Identification method for Rubus interspecific hybrid germplasm and primers used for identification

Abstract

The present invention discloses a method for identifying interspecific hybrid germplasm of Rubus rubus, comprising the following steps: using hexaploid cultivated blackberry as the female parent and diploid wild Rubus rubus as the male parent, obtaining hybrid seeds, and cultivating interspecific hybrid seedlings; designing male parent SSR primers, amplifying the DNA of the interspecific hybrid seedlings, and screening primer combinations to identify true hybrids; verifying the amplified bands of the screened SSR primer pairs in wild Rubus rubus, raspberry, and blackberry varieties, and optimizing specific primers for identifying hybrid offspring of different wild Rubus male parents. The screened SSR primers are used to identify blackberry cultivars and interspecific hybrid seedlings of Rubus rubus, and the true hybrids are identified and the parental germplasm sources are confirmed based on the amplified bands. The present invention successfully hybridizes diploid wild Rubus rubus with hexaploid cultivated blackberry varieties for the first time, uses the SSR molecular markers obtained through screening to identify hybrids, and verifies that the wild germplasm has entered the cultivated varieties through hybridization.

Description

Technical Field

[0001] The invention belongs to the technical field of plant breeding, and relates to a method and identification technology for introducing wild Rubus germplasm into cultivated varieties to create new germplasm. Background Art

[0002] Blackberry, a small berry native to Europe and the United States, has made significant contributions to agricultural and industrial development in low-lying hilly areas since its introduction to China in 1986. In recent years, the sustainable development of the blackberry industry has been plagued by a series of challenges, including delayed ripening, susceptibility to plum rains, aging varieties, subpar quality, and poor adaptability. Furthermore, intellectual property barriers hinder the targeted introduction of superior varieties. Therefore, it is urgent to utilize existing varieties and wild Rubus germplasm to develop early-maturing blackberry varieties with outstanding fresh food quality. The complex genetic backgrounds of different Rubus species not only result in low hybrid compatibility between germplasms of different ploidy, but also present significant phenotypic variation in offspring, making it difficult to determine hybrid germplasm based on phenotype, thus limiting hybrid breeding efforts.

[0003] Extensive hybrid breeding practices are crucial for creating new blackberry germplasm. In recent years, there has been an urgent need to identify parents with high hybrid affinity and methods for their hybridization. In recent years, double-season blackberries, based on the biennial blackberry fruiting pattern, have been introduced to the market, with earlier flowering and extremely ripe fruit (around May 20th). While these varieties offer significant advantages in terms of high yield and early maturity, their fruit has a rather sour taste and their edible quality needs urgent improvement. By exploring cross-ploidy hybridization techniques and screening hybrid germplasm, it is hoped that early-maturing or very early-maturing blackberry varieties with outstanding fresh fruit and nutritional qualities will be bred.

[0004] It has become possible to quickly create new interspecific hybrid germplasm of the genus Rubus through artificial hybridization and molecular marker-assisted identification methods. However, there are currently no reports of successfully hybridizing wild Rubus germplasm with cultivated varieties to achieve the integration of the traits of both parents. Summary of the Invention

[0005] The purpose of the present invention is to overcome the problems of hybrid incompatibility and lack of identification technology of Rubus rubus germplasms with different ploidy in the prior art, and to provide a technical method for successfully obtaining Rubus rubus interspecific hybrid germplasm through hybrid cultivation and identification confirmation.

[0006] In order to achieve the above object, the technical solution adopted by the present invention is as follows:

[0007] A method for identifying Rubus interspecific hybrid germplasm comprises the following steps:

[0008] (1) Obtaining hybrid seeds: using hexaploid cultivated blackberry as the female parent and diploid wild Rubus rubus as the male parent to obtain seeds through artificial hybridization; cultivating the seeds to obtain interspecific hybrid seedlings;

[0009] (2) Primer design: Young leaves of the male parent were collected for transcriptome sequencing, and several pairs of SSR primers were designed;

[0010] (3) Primer screening and germplasm identification: The SSR primers designed in step (2) are used to verify and screen all interspecific hybrid seedlings obtained in step (1), and true hybrids that have been successfully hybridized are identified based on the amplified band patterns, and specific SSR primers that can identify true hybrids are screened;

[0011] (4) Amplification verification: The SSR primers screened in step (3) are further amplified and verified in other wild Rubus, raspberry, and blackberry varieties to obtain primers that can distinguish the hybrid offspring of different wild Rubus parents;

[0012] (5) Hybrid germplasm identification: The SSR primers further screened in step (4) are used to perform molecular marker identification on interspecific hybrid seedlings. The ones that can amplify the bands are true hybrids.

[0013] The present invention successfully obtained hybrid germplasm by hybridizing hexaploid cultivated blackberry as the female parent and diploid wild Rubus rubus as the male parent, proving the feasibility of hybrid identification using SSR molecular markers. Further screening of SSR primers can verify that wild germplasm has hybridized into cultivated varieties, making the identification of true hybrids more accurate and easier to perform.

[0014] In some embodiments, the hexaploid cultivated blackberry is preferably selected from the blackberry variety 'Double Season Blackberry', and the diploid wild Rubus is selected from 'Pengsu'. By selecting the hexaploid cultivated blackberry and the diploid wild Rubus varieties, hybrid germplasm is successfully obtained.

[0015] In some embodiments, as preferred, the wild Rubus rubus used for amplification verification in step (4) is 'Pengsu', 'Raspberry', 'Raspberry palm', the raspberry varieties are yellow raspberry, red raspberry, black raspberry, and the blackberry varieties are 'Kiowa', 'Boysen', 'Hull', 'Arapaho', 'Navaho', 'Zaohei'.

[0016] The present invention further screens out primers that can distinguish hybrid offspring from different wild Rubus parental sources by amplifying and comparing the screened SSR primers with multiple varieties of wild Rubus, raspberry, and blackberry, thereby further improving the accuracy of identifying interspecific hybrid germplasm of the Rubus genus.

[0017] Based on the above identification method, the present invention also provides a set of SSR primers Rh-11, Rh-20, Rh-21, and Rh-34 for identifying true hybrids between Rubus species. The forward primer of Rh-11 is 5'-CGGTGCCTCTCTCTCTCTCT-3', and the reverse primer of Rh-11 is 5'-CGGAACCAAACCCATCATCG-3'; the forward primer of Rh-20 is 5'-AAAGACCCAGCTATGTGCCC-3', and the reverse primer of Rh-20 is 5'- The forward primer for Rh-21 is 5'-AATCCAGCTCATGTGCCTCA-3', and the reverse primer for Rh-21 is 5'-TCTGTCGGACTGCAGACAAC-3'; the forward primer for Rh-34 is 5'-CTCTCTCCTCTGCCCCTCTT-3', and the reverse primer for Rh-34 is 5'-CTCTTATCCTCGCCGTCGAC-3'. These four primers can be used to amplify codominant polymorphic SSRs that have been successfully hybridized between Rubus germplasm of different ploidy.

[0018] The present invention also provides SSR primers Rh-2, Rh-3, Rh-4 and Rh-16 for identifying interspecific hybrid germplasm of Rubus rubus, wherein the forward primer of Rh-2 is 5'-AAGCGCGACAACGAAAAGAC-3', and the reverse primer of Rh-2 is 5'-ACAAGTAAAGCATGCAACAGGT-3'; the forward primer of Rh-3 is 5'-TGGCACACAGTCACACAGAT-3', and the reverse primer of Rh-3 is 5'-T The forward primer for Rh-4 is 5'-CTCACACCCGTCTGCTTCAT-3', and the reverse primer for Rh-4 is 5'-ATTTCTGTGGTGCTCTCGCA-3'; the forward primer for Rh-16 is 5'-ACACAAACGACTTGGCTTGTG-3', and the reverse primer for Rh-16 is 5'-GACTCTTGGCCCTAGTGCTG-3'. These four primers can be used as SSR primers to distinguish wild Rubus from blackberry and raspberry germplasm.

[0019] Compared with the prior art, the present invention has the following advantages:

[0020] The present invention overcomes the previous inability to successfully obtain hybrid varieties of wild Rubus germplasm. It is the first time to successfully explore the hybridization of wild Rubus and cultivated blackberry varieties to obtain hybrid germplasm. The SSR molecular markers obtained by screening are used to identify hybrids to obtain interspecific hybrid germplasm, and the molecular markers are used to verify that the wild germplasm has entered the cultivated variety through hybridization.

[0021] Although the existing interspecific hybrid germplasm has the morphology of cultivated blackberry and wild Rubus, it is difficult to determine whether it is a true hybrid. The molecular markers of the present invention can accurately and easily identify the presence of wild Rubus germplasm components.

[0022] The method for obtaining and identifying Rubus hybrids of the present invention is of great significance for cultivating blackberry germplasm types with independent intellectual property rights by utilizing wild resources in my country. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 The process of hybridization between wild Rubus 'Pengsu' and blackberry;

[0024] In the figure, A: Flowers of the naturally distributed wild Rubus 'Pengsu'; B: Pollen collected from 'Pengsu' for hybridization; C: Fruiting after successful hybridization of 'Pengsu' and blackberry; D: Morphology of mature fruit after hybridization;

[0025] Figure 2 The process of tending from sowing to seedling formation of interspecific hybrid seeds of Rubus spp.

[0026] In the figure, A: Seedlings 40 days after sowing; B: Seedlings 60 days after sowing; C: Seedlings growing in plug trays 5 months after sowing; D: Seedlings transplanted to potting medium 5 months after sowing;

[0027] Figure 3 The leaf morphology of the seedlings of Double-Season Blackberry (A), 'Pengsu' (B), and their hybrid offspring 2-17 (C) and 2-55 (D);

[0028] Figure 4 The amplification results of four primer pairs that can identify interspecific hybrids of Rubus chinensis (A) and four primer pairs that only amplified bands in 'Pengpi' and true hybrids (B);

[0029] In the figure, A and B: M, DL2000 DNA Marker, lanes 1-7 amplified the genomic DNA of the female parent 'Shuangji Heimei', the male parent 'Pengsu', and the hybrid plants 2-17, 2-23, 2-33, 2-55, and 2-62, respectively;

[0030] Figure 5 Four pairs of primers that amplified bands only in 'Pengsu' and true hybrids were used to amplify bands in other raspberry and blackberry varieties;

[0031] In the figure, M, DL2000 DNA Marker, channels 1-12 respectively amplify the wild Rubus 'Pengsu', 'Raspberry', 'Raspberry Palm', yellow raspberry, red raspberry, black raspberry, blackberry varieties 'Kiowa', 'Boysen', 'Hull', 'Arapaho', 'Navaho', and 'Chester'.

[0032] Figure 6 These are the ploidy identification results of the female parent 'Double Season Blackberry', the male parent 'Pengsu', and the true hybrid strains 2-17 and 2-55. DETAILED DESCRIPTION

[0033] Unless otherwise specified, the terms used in the present invention generally have the meanings commonly understood by those skilled in the art.

[0034] The present invention will be further described in detail below with reference to specific preparation examples and application examples, and with reference to data. It should be understood that these examples are only for the purpose of illustrating the present invention and are not intended to limit the scope of the present invention in any way.

[0035] In the following examples, various processes and methods not described in detail are conventional methods known in the art. The primers used are indicated when they first appear, and the same primers used thereafter are the same as those first indicated.

[0036] Unless otherwise specified, the methods used in the following examples are all conventional methods.

[0037] Example 1 Artificial hybridization of blackberry and wild Rubus rubus and production of hybrid seeds

[0038] The blackberry variety 'Double Season Blackberry' is cultivated at the Lishui Baima Scientific Base of the Institute of Botany, Chinese Academy of Sciences, Jiangsu Province, under conventional field management. The wild Rubus 'Pengsu' is preserved in the wild at the Nanjing Zhongshan Botanical Garden.

[0039] In early April 2020, healthy plants and disease-free branches were selected. The petals of the double-season blackberry flowers that had not yet opened but had petals were opened were opened, and all anthers were carefully removed. The young flower buds, opened flowers and fruits on the entire branch were removed. Then, waterproof sulfuric acid paper bags were placed and fixed with paper clips to complete the detasseling and bagging. For wild Rubus 'Pengsu' growing in natural conditions, the unopened flower buds ( Figure 1 A), take it to the laboratory and use tweezers to remove the anthers, and spread them flat on a petri dish covered with sulfuric acid paper ( Figure 1 B), and then place it at room temperature at about 25℃ for 3 days. On the 4th day after bagging, collect the pollen into a 10ml centrifuge tube, and gently grind the pollen with one end of a cotton swab to make it fully dispersed. Open the fixed sulfuric acid paper bag, and you will find that the pistil has become bright green, shiny and vibrant. Use a soft brush to dip the pollen and brush it on the pistil. Put the sulfuric acid bag back on the pollinated flower branch, fix it, and mark the name and date of the hybrid combination. After 10 days, open the sulfuric acid paper bag once, remove the newly grown young inflorescence, and the formation of small drupes means that the pollination has been successful ( Figure 1 C) After the fruit has expanded, tear open the sulfuric acid paper bag from one side to allow the fruit to have sufficient ventilation and sunlight.

[0040] In late May, the ripe fruits are harvested ( Figure 1 D) 50 bagged drupes were harvested, yielding a total of 4.64g of drupes. The seeds were rinsed with running water and placed in a well-ventilated, cool place for 2 weeks, allowing them to air-dry. A total of 118 seeds weighing 0.318g were obtained. The harvested seeds were placed in sulfuric acid paper bags and sealed in ziplock bags. They were then refrigerated and stored at 4°C starting in late June.

[0041] Example 2 Hybrid seed sowing and hybrid seedling cultivation

[0042] Take out the seeds that have been refrigerated for 5 months, place them in a dry small beaker, treat them with 98% concentrated sulfuric acid for 2 to 5 minutes, and then rinse them with running water. Mix the seeds with moist fine sand, place them in a plastic ziplock bag, and bury them in fine sand at room temperature for 2 to 2.5 months. During this period, pay attention to replenishing water to the fine sand and checking the germination of the seeds. In early February 2020, the harvested interspecific hybrid seeds were sown in a hole tray filled with a substrate (peat: bark: vermiculite = 1:1:1) and placed in a greenhouse environment at about 20°C for germination. Seedlings began to emerge after two weeks, and the number of seedlings was 78 when the emergence was stable at 40 days. Figure 2 A), the emergence rate reached 66.1%.

[0043] 60 days after sowing, the seedlings were transferred outdoors to continue growing. The leaves initially showed light yellow ( Figure 2 B). In late April, the hybrid seedlings were transplanted into the plug trays. The growth was significantly accelerated. Five months after sowing, the seedlings were 15 to 20 cm tall. Figure 2 C). In early June, transplant the hybrid seedlings into a large pot with a diameter of 20 cm and a height of 30 cm and continue to cultivate them in the medium ( Figure 2 D) Apply fertilizer and water once a week according to normal management.

[0044] Example 3 Molecular marker identification of Rubus interspecific hybrid seedlings

[0045] After 2 months of cultivation in large pots of substrate, 73 seedlings were formed. The leaf morphology of different plants was observed. Among the 73 hybrid offspring (named 2-1, 2-2, 2-3, ..., 2-73), the hybrid plants 2-17, 2-23, 2-33, and 2-55 showed the characteristics of blackberry leaves in leaf shape, while the leaf morphology of the remaining 69 plants tended to be wild or intermediate. The leaf morphology of the plants 2-17 and 2-55, which were later identified as true hybrids, and their parents is shown in Figure 3 In terms of appearance, the leaves of the two individual plants are longer and narrower than those of the mother plant, and their leaf width is narrower. The leaf color is significantly lighter than that of the mother plant, and they have some similarities with the father plant, 'Pengsu'.

[0046] Young leaves from interspecific hybrid seedlings grown in large pots for two months were collected and genomic DNA was extracted using a Beijing Biotech spin-column plant genomic DNA extraction kit. PCR amplification was performed using a 15 μL reaction system consisting of 7.5 μL 2× GS Taq PCR mix, 1 μL DNA, 1 μL 5 pmol / μL primers, and 5.5 μL ddH₂O. The PCR protocol was as follows: 95°C denaturation for 5 min, 95°C denaturation for 20 s, 59°C annealing for 15 s, and 72°C extension for 20 s. Finally, 30 cycles of extension at 72°C for 5 min were performed. PCR reactions were performed on an ABI Veriti 96 gradient PCR amplifier (USA). Reaction products were separated by 2% agarose gel electrophoresis and visualized and photographed on a Shanghai Peiqing gel imaging system.

[0047] Young leaves of 'Pengsu' were collected and sent to Novogene for transcriptome sequencing to analyze and mine SSR molecular markers that can be used to identify germplasm. From the 3507 identified SSRs, partial sequences were selected for design based on the principle of 3-6 base repeats, more than 5 base repeats, and amplification products of about 200bp. A total of 40 pairs of SSR primers with high polymorphism were designed for parental polymorphism screening and hybrid individual identification. Among the 40 pairs of SSR primers, 3 pairs had no amplification bands between the parents, and 4 pairs (Rh11, Rh20, Rh21, and Rh34) had amplification bands between the parents and had good polymorphism and could identify the two successful hybrid individual plants 2-17 and 2-55 ( Figure 4 A). Among the above four pairs of SSR primers, the Rh-11 forward primer is 5'-CGGTGCCTCTCTCTCTCTCT-3', and the Rh-11 reverse primer is 5'-CGGAACCAAACCCATCATCG-3'; the Rh-20 forward primer is 5'-AAAGACCCAGCTATGTGCCC-3', and the Rh-20 reverse primer is 5'-GCTTTTGCACAAAGAGGAAGA-3'; the Rh-21 forward primer is 5'-AATCCAGCTCATGTGCCTCA-3', and the Rh-21 reverse primer is 5'-TCTGTCGGACTGCAGACAAC-3'; the Rh-34 forward primer is 5'-CTCTCTCCTCTGCCCCTCTT-3', and the Rh-34 reverse primer is 5'-CTCTTATCCTCGCCGTCGAC-3'.

[0048] It was further found that 4 pairs (Rh2, Rh3, Rh4, Rh16) had amplified bands only in the male wild Rubus 'Pengsu' and no bands in the female blackberry variety ( Figure 4B), it is easier to identify that the individual plants 2-17 and 2-55 are hybrid offspring of 'Pengsu'. Among the above four pairs of SSR primers, Rh2 (Rh-2 forward primer is 5'-AAGCGCGACAACGAAAAGAC-3', Rh-2 reverse primer is 5'-ACAAGTAAAGCATGCAACAGGT-3'), Rh3 (Rh-3 forward primer is 5'-TGGCACACAGTCACACAGAT-3', Rh-3 reverse primer is 5'-TCTGCCTCTGGTTTCCTTTCC-3'), Rh4 (Rh-4 forward primer is 5'-CTCACACCCGTCTGCTTCAT-3', Rh-4 reverse primer is 5'-ATTTCTGTGGTGCTCTCGCA-3'), and Rh16 (Rh-16 forward primer is 5'-ACACAAACGACTTGGCTTGTG-3', Rh-16 reverse primer is 5'-GACTCTTGGCCCTAGTGCTG-3').

[0049] After screening two true hybrids using eight pairs of molecular marker primers, four primer pairs (Rh2, Rh3, Rh4, and Rh16) that amplified bands only in 'Pengsu' were further validated in amplification of other wild Rubus varieties ('Shanmei' and 'Palmleaf Raspberry'), raspberry varieties (yellow raspberry, red raspberry, and black raspberry), and six other blackberry varieties ('Kiowa', 'Boysen', 'Hull', 'Arapaho', 'Navaho', and 'Zaohei'). Amplification patterns revealed that none of the four primer pairs amplified bands in any raspberry or blackberry varieties, but all amplified bands in 'Pengsu'. Rh4 and Rh16 amplified bands in all three wild Rubus varieties, while Rh2 and Rh3 amplified bands in the wild Rubus varieties 'Palmleaf Raspberry' and 'Shanmei', respectively. Therefore, these primers can easily distinguish the hybrid offspring of different wild Rubus parental sources and blackberry. The hybrid single plants 2-17 and 2-55 were analyzed for ploidy using flow cytometer ( Figure 6 ) and found that both 2-17 and 2-55 were tetraploid compared to their parents. This germplasm provides valuable insights into both the practical and theoretical aspects of creating interspecific hybrids in Rubus rubus. Consequently, we successfully created tetraploid plants through interspecific hybridization between hexaploid blackberry and diploid wild Rubus rubus.

[0050] The results show that hybridization between 'Double Season Blackberry' and the wild Rubus rubus 'Pengsu' can produce interspecific hybrid germplasm. Four pairs of highly polymorphic SSR markers developed from 'Pengsu' can effectively identify true hybrids. Furthermore, four primer pairs that amplified bands only in 'Pengsu' also amplified bands only in true hybrid progeny and wild Rubus rubus, demonstrating that these four primer pairs can distinguish between wild and cultivated Rubus parental germplasm in hybrid progenies. These primers have excellent potential for the creation of new Rubus hybrid germplasm and their efficient identification.

Claims

1. A method for identifying interspecific hybrid germplasm of Rubus rubus, characterized in that: The following steps are involved: (1) Obtaining hybrid seeds: artificially hybridizing hexaploid cultivated blackberry as the female parent and diploid wild Rubus rubus as the male parent to obtain seeds; cultivating the seeds to obtain interspecific hybrid seedlings; the hexaploid cultivated blackberry is selected from the blackberry variety 'Double Season Blackberry', and the diploid wild Rubus rubus is selected from the blackberry variety 'Pengsu'; (2) Primer design: Collect young leaves of the male parent for transcriptome sequencing and design several pairs of SSR primers; (3) Primer screening: The SSR primers designed in step (2) were used to verify and screen all the interspecific hybrid seedlings obtained in step (1), and the true hybrid germplasm that was successfully hybridized was identified using the band patterns amplified in the parents, and specific SSR primers that could identify true hybrids were screened out; the specific SSR primers screened out were Rh-11, Rh-20, Rh-21, and Rh-34. The forward primer of Rh-11 was 5'-CGGTGCCTCTCTCTCTCTCT-3', and the reverse primer of Rh-11 was 5'-CGGAACCAAACCCATCATCG-3'; the forward primer of Rh-20 was 5'-AAAGACCCAGCTATGTGCCC-3', and the reverse primer of Rh-20 was 5'-GCTTTTGCACAAAGAGGAAGA-3'; the forward primer of Rh-21 was 5'- AATCCAGCTCATGTGCCTCA-3′, the reverse primer for Rh-21 was 5′- TCTGTCGGACTGCAGACAAC-3′; the forward primer for Rh-34 was 5′- CTCTCTCCTCTGCCCCTCTT-3′, and the reverse primer for Rh-34 was 5′- CTCTTATCCTCGCCGTCGAC-3′; (4) Amplification verification: The SSR primers designed in step (2) were used to verify and screen all the interspecific hybrid seedlings obtained in step (1). The specific SSR primers that were screened out and had amplification bands only in the male parent and no bands in the female parent were further amplified and compared in other wild raspberry, raspberry, and blackberry varieties to further confirm the primers that can specifically identify hybrid germplasms from different wild raspberry parents; the SSR primers further screened out were Rh-2, Rh-3, Rh-4, and Rh-16. The forward primer of Rh-2 was 5'-AAGCGCGACAACGAAAAGAC-3', and the reverse primer of Rh-2 was 5'-ACAAGTAAAGCATGCAACAGGT-3'; the forward primer of Rh-3 was 5'-TGGCACACAGTCACACAGAT-3', and the reverse primer of Rh-3 was 5'-TCTGCCTCTGGTTTCCTTTCC-3'; The forward primer for Rh-4 is 5'-CTCACACCCGTCTGCTTCAT-3', and the reverse primer for Rh-4 is 5'-ATTTCTGTGGTGCTCTCGCA-3'; the forward primer for Rh-16 is 5'-ACACAAACGACTTGGCTTGTG-3', and the reverse primer for Rh-16 is 5'-GACTCTTGGCCCTAGTGCTG-3'; (5) Hybrid germplasm identification: The SSR primers further screened in step (4) are used to perform molecular marker identification on interspecific hybrid seedlings. The ones that can amplify the bands are true hybrids.

2. The identification method according to claim 1, wherein The wild raspberries used for amplification verification in step (4) are 'Pengsu', 'Raspberry', and 'Palm Leaf Raspberry', the raspberry varieties are yellow raspberry, red raspberry, and black raspberry, and the blackberry varieties are 'Kiowa', 'Boysen', 'Hull', 'Arapaho', 'Navaho', and 'Zaohei'.

3. SSR primers Rh-11, Rh-20, Rh-21, and Rh-34 for identifying true hybrid germplasm of Rubus interspecifics, the forward primer of Rh-11 is 5'-CGGTGCCTCTCTCTCTCTCT-3', and the reverse primer of Rh-11 is 5'-CGGAACCAAACCCATCATCG-3'; the forward primer of Rh-20 is 5'-AAAGACCCAGCTATGTGCCC-3', and the reverse primer of Rh-20 is 5'-GCTTTTGCACAAAGAGGAAGA-3'; the forward primer of Rh-21 is 5'-AATCCAGCTCATGTGCCTCA-3', and the reverse primer of Rh-21 is 5'-TCTGTCGGACTGCAGACAAC-3'; the forward primer of Rh-34 is 5'-CTCTCTCCTCTGCCCCTCTT-3', and the reverse primer of Rh-34 is 5'- CTCTTATCCTCGCCGTCGAC-3'.

4. A specific SSR primer Rh-2, Rh-3, Rh-4, and Rh-16 for identifying the parental germplasm origin of Rubus rubus, wherein the forward primer of Rh-2 is 5'-AAGCGCGACAACGAAAAGAC-3', and the reverse primer of Rh-2 is 5'- ACAAGTAAAGCATGCAACAGGT-3'; the forward primer of Rh-3 is 5'- TGGCACACAGTCACACAGAT-3', and the reverse primer of Rh-3 is 5'-TCTGCCTCTGGTTTCCTTTCC-3'; the forward primer of Rh-4 is 5'- CTCACACCCGTCTGCTTCAT-3', and the reverse primer of Rh-4 is 5'- ATTTCTGTGGTGCTCTCGCA-3'; the forward primer of Rh-16 is 5'- ACACAAACGACTTGGCTTGTG-3', and the reverse primer of Rh-16 is 5'- GACTCTTGGCCCTAGTGCTG-3'.

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