Primer pair for identifying authenticity of hybrid of lycium ruthenicum filial generation as well as application and use method of primer pair

Abstract

The invention discloses a primer pair for identifying the authenticity of a hybrid of a lycium ruthenicum filial generation as well as application and a use method of the primer pair, and belongs to the technical field of crossbreeding of lycium ruthenicum. The primer pairs comprise an LR0018 primer pair, an LR0037 primer pair, an LR0043 primer pair, an LR0112 primer pair and an LR0140 primer pair. The method for identifying the hybrid of the lycium ruthenicum based on the five pairs of SSR primers is established by combining an SSR marker with a capillary electrophoresis technology, and a new technical approach is provided for breeding a new variety of the lycium ruthenicum. By amplifying the specific expression SSR marker of the F1 generation, hybrid identification is carried out on the filial generation, the identification process is simple, convenient and rapid, the cost is low, the identification result is stable and reliable, and the research result has important significance on early selection of the authenticity of the hybrid of the filial generation among the lycium ruthenicum varieties and improvement of the breeding efficiency.

Description

Technical Field

[0001] The invention relates to the technical field of wolfberry hybrid breeding, and more particularly to a primer pair for identifying the authenticity of hybrid offspring of black wolfberry hybrids, and an application and use method thereof. Background Art

[0002] There are seven varieties of wolfberry, namely Ningxia wolfberry, Zhongning wolfberry, yellow wolfberry, red wolfberry, calyx wolfberry, black wolfberry and Xinjiang wolfberry. Among them, 'black wolfberry' belongs to the shrub of Lycium Linn. of Solanaceae. Most of the mature fruits are black berries, and a few are white berries (except for the color, the fruit phenotype is no different from the black berries). It is a drought-resistant, salt-alkali-resistant and wind-sand-resistant plant in the desert areas of northwest my country. It is of great significance to windbreak and sand fixation, soil improvement and ecological environment protection in the northwest region. The fresh fruit of black wolfberry is rich in natural flavonoid compounds, and its total anthocyanin content can be as high as 386.9mg / 100g. It has become an important characteristic economic plant in northwest my country.

[0003] With the development of the "black wolfberry" industry, the area of ​​artificial domestication has been expanding. Due to the lack of new varieties that can be promoted and applied, some production areas directly use wild seed seedlings for planting, resulting in uneven production characteristics of plantation seedlings, and problems such as different fruit sizes and large differences in anthocyanin content, which directly affects the orderly and healthy development of the "black wolfberry" industry. Anthocyanins (ACN) are glycoside compounds formed by anthocyanidins (Anthocyanidin) combined with various monosaccharides in a natural state. They are widely present in the cell sap of flowers, fruits, stems, leaves and root organs of plants, making them appear in different colors from red, purple to blue, etc. ACN gives plants colorful colors. Modern scientific research shows that ACN has antioxidant, anti-fatigue and lipid-lowering effects. At the same time, as a natural pigment, ACN is safe, non-toxic, and can be used as both medicine and food, and has great potential in the current food industry. Therefore, breeding black wolfberry varieties with high ACN content, stable yield and strong resistance has become a key issue in solving industrial development.

[0004] Hybrid breeding is one of the main approaches for plant genetic improvement and germplasm innovation. Hybrid identification and elimination of non-parental hybrids in hybrid populations is a necessary prerequisite for ensuring the accuracy of genetic law analysis and trait linkage analysis. Traditional hybrid identification mainly relies on the external morphological characteristics of plants, which not only has a long identification cycle and low efficiency, but also has the problem of low accuracy of identification results due to the development status of plants and the influence of external environmental factors. In the prior art, there is a method for identifying the genetic traits of hybrid F1 generations of hybrid breeding by using molecular marker-assisted identification, which does not need to wait until the F1 generation grows into a plant to make a judgment, and can accurately, conveniently and quickly identify the genetic traits of hybrid offspring. At present, there is only a report on the identification of interspecific distant hybridization offspring of Lycium barbarum, and no relevant report on the identification of hybrids in the hybrid population of black wolfberry is seen.

[0005] Black wolfberry is a perennial plant with a highly complex genotype and a long sexual period. It takes too long to identify and classify it through morphological markers. Therefore, how to conduct early identification of hybrid offspring and establish a molecular marker-assisted breeding system to accelerate the breeding process has become an urgent problem to be solved. Summary of the invention

[0006] In view of the above problems, the present invention provides a primer pair for identifying the authenticity of hybrid offspring of black wolfberry and its application and use method, so as to accelerate the breeding process of black wolfberry.

[0007] In order to achieve the above object, the present invention adopts the following technical solution:

[0008] A primer pair for identifying the authenticity of hybrid offspring of black wolfberry, the primer pair comprising a primer pair of LR0018, a primer pair of LR0037, a primer pair of LR0043, a primer pair of LR0112 and a primer pair of LR0140;

[0009] The sequences of the LR0018 primer pair are shown in SEQ ID NO: 1 and SEQ ID NO: 2;

[0010] The sequences of the LR0037 primer pair are shown in SEQ ID NO:5 and SEQ ID NO:6;

[0011] The sequences of the LR0043 primer pair are shown in SEQ ID NO:7 and SEQ ID NO:8;

[0012] The sequences of the LR0112 primer pair are shown in SEQ ID NO: 17 and SEQ ID NO: 18;

[0013] The sequences of the LR0140 primer pair are shown in SEQ ID NO:21 and SEQ ID NO:22.

[0014] Another object of the present invention is to provide the use of the above primer pair in identifying true and false hybrids of the hybrid offspring of Lycium ruthenicum.

[0015] Another object of the present invention is to provide a method for identifying true and false hybrids of hybrid offspring of black wolfberry, comprising the following steps:

[0016] (1) Using black berry wolfberry and white berry wolfberry of black fruit wolfberry as parents, intraspecific hybridization was performed to obtain the F1 generation, and the genomic DNA of the parents and the hybrid F1 generation was extracted respectively;

[0017] (2) modifying the primer pair LR0018, the primer pair LR0037, the primer pair LR0043, the primer pair LR0112, and the primer pair LR0140, respectively, and using the modified primer pairs to perform PCR amplification on the genomic DNA extracted from the parents and the hybrid F1 generation, respectively, to obtain amplified products;

[0018] (3) The amplified products are sequenced by capillary electrophoresis, and the bands produced by electrophoresis are compared to determine the authenticity of the hybrid offspring of black wolfberry.

[0019] Preferably, in step (1), young leaves of the parent and the intraspecific hybrid F1 hybrid thereof are extracted.

[0020] Preferably, the modification in step (2) is specifically as follows: adding the M13 universal primer sequence as shown in SEQ ID NO:45 to the 5' end of both the forward and reverse primers of the primer pair according to claim 1, and after the addition of the M13 universal primer sequence, the 5' end of the forward primer of each primer pair is connected to a FAM fluorescent marker, and the 5' end of the reverse primer is connected to a HEX fluorescent marker.

[0021] Specifically, the sequence of the LR0018 primer pair after the M13 universal primer is added is shown in SEQ ID NO:23 and SEQ ID NO:24; the sequence of the LR0037 primer pair after the M13 universal primer is added is shown in SEQ ID NO:27 and SEQ ID NO:28; the sequence of the LR0043 primer pair after the M13 universal primer is added is shown in SEQ ID NO:29 and SEQ ID NO:30; the sequence of the LR0112 primer pair after the M13 universal primer is added is shown in SEQ ID NO:39 and SEQ ID NO:40; the sequence of the LR0140 primer pair after the M13 universal primer is added is shown in SEQ ID NO:43 and SEQ ID NO:44.

[0022] Preferably, the amplification system of the PCR process in step (2) is: 1 μl of DNA template with a concentration of 50 ng / μl, 1.5 μl of 10×Buffer, 1.2 μl of dNTP with a concentration of 10 μmol / L, 0.1 μl of forward primer and 0.4 μl of reverse primer modified with a concentration of 5 μmol / L, 0.1 μl of Taq DNA polymerase with a concentration of 5 U / μl, and the mixture is supplemented to 10 μl with sterilized deionized water.

[0023] Preferably, the amplification program of the PCR process in step (2) is: 95°C for 5 min; 95°C for 30 s, 57°C for 30 s, 72°C for 30 s, 25 cycles; 95°C for 30 s, 53°C for 30 s, 72°C for 30 s, 10 cycles; and finally 60°C for 30 min.

[0024] Preferably, the judgment standard in step (3) is to compare the electrophoresis results of the F1 generation with those of the maternal parent and the paternal parent. If 2 or more pairs of primers among the 5 pairs of primers show only the maternal characteristic band or the maternal characteristic band + the newly added band in the electrophoresis results of the F1 generation, it is a false hybrid; otherwise, it is a true hybrid.

[0025] The present invention discloses a primer pair for identifying the authenticity of hybrids of black wolfberry hybrid offspring and its application and use method, wherein the primer pair includes LR0018 primer pair, LR0037 primer pair, LR0043 primer pair, LR0112 primer pair and LR0140 primer pair. The present invention adopts SSR markers in combination with capillary electrophoresis technology to establish a black wolfberry hybrid identification method based on 5 pairs of SSR primers, providing a new technical approach for breeding new black wolfberry varieties. The present invention performs hybrid identification on hybrid offspring by amplifying the specific expression SSR markers of the F1 generation. The identification process is simple, rapid, low-cost, and the identification results are stable and reliable. The research results are of great significance for the early selection of the authenticity of hybrids of hybrid offspring between black wolfberry varieties and improving breeding efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 : 0.8% agarose electrophoresis of the DNA of the leaves of the parents and F1 offspring of wolfberry, wherein, Figure A: GA population parents and F1 offspring, Figure B: GB population parents and F1 offspring, M: Marker, 1: maternal parent, 2: paternal parent, 3-24: F1 offspring;

[0027] Figure 2 : Capillary electrophoresis of primer LR0018 in labeling true and false hybrids of F1 generation;

[0028] Figure 3 : Capillary electrophoresis of primer LR0037 in labeling true and false hybrids of F1 generation;

[0029] Figure 4 : Capillary electrophoresis of primer LR0043 in labeling true and false hybrids of F1 generation;

[0030] Figure 5A : Capillary electrophoresis of primer LR0112 in marking true hybrids of F1 generation;

[0031] Figure 5B : Capillary electrophoresis of primer LR0112 in pseudohybrids of the F1 generation;

[0032] Fig. 6A : Capillary electrophoresis of primer LR0140 in marking true hybrids of F1 generation;

[0033] Figure 6B : Capillary electrophoresis of primer LR0140 in marking F1 generation pseudohybrids. DETAILED DESCRIPTION

[0034] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0035] The reagents involved in the embodiments of the present invention are all purchased from commercial channels, and the methods not mentioned are conventional experimental methods and will not be described in detail here.

[0036] Materials and Methods

[0037] The parents used in the present invention are black berry wolfberry and white pulp wolfberry in black wolfberry. The Ningxia Academy of Agriculture and Forestry Sciences began to collect and preserve black wolfberry germplasm resources in 2014, and collected and preserved more than 200 black wolfberry germplasms and natural mutants from Ningxia, Qinghai, Xinjiang, Gansu, Inner Mongolia and other places, and planted them in the experimental base of the Lycium barbarum Science Research Institute of the Ningxia Academy of Agriculture and Forestry Sciences. The natural population was continuously surveyed for agronomics, and the relevant data such as the morphological characteristics of branches, leaves, flowers, and fruits and phenological periods were statistically analyzed. In 2019, it was initially selected through a variety comparison test. From 2019 to 2021, it was asexually propagated, seed nurseries were established, and adaptability was determined through regional and production tests. It was found that its morphological characteristics were stable and consistent in Yinchuan, Zhongning and other places.

[0038] Characteristics of black berry wolfberry: strong tree vigor, strong trunk, strong branching ability (4.5 branches / plant), short and thick fruiting branches (branch length: 24.18cm, branch thickness: 0.37cm), no or occasional short thorns at each node, dark green stem tip; 2-6 leaves clustered on short branches, nearly sessile, linear oblanceolate, 0.66-4.27cm long, 2.36-9.63mm wide; 2-6 flowers born on short branches, calyx narrow bell-shaped, corolla funnel-shaped, light purple, 5 shallow lobes, and sparse hairs on the inner wall of the corolla; berries purple-black, spherical, no depression at the top, fresh fruit average longitudinal diameter 4.27mm, transverse diameter 7.36mm, 1000-grain weight: 981.85g, fresh-to-dry ratio 6.5:1, high anthocyanin content (3.94mg / g).

[0039] Characteristics of white berry wolfberry: weak tree vigor, weak trunk, weak branching ability (2.3 branches / plant), short and thin fruiting branches (branch length: 18.14cm, branch thickness: 0.21cm), short thorns on each node, yellow-green stem tip; 2-6 leaves clustered on short branches, nearly sessile, linear oblanceolate, yellow-green, 0.64-4.26cm long, 2.33-9.69mm wide; 2-6 flowers born on short branches, calyx narrow bell-shaped, corolla funnel-shaped, light purple, 5 shallow lobes, the inner wall of the corolla also has sparse villi; berries are white, spherical, with a depression at the top, the average longitudinal diameter of fresh fruit is 2.27cm, the transverse diameter is 2.37cm, 1000-grain weight: 441.85g, fresh-to-dry ratio is 7.8:1, and the anthocyanin content is low (0.09mg / g).

[0040] 1. Construction of F1 hybrid population of Lycium ruthenicum

[0041] In April 2021, the black berry wolfberry (denoted as Lr-B) and the white berry wolfberry (denoted as Lr-W) were hybridized as parents using conventional artificial controlled pollination methods. The fruits were collected in July of the same year, and pure and plump hybrid seeds were obtained after testing. In November of the same year, the hybrid seeds were treated with constant temperature germination and sown in a greenhouse in January 2022 in a medium containing a seedling medium (V 草炭 :V 蛭石 =3:1) plug tray (50 holes, 540mm×280mm), and transplant the seeds to plug trays (10cm×10cm) when the seedling height is ≥20cm; when the seedling height is ≥60cm, transplant the seedlings to the field and manage water and fertilizer uniformly. Finally, 407 hybrid F1 plants with normal growth were obtained (germplasm information is shown in Table 1), of which 301 plants were hybridized with black berry wolfberry (♀)×white berry wolfberry (♂), and were named GA 1, GA2, GA3..., GA301, and recorded as Lr-GA population; 106 plants were hybridized with white berry wolfberry (♀)×black berry wolfberry (♂), and were named GB 1, GB2, GB3..., GB 106, and recorded as Lr-GB population.

[0042]

[0043] 2. Extraction of genomic DNA from the F1 population of Lycium ruthenicum

[0044] When the height of the parent and progeny seedlings was ≥20 cm, the 5th, 7th, and 9th young leaves (about 50 mg per plant) were collected from the base, and DNA was extracted using a high-efficiency plant genomic DNA extraction kit (DP350) (Tiangen Biochemical Technology (Beijing) Co., Ltd., Germany). The DNA quality integrity was tested by 1% agarose gel electrophoresis. The results were as follows: Figure 1 As shown by Figure 1 It can be seen that the extracted DNA not only has good integrity but also has clean sample wells, indicating that when using this kit to extract the genome of wolfberry leaves, it can not only remove polysaccharides but also remove other substances that seriously interfere with the extraction of genomic DNA from wolfberry leaves.

[0045] The DNA concentration and purity were detected by ultraviolet spectrophotometer (Eppendorf Bio Photometer Plu). The OD value was about 1.5, which could meet the needs of subsequent experiments. The DNA mass concentration was diluted to 50 ng / μl and stored at -2°C for later use.

[0046] 3. SSR primer information

[0047] For the 11 pairs of highly polymorphic SSR primers developed and designed by the research group as shown in Table 2, an 18 bp M13 universal primer sequence (TGTAAAACGACGGCCAGT, SEQ ID NO: 45) was added to the 5' end of the forward and reverse primers, and then the 5' end of the forward primer was connected with a FAM fluorescent marker, the 5' end of the reverse primer was connected with a HEX fluorescent marker, and the 3' end remained unchanged. The modified 11 pairs of SSR primers are shown in Table 3. All primers were synthesized by Yingwei Jieji Trading Co., Ltd.

[0048] Table 2 Information of 11 pairs of SSR primers

[0049]

[0050] F: upstream primer; R: downstream primer; the letters in brackets are the repeating motif sequence, and the numbers outside the brackets indicate the number of repetitions of the repeating motif

[0051] Table 3 Information of 11 pairs of SSR primers after modification

[0052]

[0053] F: upstream primer; R: downstream primer

[0054] 4. PCR amplification and product detection

[0055] Using the genomic DNA of each sample as a template, PCR amplification was performed using the 11 modified SSR primer pairs in Table 3 to perform genotyping on the two parents and their offspring, including:

[0056] PCR amplification system 10μl: 50ng / μl DNA template 1μl, 10×Buffer 1.5μl, 10μmol / L dNTP 1.2μl, 5μmol / L modified forward primer 0.1μl, reverse primer 0.4μl, Taq DNA polymerase (5U / μl) 0.1μl, sterilized deionized water 5.3μl.

[0057] The PCR reaction was carried out on a Gene Amp PCR System 9600 (Perkin ELRer, USA) with the following program: 95°C for 5 min; 95°C for 30 s, 57°C for 30 s, 72°C for 30 s, 25 cycles; 95°C for 30 s, 53°C for 30 s, 72°C for 30 s, 10 cycles; and finally 60°C for 30 min.

[0058] The amplified products were detected by ABI3730XL DNA (Applied Biosystems, USA), LIZ-500 was used as the molecular weight internal standard, and the capillary electrophoresis data were statistically analyzed using Gene Marker2.20 software. The position of each peak was compared with the Gene Scan500LIZ molecular mass internal standard in the lane, the amplified fragment length was read, and the allele was recorded.

[0059] 5. Test results

[0060] The amplification results of the modified 11 pairs of SSR primers in the parents and 10 abnormal F1 generation plants are shown in Table 4.

[0061] Table 4 Amplification results of the 11 modified SSR primer pairs in the parents and abnormal F1 generation plants

[0062]

[0063]

[0064] “-”: indicates that the parental bands were amplified at this site

[0065] According to the distribution of band patterns, the results of differential bands between parents can be divided into the following three categories: parental complementary type, that is, the positions of the bands appearing in the parents are completely different. When using this primer, the F1 generation band pattern is composed of part or all of the paternal bands and part or all of the maternal bands. If the maternal or paternal band pattern appears alone, the F1 generation is non-hybrid; heterozygous complementary type, that is, the maternal band position includes but is not entirely the paternal band position. When using this primer, the F1 generation band pattern is theoretically all maternal band pattern, and it is impossible to distinguish between true and false offspring; paternal type, that is, the paternal band position includes but is not entirely the maternal band position. When using this primer, if the F1 generation has the paternal band pattern, it can be identified as a hybrid. If it is the maternal band pattern, other primers need to be used for further identification. For plant samples with missing parental specific sites or the appearance of new bands, PCR verification was repeated 3 times.

[0066] As shown in Table 4, among the 301 Lr-GA F1 generation plants, 5 F1 generation plants had abnormal genotypes at 2 to 6 loci; specifically, the Lr-GA F1 generation plant GA28 with abnormal genotype had a maternal or paternal band missing at 1 locus (383 / 399), and new bands were amplified at the other 5 loci; GA125 had a maternal or paternal band missing at 1 locus (383 / 399), and new bands were amplified at the other 3 loci; GA160 had a maternal or paternal band missing at 1 locus (383 / 399), and a new band was amplified at the other 1 locus; GA162 had a maternal or paternal band missing at 1 locus (383 / 399), and a new band was amplified at the other 1 locus; GA229 had new bands amplified at 2 loci.

[0067] Among the 106 F1 generation strains of Lr-GB, 5 F1 generation strains showed abnormal genotypes at 3 to 6 loci. Specifically, the F1 generation strain GB1 of Lr-GB with abnormal genotypes showed maternal or paternal band loss at 3 loci (399 / 399, 188 / 188, 218 / 227), and new bands were amplified at the other 3 loci; GB27 showed maternal or paternal band loss at 2 loci (399 / 399, 249 / 249), and new bands were amplified at the other two sites; GB37 had a missing maternal or paternal band at one site (249 / 249), and new bands were amplified at the other two sites; GB60 had a missing maternal or paternal band at one site (249 / 249), and new bands were amplified at the other two sites; GB105 had a missing maternal or paternal band at one site (249 / 249), and new bands were amplified at the other two sites.

[0068] 6. Analysis of the efficiency of identifying true and false hybrids

[0069] Use a, b, c, and d to mark different alleles. For example, in the Lr-GA population, the genotypes of LR0069 in the parents are 249 / 249 and 245 / 249, and the separation type of its parents is aa×ab; the genotypes of LR0081 in the parents are 271 / 271 and 264 / 307, and the separation type of its parents is aa×bc; the genotypes of LR0018 in the parents are 360 / 362 and 298 / 31 9, the separation type of its parents is ab×cd (similarly, the genotypes of LR0037, LR0043, LR0112 and LR0140 in the parental markers are all ab×cd); the genotypes of LR0050 in the parental markers are 218 / 227 and 227 / 240, respectively, and the separation type of its parents is ab×bc; the genotypes of LR0035 in the parental markers are 399 / 387 and 399 / 383, respectively, and the separation type of its parents is ab×ac.

[0070] The chi-square (χ 2 ) Goodness of fit test was used to analyze the genotype segregation of the hybrid F1 population. The distribution of different SSR loci in the F1 population offspring is shown in Table 5.

[0071] Table 5 Distribution of different SSR loci in the F1 population progeny

[0072]

[0073] As shown in Table 5, in the offspring of the Lr-GA F1 population: the genotypes of the parents of the LR0069 and LR0081 markers were paternal type, and these two pairs of markers identified 154 and 296 true hybrids in 301 plants, respectively, with true hybrid identification rates of 51.16% and 98.34%, respectively; the genotypes of the parents of 7 pairs of markers were parental complementary type, among which LR0018, LR0037, LR0043, LR0112 and LR0140 were "ab×cd" type, and LR00101 and LR0011 were "ab×cd" type. 6 was of "ab×cc" type, and the seven pairs of markers amplified 297, 296, 296, 297, 298, 298 and 300 true hybrids with parental characteristic bands in 301 individuals, respectively, with an average identification rate of 98.81%; LR0050 and LR0035 had heterozygous complementary genotypes in their parents, and these two pairs of markers identified 147 and 158 true hybrids in 301 individuals, respectively, with true hybrid identification rates of 48.83% and 52.49%, respectively.

[0074] In the offspring of the Lr-GB F1 population: the genotypes of markers LR00101 and LR0069 in the parents were paternal types. Theoretically, the offspring had both paternal and maternal bands. If they were paternal bands, it indicated that the offspring were true hybrids. If they were maternal bands, further verification was required. 43 and 46 true hybrids were identified, with identification rates of 40.57% and 46.22%, respectively. Among the 7 pairs of marker parents, LR0037, LR0043, LR0112, LR0140 and LR0018 had "ab×cd" genotypes, and LR0116 and LR0081 had "ab ×cc" type, with an average identification rate of 96.49%; LR0050 and LR0035 have heterozygous complementary genotypes in their parents and have one identical allele locus, namely "ab×bc" type. Only the offspring with the characteristic band of the mother and the c-band of the father can be identified as true hybrids, otherwise they may be parthenocarpy (the ovary does not undergo fertilization and forms a fruit without seeds), and further identification is required. These two pairs of markers identified 48 and 59 true hybrid plants in 301 plants, respectively, with true hybrid identification rates of 45.28% and 55.66%.

[0075] Based on the results of 11 pairs of marker identification, the offspring that showed maternal characteristic bands or newly added bands were repeatedly identified by multiple pairs of primers. After subtracting the single plants that did not amplify the paternal bands by 2 or more pairs of different markers, the remaining plants were true hybrids. Among the Lr-GA population (301 plants), 5 plants were subtracted, and 296 plants were determined to be true hybrids, with a true hybrid rate of 98.34%; among the Lr-GB population (106 plants), 5 plants were subtracted, and 101 plants were determined to be true hybrids, with a true hybrid rate of 95.28%. There was no difference between the positive and negative cross populations of Lycium barbarum, and there were large differences between the marker sites.

[0076] Since there is uncertainty in the identification of offspring by "paternal type" and "heterozygous complementary type" primers, while "parental complementary type" primers can accurately identify authenticity, LR0018, LR0037, LR0043, LR0112 and LR0140, whose genotypes of parents are "parental complementary type" in both GA and GB populations, were selected as primers for the identification of true hybrids in the F1 generation of black wolfberry populations, with an identification rate of more than 95.0%. The capillary electrophoresis diagrams of true and false hybrids detected by LR0018, LR0037, LR0043, LR0112 and LR0140 in the Lr-GA population and Lr-GB population of black wolfberry hybrid offspring are shown in Figure 2. Figure 2 -6.

[0077] 7. Analysis of genotype segregation rules at the loci corresponding to SSR primers

[0078] The gene segregation rules of the F1 true hybrids of the hybrid Lr-GA population and Lr-GB population of Lycium barbarum were analyzed based on the results of 11 pairs of SSR primers. The results are shown in Table 6.

[0079] Table 6 Analysis of gene segregation rules of true hybrids in hybrid F1 generation based on the amplification results of 11 pairs of SSR primers

[0080]

[0081] As shown in Table 6, the amplification results of the 11 pairs of SSR primers in these 397 F1 true hybrids all showed gene segregation. Among them, in the Lr-GA population (301 strains), the amplification results of primers LR0069 and LR0101 in the F1 generation showed two genotypes, namely aa and ab, and the amplification results of primers LR0081 and LR0116 in the F1 generation showed two genotypes, namely ab and ac and ac and bc, respectively, which were close to 1:1. 2 The results showed that none of the four loci showed skewed segregation (P>0.05). The amplification results of primers LR0018, LR0037, LR0043, LR00112 and LR00140 in the F1 generation showed four genotypes, namely ab, ac, bc and bd. The amplification results of primers LR0035 and LR0050 in the F1 generation also showed four genotypes, namely aa, ab, ac and bc and ab, ac, bc and bb, respectively. The segregation ratios of the four genotypes shown by the amplification results of these seven primer pairs in the F1 generation population were somewhat different, but the results of χ 2 The test showed that there was no deviation from the expected separation ratio of 1:1:1:1 (P>0.05).

[0082] In the Lr-GB population (106 strains), the amplification results of primers LR0069, LR0081, LR0101 and LR0116 in the F1 generation showed two genotypes, ab, bb, ac, bc, aa, ab and ab, ac, all close to 1:1. 2 The results of the PCR test showed that the LR0116 locus showed a skewed segregation phenomenon (P<0.05), while the other three loci did not show a skewed segregation phenomenon (P>0.05). The amplification results of primers LR0018, LR0037, LR0043, LR00112 and LR00140 in the F1 generation showed four genotypes, namely ab, ac, bc and bd. The amplification results of primers LR0035 and LR0050 in the F1 generation also showed four genotypes, namely aa, ab, ac and bc and ab, ac, bc and bb, respectively. There were certain differences in the segregation ratios of the four genotypes presented by the amplification results of these seven primer pairs in the F1 generation population, but the results of χ 2The test showed that there was no skewed segregation phenomenon compared with the expected segregation ratio of 1:1:1:1 (P>0.05). This showed that in the hybrid F1 generation of black berry wolfberry and white pulp wolfberry in black fruit wolfberry, the genotypes of the corresponding loci of the above 11 pairs of SSR primers all conformed to Mendel's law of segregation, with high credibility.

[0083] 8. Analysis of genotype segregation rules at the loci corresponding to SSR primers

[0084] The genetic diversity of the F1 generation true hybrids of the Lr-GA population and the Lr-GB population was analyzed. Specifically, the genetic diversity parameters such as the number of alleles (Na), the number of effective alleles (Ne), the Shannon's information index (I), the observed heterozygosity (Ho), the expected heterozygosity (He), the F test value (F) and the polymorphism information content (PIC) of 11 pairs of SSR markers in the true hybrids were calculated using POPGENE 32 software. The results are shown in Table 7.

[0085] Table 7 Polymorphism information of true hybrids in hybrid F1 generation based on 11 SSR markers

[0086]

[0087]

[0088] As shown in Table 7, in the Lr-GA population, 11 pairs of SSR markers detected a total of 64 allele loci in 296 true hybrid samples. The number of alleles detected by each marker (Na) was between 4 and 9, and the average number of alleles detected by each marker was 6; the effective number of alleles (Ne) ranged from 1.58 to 4.35, and the average number of effective alleles detected by each pair of primers was 3.20; the genetic polymorphism information content (PIC) ranged from 0.30 to 0.73, with an average of 0.59.

[0089] In the Lr-GB population, 11 pairs of SSR markers detected a total of 48 allele loci in 101 true hybrid samples. The number of alleles detected by each marker (Na) was between 2 and 6, and the average number of alleles detected by each marker was 4; the effective number of alleles (Ne) ranged from 1.55 to 4.20, and the average number of effective alleles detected by each primer pair was 3.12; the gene polymorphism information content (PIC) ranged from 0.30 to 0.72, with an average of 0.58.

[0090] When PIC>0.5, it indicates that the marker polymorphism is high and the detection ability is strong. The PIC values ​​of LR0018, LR0037, LR0043, LR0112 and LR0140 in 296 true hybrid samples of Lr-GA population are 0.71, 0.71, 0.70, 0.730 and 0.71 respectively; the PIC values ​​of LR0018, LR0037, LR0043, LR0112 and LR0140 in 101 true hybrid samples of Lr-GB population are 0.72, 0.71, 0.70, 0.70 and 0.69 respectively. This indicates that the polymorphism of LR0018, LR0037, LR0043, LR0112 and LR0140 black wolfberries in the hybrid F1 generation of black berry wolfberries and white pulp wolfberries is high, and the detection ability is strong.

[0091] The AMOVA program of GenAlEx 6.51 was used to perform AMOVA analysis on the genetic differences of the F1 true hybrids in the above Lr-GA population and Lr-GB population. The results are shown in Table 8:

[0092] Table 8 AMOVA analysis of genetic differences of true hybrids in the hybrid F1 generation between Lr-GA and Lr-GB populations

[0093]

[0094] As shown in Table 8, in the total variance of genetic variation, 2.0% of the difference comes from between populations, while 98.0% of the difference exists between individuals within a population, and the total genetic differentiation index (Fst) of the population is 0.018 (P<0.05). This shows that there is no difference in genetic differentiation between the reciprocal cross populations of black wolfberry and white wolfberry, and the genetic differentiation generated comes from the hybrid population.

[0095] In summary, the five pairs of SSR primers LR0018, LR0037, LR0043, LR0112 and LR0140 of the present invention have a high identification rate of true hybrids in the F1 generation hybrid offspring of black wolfberry. The black wolfberry hybrid identification method based on these five pairs of SSR primers is highly reliable, the identification process is simple and fast, the cost is low, and the identification results are stable and reliable. The research results are of great significance for the early selection of the authenticity of hybrids in hybrid offspring between black wolfberry varieties and for improving breeding efficiency.

[0096] The various embodiments in this specification are described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the various embodiments can be referenced to each other.

[0097] The above description of the disclosed embodiments enables those skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A primer pair for identifying the authenticity of hybrid offspring of black wolfberry, characterized in that: The primer pairs include LR0018 primer pair, LR0037 primer pair, LR0043 primer pair, LR0112 primer pair and LR0140 primer pair; The sequences of the LR0018 primer pair are shown in SEQ ID NO: 1 and SEQ ID NO: 2; The sequences of the LR0037 primer pair are shown in SEQ ID NO:5 and SEQ ID NO:6; The sequences of the LR0043 primer pair are shown in SEQ ID NO:7 and SEQ ID NO:8; The sequences of the LR0112 primer pair are shown in SEQ ID NO: 17 and SEQ ID NO: 18; The sequences of the LR0140 primer pair are shown in SEQ ID NO:21 and SEQ ID NO:

22.

2. Use of the primer pair according to claim 1 in identifying true and false hybrids of Lycium ruthenicum hybrid offspring.

3. A method for identifying true and false hybrids of black wolfberry hybrid offspring, characterized in that: The steps include: (1) After intraspecific hybridization of black berry wolfberry and white berry wolfberry of black fruit wolfberry as parents to obtain F1 generation, genomic DNA of the parents and hybrid F1 generation were extracted respectively; (2) modifying the primer pair described in claim 1, and using the modified primer pair to perform PCR amplification on the extracted genomic DNA of the parents and the hybrid F1 generation, respectively, to obtain an amplified product; (3) The amplified products are sequenced by capillary electrophoresis, and the bands produced by electrophoresis are compared to determine the authenticity of the hybrid offspring of black wolfberry.

4. A method for identifying true and false hybrids of Lycium ruthenicum hybrid offspring according to claim 3, characterized in that: In step (1), young leaves of the parent and the intraspecific hybrid F1 hybrid thereof are extracted.

5. The method for identifying true and false hybrids of Lycium ruthenicum hybrid offspring according to claim 3, characterized in that: The modification in step (2) is specifically as follows: adding the M13 universal primer sequence as shown in SEQ ID NO:45 to the 5' end of both the forward and reverse primers of the primer pair of claim 1, and after the addition of the M13 universal primer sequence, the 5' end of the forward primer of each primer pair is connected to a FAM fluorescent marker, and the 5' end of the reverse primer is connected to a HEX fluorescent marker.

6. A method for identifying true and false hybrids of Lycium ruthenicum hybrid offspring according to claim 5, characterized in that: The primer sequences after adding the M13 universal primer sequence are: The sequences of the LR0018 primer pair after the addition of the M13 universal primer are shown in SEQ ID NO:23 and SEQ ID NO:24; The sequences of the LR0037 primer pair after the addition of the M13 universal primer are shown in SEQ ID NO: 27 and SEQ ID NO: 28; The sequences of the LR0043 primer pair after the addition of the M13 universal primer are shown in SEQ ID NO:29 and SEQ ID NO:30; The sequences of the LR0112 primer pair after the addition of the M13 universal primer are shown in SEQ ID NO:39 and SEQ ID NO:40; The sequences of the LR0140 primer pair after the addition of the M13 universal primer are shown in SEQ ID NO:43 and SEQ ID NO:

44.

7. The method for identifying true and false hybrids of Lycium ruthenicum hybrid offspring according to claim 3, characterized in that: The amplification system of the PCR process in step (2) is: 1 μl of DNA template with a concentration of 50 ng / μl, 1.5 μl of 10×Buffer, 1.2 μl of dNTP with a concentration of 10 μmol / L, 0.1 μl of forward primer and 0.4 μl of reverse primer modified with a concentration of 5 μmol / L, 0.1 μl of Taq DNA polymerase with a concentration of 5 U / μl, and the mixture is supplemented to 10 μl with sterilized deionized water.

8. The method for identifying true and false hybrids of Lycium ruthenicum hybrid offspring according to claim 7, characterized in that: The amplification program of the PCR process in step (2) is: 95°C for 5 min; 95°C for 30 s, 57°C for 30 s, 72°C for 30 s, 25 cycles; 95°C for 30 s, 53°C for 30 s, 72°C for 30 s, 10 cycles; and finally 60°C for 30 min.

9. The method for identifying true and false hybrids of Lycium ruthenicum hybrid offspring according to any one of claims 3 to 8, characterized in that: The judgment standard in step (3) is: compare the electrophoresis results of the F1 generation with those of the maternal parent and the paternal parent. If 2 or more pairs of primers among the 5 pairs of primers show only the maternal characteristic band or the maternal characteristic band + the newly added band in the electrophoresis results of the F1 generation, it is a false hybrid; otherwise, it is a true hybrid.

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