellular family up to cocooning in each of the cellular families, (vi) selecting cocoons on the basis of parameters comprising high larval survival upto pupation stage, single cocoon weight, high silk content, silk filament length, cocoon shape, and floss content traits,
(vii) selecting only diapause eggs for subsequent inbreeding, (viii) inbreeding moths emerging from the cocoons of the selected families up to Fg generation, (ix) Selecting Fg generation families resistant to Bombyx mori densonucleosis virus 1 (BmDNNl), (x) inbreeding moths emerging from the cocoons of the selected families up to Fι0 generation,
(xi) extracting DNA from moths at Fι0 generation,
(xii) subjecting isolated DNA samples to fingeφrinting,
(xiϋ) selecting inbred lines showing >90% homozygosity using Polymerase Chain
Reaction (PCR),
(xiv) isolating oval and peanut shaped cocoon lines showing said degree of homozygosity,
(xv) crossing the isolated cocoons on a full diallele pattern,
(xvi) rearing resulting hybrids to retain larvae after third moult,
(xvii) evaluating the hybrid performance on the basis of total larval duration, fifth instar larval duration, larval survival upto pupal stage, single cocoon weight, cocoon shell weight, silk filament length, and floss content, and
(xviii) selecting parents giving rise to the highest hybrid vigor in the said hybrids using DNA fingeφrinting technique Fluorescent Inter-Simple Sequence Repeat anchored Polymerase Chain Reaction (FISSR-PCR), with crosses of APS9 X APSg and APS5 X APS4 showing desired characteristics.
19. A method as claimed in claim 18, wherein bivoltine hybrid strains are selected from a group comprising APSi, APS4, APS5, APSg, and APS .
20. A method as claimed in claim 18, wherein ISSR - DNA markers for testing homozygosity are selected from a group comprising CGA (ATT)4, TA(CGA)4, (CA)7, T3(ATT)4, and CT(ATT)4.
21. A method as claimed in claim 18, wherein FISSR Markers for testing heterozygosity are selected from a group comprising (CA)7, (TG)7, T(GA)9, T(GA)8, C(GA)7, and TA(CAG)4.
22. A method as claimed in claim 18, wherein Taq polymerase is used in PCR.
23. A method as claimed in claim 18, wherein homozygosity is tested on agarose gel of concentration ranging between 1-5%.
24. A method as claimed in claim 18, wherein heterozygosity is tested on polyacrylamide gel with concentration ranging between 3-8%.
PCT/IB2002/001349 2002-04-17 2002-04-17 Superior, novel, quality mulberry silkworm hybrids and a method thereof WO2003087295A2 (en)

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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101535535B (en) * 2006-09-12 2012-01-18 新加坡国立大学 Enhanced silk protein material having improved mechanical performance and method of forming the same.
CN103070138A (en) * 2013-02-04 2013-05-01 江苏科技大学 Method for breeding hypersilk, bombyx mori densovirus-resistant and fluoride-resistant silkworm variety
CN107751122A (en) * 2017-12-12 2018-03-06 四川省农业科学院蚕业研究所 With the method for silkworm place soil species seed selection trimolter new material
CN109929943A (en) * 2019-01-08 2019-06-25 贵阳中医学院 Radix Glycyrrhizae ISSR fluorescent molecule labeled primer and its application
CN110352918A (en) * 2019-08-15 2019-10-22 重庆三峡农业科学院(重庆市万州区甘宁蚕种场) A kind of raising quality of silkworm eggs method
CN110665842A (en) * 2019-11-14 2020-01-10 广西立盛茧丝绸有限公司 Near infrared spectrum cocoon selection method
CN114190339A (en) * 2021-08-03 2022-03-18 西南大学 Restricted white-egg silkworm variety breeding method suitable for separate breeding of male and female silkworms and separate reeling of male and female cocoons

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