Primer combination of ssr molecular marker of sabina vulgaris and application thereof

Abstract

The application discloses a Sabina SSR molecular marker primer combination and application thereof. The application belongs to the technical field of biology and particularly relates to a Sabina SSR molecular marker primer combination and application thereof. The developed SSR molecular marker primer combination of the Sabina genus comprises at least one of 17 pairs of primers, i.e., SSR-G34, SSR-G46, SSR-G102, SSR-G104, SSR-G108, SSR-G109, SSR-G111, SSR-G124, SSR-G149, SSR-G161, SSR-G164, SSR-G165, SSR-G173, SSR-G193, SSR-G197, SSR-G223 and SSR-G245, and the nucleotide sequences of the 17 pairs of primers are shown in SEQ ID No. 1-SEQ ID No. 34.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biotechnology, and particularly relates to a Sabina SSR molecular marker primer combination and application thereof. BACKGROUND

[0002] Sabina chinensis (Linn.) Ant., also known as "Jun" in ancient times and "Jun Bai" in Beijing and other places, belongs to the plant of Sabina in Cupressaceae and is widely distributed in China and is an important landscaping tree species. Meanwhile, Sabina chinensis is drought-resistant and thin-tolerant and is an important ecological and stress-resistant tree species in China. Sabina chinensis has a long cultivation history in China and has rich germplasm resources. There are more than 6000 Sabina chinensis first-class and second-class ancient trees in Beijing. The collection and preservation of Sabina chinensis germplasm resources in China started in the 1980s, and the evaluation technology of the germplasm resources is still mainly morphological evaluation. The morphological differences between different varieties are identified through the phenotypic traits in the DUS test guide for new plant varieties. With the continuous development of Sabina chinensis germplasm innovation research, new varieties and new germplasms are increasing, and the application and protection of new Sabina chinensis varieties are facing the severe challenge of similar phenotypic traits.

[0003] As the core technology of the rapid test of the DUS test of new plant varieties, DNA molecular detection technology is gradually valued. Microsatellite (Simple Sequence Repeats, SSR) and SNP (single nucleotide polymorphism) are recommended by the UPOV molecular test guide as the preferred markers for variety authenticity identification and database construction. However, there is a lack of microsatellite molecular markers for Sabina plants, and there is no application of efficient and stable SSR molecular markers in the identification of Sabina plant germplasm and the construction of fingerprint atlas. In order to better carry out the breeding of new Sabina varieties, and in order to better serve the protection and protection of the rights and interests of the breeding of new Sabina varieties, it is urgent to develop more efficient, stable and polymorphic specific microsatellite molecular marker primers. SUMMARY

[0004] The main problem to be solved by the application is how to identify and evaluate Sabina variety resources.

[0005] In order to solve the above problems, the application provides a Sabina SSR molecular marker primer combination.

[0006] The application provides a SSR molecular marker primer set of Sabina, which comprises at least one of 17 pairs of primers, i.e., SSR-G34, SSR-G46, SSR-G102, SSR-G104, SSR-G108, SSR-G109, SSR-G111, SSR-G124, SSR-G149, SSR-G161, SSR-G164, SSR-G165, SSR-G173, SSR-G193, SSR-G197, SSR-G223 and SSR-G245, and the nucleotide sequences of the 17 pairs of primers are shown as SEQ ID No.1-SEQ ID No.34.

[0007] Further, in the 17 pairs of primer set, the 6-FAM fluorescent group can be used to modify the 5' end of the forward primer sequence, and 17 pairs of fluorescent SSR primers are synthesized to facilitate identification.

[0008] The application further provides a kit comprising the SSR molecular marker primer set.

[0009] The application further provides a DNA chip comprising the SSR molecular marker primer set.

[0010] The application further provides a screening method of the SSR molecular marker primer set, and the method comprises the following steps:

[0011] B1) taking the transcriptome sequence of Sabina species 'Fengjia' as a reference sequence, detecting the SSR site of a gene sequence (Unigene) and designing primers to obtain an SSR marker;

[0012] B2) screening the diversity of the SSR marker in germplasm resources materials according to B1);

[0013] B3) designing primers for the SSR marker screened in B2) to obtain the SSR molecular marker primer set.

[0014] In the method, the screening standard in B3) is that the single nucleotide, the double nucleotide, the triple nucleotide, the quadruple nucleotide, the quintuple nucleotide and the sextuple nucleotide are repeated for 12 times, 6 times, 5 times, 5 times, 4 times and 4 times, respectively.

[0015] The application further provides a method for analyzing the genetic diversity or the genetic relationship of different Sabina species, which comprises the following steps: taking the genomic DNA of a Sabina species to be tested as a template, performing PCR amplification by using the SSR molecular marker primer set, performing electrophoretic detection on the PCR amplification product, and analyzing the genetic diversity or the genetic relationship of Sabina according to the electrophoretic detection result.

[0016] Further, the genetic diversity analysis or the genetic relationship analysis of Sabina according to the electrophoresis detection result comprises: if a band is recorded as "1" at the same electrophoretic mobility position, and if no band is recorded as "0". Further, cluster analysis is performed to construct a genetic relationship tree diagram, and the genetic diversity analysis or the genetic relationship analysis of Sabina is performed according to the statistical result.

[0017] Further, the genetic diversity is statistically analyzed by using Popgene 32, and the genetic relationship cluster diagram is constructed by using MEGA 11.

[0018] The application further provides a method for fingerprint analysis of Sabina varieties to identify the Sabina varieties, which comprises: taking the genomic DNA of a to-be-tested Sabina variety and a standard Sabina variety as templates respectively, performing PCR amplification by using the SSR molecular marker primer group described above, and performing electrophoresis detection on the PCR amplification product to obtain a to-be-tested Sabina germplasm resource fingerprint and a standard Sabina variety fingerprint, and identifying the Sabina varieties according to the fingerprint comparison and analysis result.

[0019] In the above method, the PCR amplification reaction system (20 μL) comprises: 10 μL of PCR mix, 0.3 μL (20 μM) of a forward primer with a fluorescent label, 0.3 μL (20 μM) of a reverse primer, 2 μL of template DNA, and 7.4 μL of ddH2O.

[0020] The PCR amplification program is as follows: 94 ℃ pre-denaturation for 5 min, 1 cycle; 94 ℃ denaturation for 30 s, 60 ℃ annealing for 40 s, and 72 ℃ extension for 50 s, 10 cycles; 94 ℃ denaturation for 30 s, 53 ℃ annealing for 40 s, and 72 ℃ extension for 50 s, 27 cycles; and finally 72 ℃ extension for 10 min.

[0021] In the above method, the electrophoresis detection is agarose gel electrophoresis detection, polyacrylamide gel electrophoresis detection, or / and capillary electrophoresis detection.

[0022] Further, the SSR molecular marker primer group can be: 9 pairs of SSR primers of SSR-G34, SSR-G46, SSR-G104, SSR-G124, SSR-G149, SSR-G164, SSR-G197, SSR-G223, and SSR-G245.

[0023] The application further provides any one of the following applications of the SSR molecular marker primer group described above, and / or the kit, and / or the DNA chip:

[0024] A1) application in genetic diversity analysis of Sabina;

[0025] A2) Application in the diversity analysis of Juniperus species;

[0026] A3) Application in the analysis of phylogenetic relationships in the genus Juniperus;

[0027] A4) Application in the identification of Juniperus species;

[0028] A5) Application in constructing genetic maps or DNA fingerprint maps of the Juniperus genus;

[0029] A6) Application in the improvement of germplasm resources of Juniperus spp.;

[0030] A7) Application in gene localization or functional gene mining of Juniperus chinensis;

[0031] Application of A8 in molecular marker-assisted breeding of Juniperus chinensis.

[0032] In this article, the juniper varieties mentioned may be: 'Fengjun', 'Rongtuan', 'Lanta', 'Menglinlanbai' and 'Menglinzhongbai', as well as 'Jinhua', 'Yunmi' and 'Diecui'.

[0033] This invention utilizes the transcriptome sequence of the Chinese juniper cultivar 'Fengjun' to develop a set of SSR primers. The SSR primer set developed in this invention has the advantages of stable amplification, high efficiency and polymorphism, and can effectively realize the germplasm identification, differentiation and fingerprinting of Chinese juniper and its closely related species. Attached Figure Description

[0034] Fig. 1 The results of polyacrylamide gel electrophoresis using some of the SSR primers described in this invention are shown.

[0035] Fig. 2 This is a phylogenetic clustering diagram of the genome amplification results of 77 Juniperus chinensis varietal resources using the complete set of SSR primers described in this invention. Detailed Implementation

[0036] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0037] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0038] Unless otherwise specified, the quantitative experiments in the following examples are all repeated three times, and the results are averaged.

[0039] The test materials in the following examples are 8 Sabina species, of which 5 are female: 'Fengjia', 'Rongtuan', 'Lantai', 'Menglin Land Cypress' and 'Menglin Zhongbai', and 3 are male: 'Jinhua', 'Yunmi' and 'Diecui'; the materials come from the Important Tree Species Germplasm Resource Bank (Platform Number Details are shown in Table 1) of Beijing Academy of Agriculture and Forestry Sciences, and are also included in the National Forestry and Grassland Germplasm Resource Bank (https: / / www.nfgrp.cn / ). The public can obtain the biological material from the National Forestry and Grassland Germplasm Resource Bank or the applicant, and the biological material is only used for repeating the experiments of the present application and cannot be used for other purposes.

[0040] Table 1, information of test germplasm resources

[0041]

[0042]

[0043] Table 2, information of test 9 related species germplasm resources

[0044]

[0045]

[0046] Example 1, development and screening method of Sabina SSR primer set

[0047] 1. Primer development and design method

[0048] According to the transcriptome sequencing data of the needle leaves, female flowers and cones of the Sabina species 'Fengjia', the Unigene was detected by using MISA (Microsatellite) software, and the detection standard was set as follows: mononucleotide, dinucleotide, trinucleotide, tetranucleotide, pentanucleotide and hexanucleotide, and the minimum number of repeats of each nucleotide was 12, 6, 5, 5, 4 and 4 respectively. Then, the detected SSR primers were designed by using Primer 3.0.

[0049] 2. Polymorphic primer screening method

[0050] The test materials are 8 Sabina species, of which 5 are female: 'Fengjia', 'Rongtuan', 'Lantai', 'Menglin Land Cypress' and 'Menglin Zhongbai', and 3 are male: 'Jinhua', 'Yunmi' and 'Diecui' (Table 1).

[0051] 1) DNA extraction and detection

[0052] The Sabina needle leaf DNA was extracted by using a plant genomic DNA extraction kit (Tiangen Biochemical Technology (Beijing) Co., Ltd., DP305) according to the instruction steps, and the DNA purity was detected by using 2.0% agarose gel electrophoresis.

[0053] 2) PCR amplification

[0054] According to the primer design results of step 1, 250 pairs of primers were synthesized for polymorphic primer screening, mainly dinucleotides and trinucleotides.

[0055] The reaction system used for primer screening was 20 μL, including PCR mix 10 μL, forward primer 0.3 μL (20 μM), reverse primer 0.3 μL (20 μM), template DNA 2 μL, and ddH2O 7.4 μL.

[0056] The amplification program used for primer screening was: 94°C pre-denaturation for 5 min, 1 cycle; 94°C denaturation for 30 s, 60°C annealing for 40 s, 72°C extension for 50 s, 10 cycles; 94°C denaturation for 30 s, 53°C annealing for 40 s, 72°C extension for 50 s, 27 cycles; final 72°C extension for 10 min.

[0057] 3) Screening of polymorphic SSR primers by triple detection method

[0058] Screening was performed by agarose gel electrophoresis detection, polyacrylamide gel electrophoresis detection, and capillary electrophoresis detection, respectively.

[0059] Agarose gel electrophoresis detection was used to detect the PCR amplification products of 8 samples of Sabina chinensis, and primers with target genes were initially selected.

[0060] Polyacrylamide gel electrophoresis detection was used to detect the PCR amplification products of 8 samples of Sabina chinensis on the basis of initial selection, and primers containing clear target bands and polymorphisms were reselected.

[0061] Capillary electrophoresis detection was used to further detect the PCR amplification products of 8 samples of Sabina chinensis on the basis of reselection, and high-efficiency primers with rich loci, clear peak type, and stable amplification were screened out.

[0062] 3. Obtaining of polymorphic primers

[0063] According to the primer development and screening methods of steps 1 and 2, a total of 86 resources of Sabina chinensis and its relatives (Y1-Y77 in Table 1; B02-B11 in Table 2) were subjected to polymorphism analysis, and a set of high-efficiency microsatellite molecular marker primers based on the transcriptome sequence of Sabina chinensis was finally obtained, totaling 17 pairs (Table 3). As shown in Table 4, the total band number amplified by the 17 pairs of SSR primers obtained according to steps 1 and 2 was between 3 and 22, the polymorphic band number was between 3 and 22, and the polymorphism percentage of each pair of primers reached 100%.

[0064] Table 3, 17 pairs of juniper SSR marker primer group information

[0065]

[0066] Table 4, SSR primer pair 86 pairs of juniper and its related species resources amplified polymorphism results

[0067]

[0068]

[0069] Example 2, a method for identifying juniper germplasm using SSR marker primers

[0070] Using the juniper SSR primers screened in Example 1, numbered SSR-G34, SSR-G46, SSR-G102, SSR-G104, SSR-G108, SSR-G109, SSR-G111, SSR-G124, SSR-G149, SSR-G161, SSR-G164, SSR-G165, SSR-G173, SSR-G193, SSR-G197, SSR-G223, SSR-G245, fluorescence primer capillary electrophoresis method was used to amplify 77 pairs of juniper germplasm resources collected and preserved in the national forestry and grassland germplasm resource library (https: / / www.nfgrp.cn / , see Table 1 for details). The size of the fluorescence PCR product was detected by capillary electrophoresis, the genetic diversity was statistically analyzed by Popgene 32, and the genetic relationship clustering diagram was constructed by MEGA 11.

[0071] The specific steps are as follows:

[0072] 1. DNA extraction and detection

[0073] The genomic DNA of the needle leaves of the juniper samples to be tested was extracted using a DNA extraction kit. The DNA purity and integrity were detected by 2.0% agarose gel electrophoresis.

[0074] 2. Fluorescent primer synthesis

[0075] The forward primer sequence of the 17 pairs of juniper SSR marker primers developed in Example 1 was modified at the 5' end with a 6-FAM fluorescent group, and 17 pairs of fluorescent SSR primers were synthesized.

[0076] 3. Microsatellite fluorescence primer PCR amplification

[0077] PCR amplification reaction system was 20 μL, including PCR mix 10 μL, forward primer with fluorescent label 0.3 μL (20 μM), reverse primer 0.3 μL (20 μM), template DNA 2 μL, ddH2O 7.4 μL.

[0078] PCR amplification procedure was: 94 ℃ pre-denaturation 5 min, 1 cycle; 94 ℃ denaturation 30 s, 60 ℃ annealing 40 s, 72 ℃ extension 50 s, 10 cycles; 94 ℃ denaturation 30 s, 53 ℃ annealing 40 s, 72 ℃ extension 50 s, 27 cycles; final 72 ℃ extension 10 min.

[0079] 4. Capillary fluorescence electrophoresis detection

[0080] After mixing formamide and molecular weight marker in a volume ratio of 100:1, 9 μL was taken and added to the loading plate, and then 1 μL of 10-fold diluted PCR product obtained in step 3 was added. Then capillary electrophoresis was carried out by using ABI 3730XL full-automatic DNA sequencer, and the raw data obtained by the sequencer were analyzed by using Fragment (Plant) fragment analysis software in Genemarker, and the positions of the molecular weight markers in each lane were compared with the positions of the peak values of each sample to obtain the fragment size.

[0081] 5. Germplasm resource identification

[0082] Format conversion was carried out by using Convert 1.31 software, genetic identity and genetic distance were statistically analyzed by using Popgene32 software, UPGMA method was used for germplasm resource clustering analysis, and a phylogenetic tree was constructed.

[0083] According to the clustering results of 77 Jumperia germplasm resources (see Table 1 for details) by using 17 pairs of SSR primers with fluorescent labels, Fig. 2 ) except Y32, Y46, Y32, Y48, Y49 and Y53, Y17 and Y33, Y16 and Y25, the remaining 67 germplasms can be completely distinguished and identified; among the 10 germplasms that cannot be completely distinguished, Y32, Y46, Y32, Y48, Y49 and Y53 belong to the same clone, Y17 and Y33 belong to the same clone, and Y16 and Y25 belong to the same clone. Further prove the reliability of the developed SSR primer in the aspect of germplasm identification.

[0084] Example 3, application of the SSR marker primer set in the construction of DNA molecular identity card of Jumperia approved variety

[0085] Using the 17 pairs of juniper SSR primer groups screened out in Example 1, namely SSR-G34, SSR-G46, SSR-G102, SSR-G104, SSR-G108, SSR-G109, SSR-G111, SSR-G124, SSR-G149, SSR-G161, SSR-G164, SSR-G165, SSR-G173, SSR-G193, SSR-G197, SSR-G223, and SSR-G245, the 4 local juniper varieties, 'Fengjia', 'Jinhua', 'Rongtuan', and 'Lantai', collected and preserved in the National Forestry and Grassland Germplasm Bank, were amplified by using the fluorescent primer capillary electrophoresis method. The size of the fluorescent PCR product was detected by using capillary electrophoresis.

[0086] 1. DNA extraction and detection

[0087] The genomic DNA of the four juniper needle leaves to be tested was extracted by using a DNA extraction kit. The DNA purity and integrity were detected by using 2.0% agarose gel electrophoresis.

[0088] 2. Synthesis of fluorescent primers

[0089] The 5' end of the forward primer sequence of the above-mentioned 17 pairs of juniper SSR marker primers was modified by using a 6-FAM fluorescent group to synthesize 17 pairs of fluorescent SSR primers.

[0090] 3. Microsatellite fluorescent primer PCR amplification

[0091] The PCR amplification reaction system was 20 μL, including PCR mix 10 μL, forward primer with fluorescent label 0.3 μL (20 μM), reverse primer 0.3 μL (20 μM), template DNA 2 μL, and ddH2O 7.4 μL.

[0092] The PCR amplification program was as follows: 94℃ pre-denaturation for 5 min, 1 cycle; 94℃ denaturation for 30 s, 60℃ annealing for 40 s, 72℃ extension for 50 s, 10 cycles; 94℃ denaturation for 30 s, 53℃ annealing for 40 s, 72℃ extension for 50 s, 27 cycles; and finally 72℃ extension for 10 min.

[0093] 4. Capillary fluorescent electrophoresis detection

[0094] Mix formamide and molecular weight marker in the volume ratio of 100:1, then take 9 μL and add into the loading plate, and then add 1 μL of 10-fold diluted PCR product obtained in step 3. Then use ABI 3730XL automatic DNA sequencer for capillary electrophoresis, and use Fragment (Plant) fragment analysis software in Genemarker to analyze the raw data obtained by the sequencer, compare the position of the molecular weight marker in each lane with the position of the peak value of each sample, and obtain the fragment size.

[0095] 5. DNA molecular identity card construction

[0096] Statistical 17 pairs of SSR primer pairs respectively in four Juniperus formosana breeding on the amplification of fragment size, the size of each material in the SSR site amplification fragment is arranged in order from small to large, and the amplification of different varieties of resources at the site is counted, with "1" and "0" without.

[0097] The number of amplified bands of 17 pairs of SSR primers in four Juniperus formosana breeding varieties ranged from 1 to 4, suggesting that the test sample was a polyploid. From the amplification results (Table 5), it can be seen that among the selected 17 pairs of SSR primers, any one of SSR-G34, SSR-G46, SSR-G104, SSR-G124, SSR-G149, SSR-G164, SSR-G197, SSR-G223, and SSR-G245 can identify and distinguish the four breeding varieties.

[0098] Table 5, 17 pairs of SSR primers in four Juniperus formosana breeding varieties

[0099]

[0100]

[0101] Further, using 9 pairs of SSR primers SSR-G34, SSR-G46, SSR-G104, SSR-G124, SSR-G149, SSR-G164, SSR-G197, SSR-G223, and SSR-G245, a DNA molecular identity card composed of 42 codes for four Juniperus formosana breeding varieties was constructed according to the 0,1 method (Table 6 and Table 7).

[0102] Table 6, 9 pairs of SSR primers in four Juniperus formosana breeding varieties and corresponding codes

[0103]

[0104]

[0105] Table 7, DNA molecular identity card of four Juniperus formosana breeding varieties

[0106] Improved Sabina vulgaris DNA molecular identity coding ‘Golden Flower Sabina’ 110011001110111000100011101110110100110011 ‘Fluffy Ball Sabina’ 001010010101000100011100110011010111111000 ‘Blue Tower Sabina’ 010010111000010011010001010111111100110100 ‘Peak Sabina’ 011110000110111010110010110110000111100010

[0107] The application has been described in detail. Those skilled in the art will understand that they can make modifications and alterations to this application without departing from the spirit and scope of the application. Although this application presents specific examples, it is to be understood that further modifications can be made. In general, the application is intended to cover any adaptations or variations of the present application including modifications based on the disclosure in the patent application as filed, which are apparent to those skilled in the art to which the present application pertains.

Claims

1. A set of SSR molecular marker primers for the genus Sabina, characterized in that, The SSR molecular marker primer set comprises 17 pairs of primers numbered SSR-G34, SSR-G46, SSR-G102, SSR-G104, SSR-G108, SSR-G109, SSR-G111, SSR-G124, SSR-G149, SSR-G161, SSR-G164, SSR-G165, SSR-G173, SSR-G193, SSR-G197, SSR-G223 and SSR-G245, and the nucleotide sequences of the 17 pairs of primers are shown as SEQ ID No. 1-SEQ ID No.

34.

2. A kit characterized in that, The kit comprises the SSR molecular marker primer set of claim 1.

3. A DNA chip characterized by The DNA chip comprises the SSR molecular marker primer set of claim 1.

4. A method for genetic diversity analysis or genetic relationship analysis of Sabina germplasm resources, the method comprising: The genomic DNA of the Sabina vulgaris germplasm resource to be detected is used as a template, and the SSR molecular marker primer set of claim 1 is used for PCR amplification to obtain PCR amplification products, and the PCR amplification products are subjected to electrophoretic detection, and the genetic diversity of the Sabina vulgaris germplasm resource is analyzed or the genetic relationship is analyzed according to the electrophoretic detection results.

5. A method of performing a fingerprint analysis of a Sabina variety to identify a Sabina variety, the method comprising: The genomic DNA of the Sabina vulgaris variety to be detected and the genomic DNA of the standard Sabina variety are used as templates, respectively, and the SSR molecular marker primer set of claim 1 is used for PCR amplification, and the PCR amplification products are subjected to electrophoretic detection to obtain the fingerprint spectrum of the Sabina vulgaris germplasm resource to be detected and the fingerprint spectrum of the standard Sabina variety, and the variety is identified according to the comparison and analysis results of the fingerprint spectrum.

6. The method according to claim 4 or 5, characterized in that, The PCR amplification reaction system is 20 μL: 10 μL of PCRmix, 0.3 μL of 20 μM forward primer with a fluorescent label, 0.3 μL of 20 μM reverse primer, 2 μL of template DNA, and 7.4 μL of ddH2O; The PCR amplification program is: 94℃ pre-denaturation for 5 min, 1 cycle; 94℃ denaturation for 30 s, 60℃ annealing for 40 s, and 72℃ extension for 50 s, 10 cycles; 94℃ denaturation for 30 s, 53℃ annealing for 40 s, and 72℃ extension for 50 s, 27 cycles; and finally 72℃ extension for 10 min.

7. The method of any of claims 4 or 5, wherein, The electrophoretic detection is agarose gel electrophoresis detection, polyacrylamide gel electrophoresis detection, or / and capillary electrophoresis detection.

8. The SSR molecular marker primer set of claim 1, and / or the kit of claim 2, and / or the DNA chip of claim 3 is used in the following applications: A1) in the genetic diversity analysis of Sabina; A2) in the genetic diversity analysis of Sabina variety resources; A3) in the genetic relationship analysis of Sabina; A4) in the variety identification of Sabina; A5) in the construction of a genetic map or a DNA fingerprint spectrum of Sabina; A6) in the improvement of Sabina germplasm resources; A7) in the gene positioning or functional gene mining of Sabina; A8) in the molecular marker assisted breeding of Sabina.

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