CLIAMS:I/We claim:
1. A process of determining the sex of Casuarina junghuhniana comprising:
a. providing a forward DNA primer having polynucleotide sequence as set forth in SEQ ID NO: 3 and a reverse DNA primer having polynucleotide sequence as set forth in SEQ ID NO: 4;
b. carrying out a PCR amplification reaction with a DNA sample from Casuarina junghuhniana; and
c. determining the size of the amplicon product from said PCR reaction,
wherein a 1191bp band is indicative that the Casuarina junghuhniana plant in male.
2. The process as claimed in claim 1, wherein the amplicon obtained from the PCR amplification reaction using DNA primers having polynucleotide sequence as set forth in SEQ ID NO: 3 and 4 has a polynucleotide sequence as set forth in SEQ ID NO: 1.
3. A process of determining the sex of Casuarina junghuhniana comprising:
a. providing a forward DNA primer having polynucleotide sequence as set forth in SEQ ID NO: 3 and a reverse DNA primer having polynucleotide sequence as set forth in SEQ ID NO: 4;
b. carrying out a PCR amplification reaction with a DNA sample from Casuarina junghuhniana; and
c. determining the size of the amplicon product from said PCR reaction,
wherein a 971bp band is indicative that the Casuarina junghuhniana plant in female.
4. The process as claimed in claim 3, wherein the amplicon obtained from the PCR amplification reaction using DNA primers having polynucleotide sequence as set forth in SEQ ID NO: 3 and 4 has a polynucleotide sequence as set forth in SEQ ID NO: 2.
,TagSPECI:FIELD OF INVENTION
[0001] The present disclosure relates to the field of plant molecular biology. Particularly, the present disclosure relates to a process of sex determination of Casuarina plants, involving detection of PCR amplified plant sex specific DNA sequences.

BACKGROUND OF THE INVENTION
[0002] Eucalyptus meets major wood requirement of the paperboards and specialty papers division (PSPD) in the mill industry, and to a lesser extent, Subabul (Leucaena leucocephala) and Casuarinas.
[0003] Casuarina junghuhniana has properties that are comparable to Eucalyptus in terms of pulp yield, fiber length, and short rotation cycles. Further, unlike Eucalyptus varieties, Casuarina species are not susceptible to gall-midge infestation. Thus, C. junghuhniana can be used as a replacement for Eucalyptus in the PSPD mill industry. C. junghuhniana has other useful characteristics such as fast growing, can be found in both coastal and inland sites, nitrogen fixing capacity, shorter gestation period of only four years, good fuel wood, drought tolerance, preference in the construction industry for scaffolding, and high calorific value of wood (preferred crop for biomass power plant fuel).
[0004] In C. junghuhniana, inter and intra-specific hybridization is one of the important breeding activities to develop high yielding genotypes. C. junghuhniana is a dioecious species where the male and female flowers arise from different trees. In a hybridization orchard, the female and male tress are kept in a defined ratio of 3:1. For, every three rows of female trees, one row of male trees are planted. A current drawback in sex determination of C. junghuhniana is that, sex determination is not possible until the tree flowers. This mode of identification of male or female tree through morphological observation is feasible only after two years, when the plant starts flowering. Therefore, the current practice in hybridization orchards is to plant C.junghuhniana in high density and weed out the unwanted trees after two years. This process is not only time consuming, but also is a waste of time, and resources.
[0005] There is a need for an improved and efficient process for identification and differentiation of male and female plants of Casuarina at an early stage of development, thereby saving time, and resources and helping the hybridization program.
[0006] US patent 6,037,128 describes a process for the preparation of semi-synthetic amplicon useful for sex determination of the papaya plant.
[0007] US patent 5,418,133 describes a nucleic acid isolate capable of hybridizing only to Y-chromosome specific DNA sequences of cattle, sheep, goats and other ruminants.

SUMMARY OF THE INVENTION
[0008] An aspect of the present disclosure relates to a process of determination of sex of a Casuarina junghuhniana plant.
[0009] An aspect of the present disclosure relates to a process of determining the sex of a Casuarina junghuhniana plant, said process comprising: (a) providing a forward DNA primer having a polynucleotide sequence as set forth in SEQ ID NO: 3 and a reverse DNA prime having a polynucleotide sequence as set forth in SEQ ID NO: 4; (b) carrying out a PCR amplification reaction with a DNA sample from Casuarina junghuhniana; and (c) determining the size of the amplicon product from said PCR amplification reaction, wherein a 1191bp band is indicative that the Casuarina junghuhniana plant is male.
[0010] An aspect of the present disclosure relates to a process of determining the sex of a Casuarina junghuhniana plant, said process comprising: (a) providing a forward DNA primer having a polynucleotide sequence as set forth in SEQ ID NO: 3 and a reverse DNA prime having a polynucleotide sequence as set forth in SEQ ID NO: 4; (b) carrying out a PCR amplification reaction with a DNA sample from Casuarina junghuhniana; and (c) determining the size of the amplicon product from said PCR amplification reaction, wherein a 971bp band is indicative that the Casuarina junghuhniana plant is female.
[0011] This summary is provided to introduce concepts related to a process of determining the sex of a Casuarina junghuhniana plant. This summary is not intended to identify essential features of the claimed subject matter nor is it intended for use in determining or limiting the scope of the claimed subject matter.

BRIEF DESCRIPTION OF ACCOMPANYING DRAWINGS
[0012] The following drawings form part of the present specification and are included to further illustrate aspects of the present disclosure. The disclosure may be better understood by reference to the drawings in combination with the detailed description of the specific embodiments presented herein.
[0013] Figure 1 shows the electropherogram of unique fragments produced by RAPD primers using male or female Casuarina junghuhniana plant DNA, in accordance with an embodiment of the present disclosure.
[0014] Figure 2 shows the electropherogram of Casuarina junghuhniana plant sex specific fragment using IMC3015 primers, in accordance with an embodiment of the present disclosure.
[0015] Figure 3 shows the electropherogram of validation of IMC3015 primers in determining sex of Casuarina junghuhniana plants, in accordance with an embodiment of the present disclosure.

DETAILED DESCRIPTION OF THE INVENTION
[0016] Those skilled in the art will be aware that the invention described herein is subject to variations and modifications other than those specifically described. It is to be understood that the invention described herein includes all such variations and modifications. The invention also includes all such steps, features, compositions and methods referred to or indicated in this specification, individually or collectively, and any and all combinations of any two or more of said steps or features.
Definitions
[0017] For convenience, before further description of the present invention, certain terms employed in the specification, examples are collected here. These definitions should be read in light of the remainder of the disclosure and understood as by a person of skill in the art. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by a person of ordinary skill in the art. The terms used throughout this specification are defined as follows, unless otherwise limited in specific instances.
[0018] As used in the specification and the claims, the singular forms “a”, “an”, and “the” include plural referents unless the context clearly dictates otherwise.
[0019] The term “oligonucleotide” as used herein, refers to a molecule comprised of two or more deoxyribonucleotides or ribonucleotides. Oligonucleotides may include, but are not limited to, primers, probes, nucleic acid fragments to be detected, and nucleic acid controls. The exact size of an oligonucleotide depends on many factors and the ultimate function or use of the oligonucleotide.
[0020] The term “primer” as used herein, refers to an oligonucleotide, whether natural or synthetic, capable of acting as a point of initiation of DNA synthesis under conditions in which synthesis of a primer extension product complementary to a nucleic acid strand is induced, i.e., in the presence of four different nucleoside triphosphates and an agent for polymerization (i.e., DNA polymerase or reverse transcriptase) in an appropriate buffer and at a suitable temperature. A primer is preferably a single-stranded oligodeoxyribonucleotide. The appropriate length of a primer depends on the intended use of the primer but typically ranges from 15 to 25 nucleotides. Short primer molecules generally require cooler temperatures to form sufficiently stable hybrid complexes with the template. A primer need not reflect the exact sequence of the template but must be sufficiently complementary to hybridize with a template.
[0021] The term “PCR primers” as used herein, refers to a pair of PCR primers whose sequences are complementary to DNA subsequences immediately flanking the DNA subsequence (target sequence) which it is desired to amplify. The primers are chosen to specifically bind those particular flanking subsequences and no other sequences present in the sample. The PCR primers are thus chosen to amplify the unique target sequence and no other.
[0022] The term “nucleic acid” as used herein, refers to a deoxyribonucleotide or ribonucleotide polymer in either single- or double-stranded form, and unless otherwise limited, would encompass known analogs of natural nucleotides that can function in a similar manner as naturally occurring nucleotides.
[0023] The terms “hybridize” or “hybridizing” as used herein, refer to the binding of two single stranded nucleic acids via complementary base pairing.
[0024] The terms “amplifying” or “amplification” as used herein, refers to an “exponential” increase in target nucleic acid, are being used herein to describe both linear and exponential increases in the numbers of a select target sequence of nucleic acid.
[0025] RAPD methods are based on polymerase chain reaction (PCR) techniques. DNA fragments are amplified by PCR, based on the presence of short inverted DNA repeats. RAPD markers are typically synthesized by PCR from 10-base oligonucleotide primers of arbitrary sequence. Any short sequence of 10 nucleotides is likely to find many complementary sites in a eukaryotic genome. When two sites are nearby and in inverted orientation, PCR can amplify the DNA between the sites; this DNA fragment is identified by its size which is determined by the distance between the short inverted repeats.
[0026] The present invention is not to be limited in scope by the specific embodiments described herein, which are intended for the purposes of exemplification only
[0027] Functionally-equivalent processes and methods are clearly within the scope of the invention, as described herein.
BRIEF DESCRIPTION OF SEQUENCES
[0028] SEQ ID NO: 1 shows a DNA fragment specific to a male Casuarina junghuhniana plant.
[0029] SEQ ID NO: 2 shows a DNA fragment that specific to a female Casuarina junghuhniana plant.
[0030] SEQ ID NO: 3 shows the oligonucleotide sequence of forward primer.
[0031] SEQ ID NO: 4 shows the oligonucleotide sequence of the reverse primer.
[0032] In an embodiment of the present disclosure, there is provided a process of determining the sex of a Casuarina junghuhniana plant, said process comprising: (a) providing a forward DNA primer having a polynucleotide sequence as set forth in SEQ ID NO: 3 and a reverse DNA prime having a polynucleotide sequence as set forth in SEQ ID NO: 4; (b) carrying out a PCR amplification reaction with a DNA sample from Casuarina junghuhniana; and (c) determining the size of the amplicon product from said PCR amplification reaction, wherein a 1191bp band is indicative that the Casuarina junghuhniana plant is male.
[0033] In an embodiment of the present disclosure, there is provided a process of determining the sex of a Casuarina junghuhniana plant, said process comprising: (a) providing a forward DNA primer having a polynucleotide sequence as set forth in SEQ ID NO: 3 and a reverse DNA prime having a polynucleotide sequence as set forth in SEQ ID NO: 4; (b) carrying out a PCR amplification reaction with a DNA sample from Casuarina junghuhniana; and (c) determining the size of the amplicon product from said PCR amplification reaction, wherein a 971bp band is indicative that the Casuarina junghuhniana plant is female.
[0034] In an embodiment of the present disclosure, the polynucleotide sequence of the DNA fragment having length of 1191bp is as set forth in SEQ ID NO: 1.
[0035] In an embodiment of the present disclosure, the DNA fragment having polynucleotide sequence as set forth in SEQ ID NO: 1 is specific to male Casuarina junghuhniana plant.
[0036] In an embodiment of the present disclosure, the polynucleotide sequence of the DNA fragment having length of 971bp is as set forth in SEQ ID NO: 2.
[0037] In an embodiment of the present disclosure, the DNA fragment having polynucleotide sequence as set forth in SEQ ID NO: 2 is specific to female Casuarina junghuhniana plant.
[0038] In an embodiment of the present disclosure, the PCR amplification reaction is carried out using a thermostable DNA polymerase enzyme.
[0039] In an embodiment of the present disclosure, the RAPD PCR is carried out using one or more single stranded Oligonucleotide primers, such as known decamers.
[0040] In an embodiment of the present disclosure, separation of products of PCR amplification reaction is carried out using conventional electrophoresis methods using agarose gel, polyacrylamide gel, and mixtures thereof.
[0041] In an embodiment of the present disclosure, there is provided a DNA forward primer having polynucleotide sequence as set forth in SEQ ID NO: 3.
[0042] In an embodiment of the present disclosure, there is provided a DNA reverse primer having polynucleotide sequence as set forth in SEQ ID NO: 4.
[0043] In an embodiment of the present disclosure, there is provided a DNA forward primer and a DNA reverse primer for determination of sex of a Casuarina junghuhniana plant.
[0044] The process of sex determination as described herein is non-destructive, amenable for large-scale application in hybridization orchards.
[0045] Although the subject matter has been described in considerable detail with reference to certain preferred embodiments thereof, other embodiments are possible.

EXAMPLES
[0046] The disclosure will now be illustrated with working examples, which is intended to illustrate the working of disclosure and not intended to take restrictively to imply any limitations on the scope of the present disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this disclosure belongs.
Example 1
Identification of sex specific DNA fragments in Casuarina junghuhniana
[0047] DNA was isolated from 50 male Casuarina junghuhniana trees and 45 female Casuarina junghuhniana trees using Qiagen mini plant DNA isolation kit (Qiagen) as per manufacturer’s instructions. The isolated DNA was quantified by agarose gel electrophoresis using control DNA of known quantity. Bulking of DNA samples with DNA from each male or female sample was made to a final concentration of 20ng/µL or 25ng/µL. Thus, five male bulk (MB) and five female bulk (FB) preparations were made. The purpose of making bulk preparations was to reduce the number of samples for random amplified polymorphic DNA (RAPD) analysis and also to avoid the occurrence of specific band due to genotypic differences.
[0048] The RAPD method is based on polymerase chain reaction (PCR) techniques. DNA fragments are amplified by PCR based on the presence of short inverted DNA repeats. RAPD markers are typically synthesized by PCR from 10-base oligonucleotide primers of arbitrary sequence. Any short sequence of 10 nucleotides is likely to find many complementary sites in a genome. When two sites are nearby and in inverted orientation, PCR can amplify the DNA between these sites. This DNA fragment is identified by its size which is determined by the distance between the short inverted repeats. RAPD was conducted on these different bulk samples. PCR amplification was performed in 20µL reaction mixture containing 2µL template DNA (10ng); 0.2µL Taq DNA polymerase (5U); 0.9µL dNTPs (2.5mM of dATP, dCTP, dGTP, and dTTP); 2µL 10X PCR buffer; 2µL Mgcl2 (25mM); 1.5µL random primer (5pmoles); and 11.4µL double distilled water. The amplification was carried out in DNA thermal cycler (Eppendorf) as outlined in Table 1.
Table 1
Number of cycles Step Temp. Time
1 Initial denaturation 95°C 5 minutes
40 Denaturation 95°C 1 minute
Annealing 36°C 1 minute
Extension 72°C 2 minutes
1 Final extension 72°C 8 minutes
The products were separated on a 1.7% agarose gel. The gel was electrophoresed at 100V for 2 hours.
[0049] Figure 1 shows the electropherogram of unique fragments produced by RAPD primers with male bulk and female bulk DNA samples. 41 RAPD primers were screened, out of which, 11 primers were found to be male specific, and 11 primers were found to be female specific.
[0050] Unique sex-specific DNA bands of various sizes as shown in Figure 1 were excised and purified using MN gel extraction kit as per manufacturer’s instructions. PCR products were subsequently ligated in to pJET1.2 vector as per manufacturer’s instructions (MBI, Fermentas, USA) (Catalog #k1214). The ligated mixture was transformed in to E.coli DH5a cells. Plasmids from DH5a were isolated and PCR reaction was carried out to amplify the sex-specific DNA fragments. PCR amplification was performed in 20µL reaction mixture containing 2µL template DNA (25ng); 0.2µL Taq DNA polymerase (5U); 0.6µlL dNTPs (2.5mM of each dATP, dCTP, dGTP, dTTP); 2µL 10X PCR buffer; 0.2µL of pJET1.2 forward primer (10pmole); 0.2µL of pJET1.2 reverse primer (10pmole); and 15.76µL double distilled water. The amplification was carried out in DNA thermal cycler (Eppendorf) as outlined in Table 2.
Table 2
Number of cycles Step Temp. Time
1 Initial denaturation 95°C 5 minutes
35 Denaturation 94°C 30 seconds
Annealing 55°C 30 seconds
Extension 72°C 2 minutes
1 Final extension 72°C 5 minutes
The amplicons cloned in pJET1.2 were sequenced using pJET1.2 primers, at Eurofins, Bangalore.
[0051] Vector comprising the RAPD primer product of interest was sequenced and analyzed. Forward and reverse primers were designed for this RAPD primer in such a way that RAPD site was included at the beginning. This DNA fragment herein after referred as IMC3015 marker was initially screened in Male Bulk, Female Bulk, male individuals and female individuals. A unique band of size 971bp was observed in Female Bulk and a unique band of size 1191bp was observed in Male Bulk with the IMC3015 marker.
[0052] Figure 2 shows the electropherogram showing a unique band of size 971bp in Female Bulk, and a unique band of size 1191bp in Male Bulk.
[0053] The 971bp band specific to female Casuarina junghuhniana plants has a nucleotide sequence as set forth in SEQ ID NO: 2, and can be used for hybridization with DNA from Casuarina junghuhniana of undetermined sex to ascertain the sex of the plant.
[0054] The 1191bp band specific to male Casuarina junghuhniana plants has a nucleotide sequence as set forth in SEQ ID NO: 1, and can be used for hybridization with DNA from Casuarina junghuhniana of undetermined sex to ascertain the sex of the plant.
Example 2
Validation of IMC3015 sex-specific DNA fragment in Casuarina junghuhniana
[0055] The IMC3015 marker was validated in a population containing 20 female and 8 male plants collected from Ongole, Dist. Prakasam, Andhra Pradesh, INDIA. DNA was isolated from needles of known male and female individuals using modified CTAB method. Quality and quantity of DNA was checked using agarose gel electrophoresis (1.0%) and Nanodrop respectively. PCR amplification was performed in 20 µl reaction mixture containing 3µL template DNA (15ng); 0.2µL Taq DNA polymerase (5U); 1.6µL dNTPs (2.5mM of each dATP, dCTP, dGTP, dTTP); 2µL 10X PCR buffer; 1µL Forward primer (5pmole); 1µL Reverse primer (5pmole); and 11.2µL double distilled water. The amplification was carried out in DNA thermal cycler (Eppendorf) as outlined in Table 3.
Table 3
Number of cycles Step Temp. Time
1 Initial denaturation 95°C 5 minutes
35 Denaturation 94°C 30 seconds
Annealing 58°C 30 seconds
Extension 72°C 2 minutes
1 Final extension 72°C 5 minutes
[0056] Figure 3 shows the electropherogram showing the band of size 971 bp in known female population and a band of size 1191 bp in known male population. This confirms that the IMC3015 marker clearly differentiates both male and female plants in a given Casuarina junghuhniana population.