Identification of ssr primers for 19 chromosomes of populus and their application in breeding of poplar primary trisomics

Abstract

This invention discloses SSR primers for identifying the 19 chromosomes of poplar and their application in the breeding of primary trisomy in poplar. From 180 pairs of SSR primers, this invention screened 19 specific SSR primer pairs that amplified stable bands, exhibited good polymorphism, and identified all 19 chromosomes of poplar. These 19 primer pairs can only amplify the alleles of the corresponding chromosomes; therefore, they can be used for chromosome identification and number determination in aneuploid progeny populations. This invention utilizes the extreme imbalance of homologous chromosome segregation during meiosis in triploid poplar pollen mother cells to obtain aneuploid pollen, which is then pollinated and crossed with normal haploid female gametes to establish an aneuploid progeny population. The selected 19 specific SSR primer pairs are then used to identify the chromosome number of aneuploid progeny individuals. Finally, chromosome counting is used to confirm whether the obtained primary trisomy of poplar is a true trisomy. This invention has promising applications in poplar aneuploid breeding.

Description

Technical Field

[0001] This invention relates to SSR primers for identifying poplar chromosomes and their applications, particularly to SSR primers for identifying all 19 chromosomes of poplar and their application in the breeding of primary trisomy of poplar, belonging to the field of poplar aneuploid breeding. Background Technology

[0002] Most plants in nature exist as euploids, where the number of chromosomes in their somatic cells is an integer multiple of the chromosome set (x), such as diploids (2n = 2x), triploids (2n = 3x), and tetraploids (2n = 4x). Trisomy (2n+1) refers to a plant individual with an extra chromosome in its diploid chromosome set, classifying it as an aneuploid plant. Due to differences in the nature of the extra chromosome, plant trisomy can be divided into four types: primary trisomy, secondary trisomy, tertiary trisomy, and telomere trisomy (Liu Qingchang. Genetics (3rd Edition). 2015, Science Press), with primary trisomy being the most widely used.

[0003] Primary trisomy is an extremely important genetic tool. It can be used to replace and separate target chromosomes, and to locate genes and linkage groups onto specific chromosomes, thereby constructing a physical map of genes (Shen Shuxing. Creation and Characteristic Study of Complete Primary Trisomy in Chinese Cabbage. 2007, Nanjing Agricultural University Library). Therefore, plant breeders both domestically and internationally attach great importance to the creation, identification, and research of primary trisomy in plants.

[0004] Currently, there are four main methods for breeding primary trisomy in plants: The first is to directly screen natural plant trisomy from nature. Because chromosome segregation errors easily occur during meiosis in plant pollen mother cells, if a pair of homologous chromosomes fails to separate in anaphase I, it will result in two types of gametes, n+1 and n-1, which will then fertilize with normal (n) gametes to produce trisomy and monosomy. Blakeslee isolated a primary trisomy with 12 chromosomes from Datura (Blackslee AF, Avery B T. Mutations in Jimson Weed[J]. Journal of Heredity, 1987, 74:346-360), and these Datura trisomy plants were all spontaneously generated. Although primary trisomy in plants can occur spontaneously, this phenomenon is extremely rare. Furthermore, the frequency of natural primary trisomy generation in plants is extremely low. The second method for selecting primary trisomy in plants is to utilize the irregular distribution of chromosomes during meiosis in homologous triploids to screen trisomy plants from the offspring of 3x×2x or 2x×3x hybrids.To date, some important crops, such as tomato (Rick CM, Barton DW. Cytological and genetical identification of the primary trisomics of the tomato[J]. Genetics, 1954, 39: 640-666), maize (Mcclintock B. Chromosome morphology of zea mays[J]. Science, 1929, 69: 629-630), rice (Zhang Tingbi, Zhu Huan, Shu Lihui et al. Research on aneuploidy of Chinese indica rice varieties - plant morphology[J]. Acta Genetica Sinica, 1987, 14(4): 255-261), sugar beet (Guo Dedong, Li Shanyuan, Bai Qingwu et al. Preliminary report on the establishment and identification of sugar beet trisomic series[J]. Acta Genetica Sinica, 1996, 13(1): 27-34), barley (Tsuchiga T. Cytogenetic studies of trisomics in barley[J]. Japanese Journal of Agriculture and Rural Development, 1996, 13(1): 27-34), and barley (Tsuchiga T. Cytogenetic studies of trisomics in barley[J]. Japanese Journal of Agriculture and Rural Development, 1996 ... Primary trisomy of species such as rapeseed (Tang K, Hill CB, Williams P H. Development of trisomics of rapid-cycling Brassica rapa[J]. Eucarpla Cruciferae Newsletter, 1998, 13: 60-61) and millet (Wang Runqi, Gao Junhua, Wang Zhixing et al. Establishment of millet trisomy series[J]. Acta Botanica Sinica, 1994, 36(9): 690-695) was successfully created through this method. The third method for breeding primary trisomy of plants is to use various ionizing rays to irradiate and cause abnormal chromosome division, thereby increasing the frequency of aneuploidy and screening for primary trisomy. Hu Baomin et al. (1996) treated upland cotton seeds with γ-rays, which significantly increased the frequency of aneuploidy in the self-pollinated progeny population and isolated 3 upland cotton trisomy and 2 tetrasomy (Hu Baomin, Zhang Tianzhen, Pan Jiaju. Induction, identification, research and utilization of upland cotton trisomy I: Origin, identification and morphology of 5 trisomy. Acta Agronomica Sinica, 1996, 22(2):147-153). Homotetraploid microspore culture is the fourth approach to selecting plant primary trisomy. Shen Shuxing et al. (2006) created a complete set of primary trisomy of Chinese cabbage by in vitro culture of free microspores of homotetraploid Chinese cabbage (Shen Shuxing, Hou Xilin, Zhang Chenghe. Research on the creation of primary trisomy of Chinese cabbage by microspore culture. Acta Horticulturalis Sinica, 2006, 33(6):1209-1214).Liu Zongxian et al. (1995) identified 272 primary trisomy plants of rice from pollen plants of tetraploid rice (Liu Zongxian et al. Study on screening primary trisomy by anther culture of tetraploid rice. Acta Botanica Sinica, 1995, 37(2):125-133).

[0005] Currently, the identification methods for primary trisomics in plants include: (1) morphological identification (Nobuo I, Takeshi O, Masahiro N. Studies on the trisomics in rice plants (Oryza sativa L.) Ⅰ. Morphological classification of trisomics[J]. Japanese journal of breeding, 1970, 20(4): 230-236); (2) somatic cell mitotic chromosome karyotype analysis (Guo Dedong, Li Shanyuan, Bai Qingwu et al. Preliminary report on the establishment and identification of sugar beet trisomic series[J]. Acta Genetica Sinica, 1996, 13(1): 27-34); (3) pachytene karyotype analysis of geron cells (Sharstry SNS, Ranga RDK, Misra RN, et al. Pachytene analysis in Oryza.Ⅰ. Chromosome morphology in Oryza sativa L. India[J]. Journal of genetics and plant (4) Chromosome banding karyotype analysis (Justus I, Kevin BJ, David H. Production and identification of primary trisomics in diploid Agropyron cristatum (crested wheatgrass) J). Genome, 994, 7: 469-476); (5) Identification of trisomy using chromosome translocation lines (Guo Wangzhen, Yi Chengxin, Tang Canming et al. Induction, identification, research and utilization of upland cotton trisomy IV. Discovery and identification of chromosome 19 [J]. Acta Agronomica Sinica, 1999, 25(2): 145-149). These methods have been widely used for trisomy identification in species such as rice, tomato, maize and upland cotton.

[0006] Poplar is a general term for tree species in the genus *Populus* of the family Salicaceae. It is characterized by rapid growth, strong resistance to adverse conditions, and wide adaptability, and is one of the five major afforestation tree species in northern China. To date, no research reports have been found on the creation and identification of primary trisomy in poplar. Although artificially induced triploid poplars have entered their reproductive growth stage and a certain amount of pollen can be obtained, hybridization of diploid and triploid poplars can produce a large number of aneuploid offspring. However, using traditional morphological methods, karyotype analysis, chromosome banding techniques, and fluorescence in situ hybridization (FISH) for screening primary trisomy plants is time-consuming, labor-intensive, and inefficient. Therefore, there is an urgent need to develop a more efficient method for screening and identifying poplar trisomy. Summary of the Invention

[0007] One of the objectives of this invention is to provide an SSR molecular marker that can accurately identify all 19 chromosomes covering the poplar genome;

[0008] The second objective of this invention is to use the screened SSR molecular markers to screen primary trisomy from aneuploid poplar progeny.

[0009] The above-mentioned objective of the present invention is achieved through the following technical solution:

[0010] This invention first provides 19 pairs of SSR primers that accurately identify 19 chromosomes covering the poplar genome; wherein, the nucleotide sequences of the first SSR primer pair are shown in SEQ ID No. 1-2, the second SSR primer pair are shown in SEQ ID No. 3-4, the third SSR primer pair are shown in SEQ ID No. 5-6, the fourth SSR primer pair are shown in SEQ ID No. 7-8, the fifth SSR primer pair are shown in SEQ ID No. 9-10, the sixth SSR primer pair are shown in SEQ ID No. 11-12, the seventh SSR primer pair are shown in SEQ ID No. 13-14, the eighth SSR primer pair are shown in SEQ ID No. 15-16, the ninth SSR primer pair are shown in SEQ ID No. 17-18, and the tenth SSR primer pair are shown in SEQ ID No. 10. As shown in SEQ ID Nos. 19-20, the nucleotide sequences of the 11th SSR primer pair are shown in SEQ ID Nos. 21-22, the 12th SSR primer pair is shown in SEQ ID Nos. 23-24, the 13th SSR primer pair is shown in SEQ ID Nos. 25-26, the 14th SSR primer pair is shown in SEQ ID Nos. 27-28, the 15th SSR primer pair is shown in SEQ ID Nos. 29-30, the 16th SSR primer pair is shown in SEQ ID Nos. 31-32, the 17th SSR primer pair is shown in SEQ ID Nos. 33-34, the 18th SSR primer pair is shown in SEQ ID Nos. 35-36, and the 19th SSR primer pair is shown in SEQ ID Nos. 37-38.

[0011] Based on known Populus tomentosa SSR primer sequences, this invention identified 180 primer pairs through a database and screened for specific primers. During the screening process, DNA samples from parents of different ploidy were used for PCR amplification using TP-M13-SSR PCR technology. According to Schuelke's method (Schuelke M. An economic method for the fluorescent labeling of PCR fragments. Nature biotechnology, 2000, 18(2):233-234.), SSR requires three types of primers: an upstream primer, a downstream primer, and fluorescent primers labeled with fluorescence (ROX, FAM, TAMRA, HEX). Genomic DNA from parents of different ploidy was used to amplify 180 pairs of polymorphic SSR primers. The data results were analyzed using GeneMarker V2.2.0 software, and CERVUS 3.0 software was used to screen for SSR primer pairs that could be used for the identification of the 19 chromosomes of the Populus tomentosa genome. Finally, 19 pairs of SSR primer pairs with stable amplification bands and good polymorphism were selected.

[0012] Of the 19 effective SSR primer pairs selected, primer pair PTSSR2102 is located on chromosome 1; primer pair Ptr-2-SSR56 is located on chromosome 2; primer pair GCPM-2151 is located on chromosome 3; primer pair MB71513 is located on chromosome 4; primer pair LG-V-2 is located on chromosome 5; primer pair 12 is located on chromosome 6; primer pair LG-III is located on chromosome 7; primer pair Ptr-8-SSR56 is located on chromosome 8; primer pair PTSSR646 is located on chromosome 9; and primer pair Ptr-10-SS... R12 is located on chromosome 10; primer pair SSR16 is located on chromosome 11; primer pair Ptr-12-SSR53 is located on chromosome 12; primer pair Ptr-13-SSR40 is located on chromosome 13; primer pair PTSSR1259 is located on chromosome 14; primer pair PTSSR1817 is located on chromosome 15; primer pair SSR8 is located on chromosome 16; primer pair MB70257 is located on chromosome 17; primer pair GCPM-1577 is located on chromosome 18; and primer pair PTSSR1526 is located on chromosome 19. Each of these 19 primer pairs is specific, amplifying only the alleles of the corresponding chromosome. Therefore, these 19 selected SSR primer pairs can be used for chromosome identification and number determination in poplar aneuploid progeny populations.

[0013] Based on this, the present invention further provides a method for selecting poplar primary trisomy using the 19 pairs of SSR primers described above, comprising the following steps:

[0014] (1) Collect fresh fresh pollen from triploid male poplar clones and cross them with fertile female poplar plants to obtain aneuploid offspring populations;

[0015] (2) The chromosome number of individuals in the aneuploid progeny population was identified by using the 19 pairs of SSR primers selected, and suspected poplar trisomy plant offspring were preliminarily screened.

[0016] In a preferred embodiment of the present invention, the poplar species is Populus tomentosa, Populus adenlpodn, Populus tomentosa × Populus balleana, Populus alba × Populus glandulosa, or a hybrid thereof.

[0017] As a preferred embodiment of the present invention, the triploid poplar male clones include: 'Beilin Male No. 1', 'Beilin Male No. 2', or 'Sanmao Yang No. 8'.

[0018] In a preferred embodiment of the present invention, the female poplar trees include *Populus simonii* or *Populus pubescens*.

[0019] As a preferred embodiment of the present invention, the identification in step (3) includes: using the genomic DNA of the aneuploid progeny population as a template, performing PCR amplification using the selected SSR primer pairs, and performing capillary electrophoresis on the PCR amplification products; analyzing the data results using GeneMarker V2.2.0 software, screening using CERVUS3.0 software, determining the chromosome number of hybrid progeny based on the position and peak size of the capillary electrophoresis bands, and initially screening out suspected poplar trisomic progeny.

[0020] In a preferred embodiment, the present invention further includes: using chromosome counting methods such as the Carbopol smear method to count the chromosome number of the preliminarily screened suspected poplar primary trisomy plants, and finally determining whether the suspected poplar primary trisomy is a true poplar primary trisomy.

[0021] The present invention further provides a PCR kit for identifying chromosomes in aneuploid poplar progeny plants, comprising: 19 selected SSR primer pairs, PCR Mix, fluorescent primers, and ddH2O.

[0022] This invention utilizes the genomic DNA of parents with different ploidy to screen SSR primer pairs from 180 pairs of polymorphic SSR primers that can be used to identify the 19 chromosomes of the poplar genome. Finally, 19 pairs of specific SSR primer pairs with stable amplification bands and good polymorphism were selected. These 19 pairs of SSR primer pairs can only amplify the alleles of the corresponding chromosomes. Therefore, these 19 pairs of SSR primers can be used for chromosome identification and number determination in aneuploid offspring populations.

[0023] This invention further utilizes the extreme imbalance of homologous chromosome segregation during meiosis in triploid poplar pollen mother cells to obtain aneuploid pollen and pollinate it with normal haploid female gametes to establish an aneuploid progeny population. Then, PCR amplification is performed using 19 selected SSR primer pairs. Based on the chromosome number of the aneuploid progeny individuals, poplar trisomy progeny are initially screened. Finally, the chromosome number of the initially screened trisomy plants is confirmed by chromosome counting to determine whether the initially screened suspected primary poplar trisomy plants are true poplar trisomy plants. Attached Figure Description

[0024] Figure 1 The amplification effects of three types of SSR primers in parents and offspring are shown.

[0025] Figure 2 The amplification effect of 19 pairs of specific primers in progeny 778 is shown.

[0026] Figure 3 The amplification effect of 19 pairs of specific primers in progeny 787 is shown.

[0027] Figure 4 The chromosome number of suspected poplar trisomic offspring; (a) chromosome number of diploid offspring; (b) chromosome number of trisomic offspring 778; (c) chromosome number of trisomic offspring 787. Detailed Implementation

[0028] The present invention will be further described below with reference to specific embodiments, and the advantages and features of the present invention will become clearer as a result. However, these embodiments are merely exemplary and do not constitute any limitation on the scope of the present invention. Those skilled in the art should understand that modifications or substitutions to the details and form of the present invention can be made without departing from the spirit and scope of the invention, but all such modifications and substitutions fall within the protection scope of the present invention.

[0029] Example 1: Screening for SSR molecular markers covering 19 chromosomes of the poplar genome and their application in the screening and identification of primary trisomic progeny of poplar.

[0030] 1. Materials and Methods

[0031] 1.1 Materials

[0032] After the leaves fall in winter, male Populus tomentosa trees with no diseases or pests and full flower buds, known as 'Beilin Male Tree No. 1', were collected from Weixian County, Hebei Province, while female Populus yunnanensis trees with flower branches were collected from Guanxian County, Shandong Province. The collected flowers were wrapped in plastic sheeting and transported to a greenhouse for later use.

[0033] 1.2 Pollen Collection

[0034] Hydroponically culture the flower branches of 'Beilin Male No. 1' in a greenhouse, changing the water every 2-3 days. After the anthers mature and dehisce, collect the pollen of 'Beilin Male No. 1', store it in 10mL centrifuge tubes, add an appropriate amount of silica gel, label the tubes, seal them with plastic wrap, and store them in a -20℃ refrigerator for later use.

[0035] 1.3 Hybridization

[0036] The flowering branches of *Populus amurensis* were hydroponically cultured in a greenhouse, with the water changed every 2-3 days. When the stigma of the pistil of *Populus amurensis* reached the optimal pollination period, pollination with pollen from 'Beilin Male Plant No. 1' was performed on the stigma. After pollination, the female flowering branches of *Populus amurensis* continued to be hydroponically cultured in the greenhouse.

[0037] 1.4 Seed Collection and Sowing

[0038] When the capsules of *Populus alba* mature and split open, promptly bag them, collect the seeds, and store them in 10mL centrifuge tubes containing an appropriate amount of silica gel. Mix peat moss, perlite, and vermiculite evenly in a 5:2:1 ratio, fill the mixture into seedling trays, compact it, and water thoroughly with tap water. Sow the seeds in the trays and label them with sticks. Seeds will begin to germinate 3-5 days after sowing. During seedling growth, ensure adequate moisture and promptly remove weeds.

[0039] 1.5 Genomic DNA Extraction

[0040] Once the seedlings have grown to 5-10cm, fresh leaves are picked, and genomic DNA is extracted from the female parent plant of *Populus alba*, the male parent plant of 'Beilin Male Plant No. 1', and the hybrid offspring plants using the plant genomic DNA extraction kit from Tiangen Biotech (Beijing) Co., Ltd.

[0041] 1.6 Polymorphic primer screening

[0042] Based on the known Populus tomentosa SSR primer sequences, 180 primer pairs (listed in Table 2) were selected from the database (http: / / phytozome.jgi.doe.gov / pz / portal.html) for primer screening. During the screening process, TP-M13-SSR PCR was performed using parental DNA samples. Following the method of Schuelke (Schuelke M. An economic method for the fluorescent labeling of PCR fragments. Nature biotechnology, 2000, 18(2):233-234.), SSR requires three types of primers: an upstream primer, a downstream primer, and fluorescent primers labeled with fluorescence (ROX, FAM, TAMRA, HEX). The PCR reaction system is shown in Table 1.

[0043] Table 1 PCR amplification reaction system

[0044]

[0045] The specific procedure is as follows: 94℃ for 5 min; 94℃ for 30 sec, followed by annealing at the optimal temperature of the primers for 30 sec, and then 72℃ for 30 sec for a total of 25 cycles; 94℃ for 30 sec, 53℃ for 30 sec, and 72℃ for 30 sec for a total of 8 cycles; 72℃ for 8 min, and then stored at 4℃. The obtained PCR amplification products were detected by capillary electrophoresis on an ABI-3730xl gene analyzer; the data results were analyzed using GeneMarker V2.2.0 software, and SSR primer pairs suitable for paternal identification of offspring individuals were screened using CERVUS 3.0 software.

[0046] 1.7 Screening and Identification of Trisomic Offspring

[0047] Using progeny genomic DNA as a template, amplification was performed using selected SSR polymorphic primers. Analysis was performed using CERVUS 3.0 software, and the chromosome number of hybrid progeny was determined based on capillary electrophoresis band positions and peak sizes, allowing for the preliminary screening of trisomic progeny. Shoot tip tissues from suspected trisomic plants were collected, pretreated in a saturated p-dichlorobenzene solution for 2-3 hours, and then transferred to Carnoy fixative and fixed at 4°C for 24 hours. The fixed shoot tip material was rinsed 2-3 times with distilled water for 10 minutes each time. After blotting the surface moisture with filter paper, the material was dissociated in concentrated hydrochloric acid for 25 minutes, washed twice with distilled water for 10 minutes each time, and then softened in 45% acetic acid for later use. A small amount of softened stem tip material was placed on a glass slide, and one drop of modified carbofuran staining solution was added. The material was crushed with tweezers and pressed into a slide. Chromosomes were observed and photographed using an Olympus BX 51 optical microscope to determine the chromosome number of suspected trisomic offspring.

[0048] 2. Experimental Results

[0049] 2.1 Screening of Polymorphic Primers

[0050] Using the genomic DNA of the parents, 180 pairs of SSR primers with good polymorphism (Table 2) were amplified. Finally, 19 pairs of SSR primers with stable amplification bands and good polymorphism were screened out. Detailed information of these 19 effective SSR primers is shown in Table 3, and their locus information in the parents is shown in Table 4.

[0051] Table 2 180 Primer Sequences

[0052]

[0053]

[0054]

[0055]

[0056]

[0057]

[0058] Table 4 Parental loci of 19 pairs of SSR primers

[0059]

[0060] Further analysis of the 19 polymorphic SSR primers selected revealed that these primers can be divided into three types: the first type is where the parents do not share a common allele, taking primer 12 on chromosome 6 as an example ( Figure 1-a) In the maternal parent, *Populus simonii*, the allele configuration is heterozygous, with two loci at 248.0 bp and 298.5 bp. The paternal parent, 'Beilin Male No. 1', has a different allele configuration, with three loci at 235.6 bp, 237.2 bp, and 243.4 bp. Therefore, the genetic information of the hybrid offspring can be determined based on the band positions. The second type is a heterozygous genotype with polymorphism detected in the maternal parent, but the maternal and paternal parents share a common pair of alleles. Taking primer PTSSR646 as an example (…). Figure 1 -b) In the maternal parent, the allele configuration is heterozygous, with two loci at 185.4 bp and 194.1 bp, while the paternal parent has the same 194.1 bp as the maternal parent (Populus alba), plus two more loci at 196.7 bp and 202.5 bp; the third type is where the maternal parent shows a heterozygous genotype, but the paternal parent has two pairs of common alleles with both the maternal parent, taking primer Ptr-12-SSR53 as an example ( Figure 1 -c) This primer amplified two bands in the maternal parent, located at 232.2 bp and 244.9 bp, and three bands in the paternal parent, located at 226.0 bp, 232.2 bp, and 244.9 bp, respectively. Two bands were located at the same positions in both parents. When using the second and third types of SSR primers to screen trisomic offspring, in addition to referring to the band positions, it is also necessary to analyze the genetic configuration based on the dose-response relationship of the primers in the offspring.

[0061] from Figure 2 It can be seen that primers PTSSR2102, Ptr-8-SSR56, PTSSR1817, SSR8, and PTSSR1526 showed only one band in progeny 778, indicating that the progeny inherited common alleles from both the maternal and paternal parents on chromosomes 1, 8, 15, 16, and 19, thus exhibiting a dose-effect relationship. Primers Ptr-2-SSR56, GCPM-2151, LC-V-2, 12, LG-Ⅲ, PTSSR646, Ptr-10-SSR12, Ptr-12-SSR53, Ptr-13-SSR40, PTSSR1259, MB70257, and GCPM-1577 showed heterozygous genotypes in progeny 778, with one from the maternal parent and the other inherited from the paternal parent. Primer SSR16 on chromosome 11 amplified two loci in offspring 778. Based on the dose-response relationship, it can be easily determined that offspring 778 inherited both the maternal locus 195.7 and the paternal heterozygous locus 195.7 / 201.7. The 19 selected primer pairs are all specific, amplifying only the alleles of their corresponding chromosomes. Therefore, these 19 SSR primer pairs can be used for chromosome identification and number determination in aneuploid offspring populations. In summary, offspring 778 can be preliminarily identified as a trisomic offspring of chromosome 11.

[0062] Similarly, using the 19 selected specific primer pairs and the same discrimination method, it was determined that offspring 787 has three copies of chromosome 17. Therefore, offspring 787 is a trisomic offspring with chromosome 17. Figure 3 As shown.

[0063] Further microscopic examination of two suspected trisomic plants, 778 and 787, using somatic cell chromosome counting was performed. The control diploid plant had 38 chromosomes. Figure 4 -a), the trisomic plants 778 and 787 have 39 chromosomes. Figure 4 -b, c) indicates that both suspected trisomy plants are true trisomy.

Claims

1. 19 pairs of SSR primer pairs for identifying 19 chromosomes of Populus genome, characterized in that, The nucleotide sequences of the first SSR primer pair are shown in SEQ ID No. 1-2, the second SSR primer pair is shown in SEQ ID No. 3-4, the third SSR primer pair is shown in SEQ ID No. 5-6, the fourth SSR primer pair is shown in SEQ ID No. 7-8, the fifth SSR primer pair is shown in SEQ ID No. 9-10, the sixth SSR primer pair is shown in SEQ ID No. 11-12, the seventh SSR primer pair is shown in SEQ ID No. 13-14, the eighth SSR primer pair is shown in SEQ ID No. 15-16, the ninth SSR primer pair is shown in SEQ ID No. 17-18, the tenth SSR primer pair is shown in SEQ ID No. 19-20, and the eleventh SSR primer pair is shown in SEQ ID No. 19-20. As shown in Nos. 21-22, the nucleotide sequences of the 12th SSR primer pair are shown in SEQ ID Nos. 23-24, the nucleotide sequences of the 13th SSR primer pair are shown in SEQ ID Nos. 25-26, the nucleotide sequences of the 14th SSR primer pair are shown in SEQ ID Nos. 27-28, the nucleotide sequences of the 15th SSR primer pair are shown in SEQ ID Nos. 29-30, the nucleotide sequences of the 16th SSR primer pair are shown in SEQ ID Nos. 31-32, the nucleotide sequences of the 17th SSR primer pair are shown in SEQ ID Nos. 33-34, the nucleotide sequences of the 18th SSR primer pair are shown in SEQ ID Nos. 35-36, and the nucleotide sequences of the 19th SSR primer pair are shown in SEQ ID Nos. 37-38.

2. The 19 pairs of SSR primers according to claim 1, characterized in that, The poplar is selected from the group consisting of Populus tomentosa Carr. Populus tomentosa ) or Populus alba L. P. alba × P. glandulosa .

3. Use of the 19 pairs of SSR primer pairs of claim 1 in identifying or characterizing the chromosome number of a poplar aneuploid progeny plant; said poplar being selected from the group consisting of Populus tomentosa Carr. ( Populus tomentosa ) or Populus alba L. x Populus tremula L. ( P. alba × P. glandulosa ).

4. The application according to claim 3, characterized in that, Includes the following steps: (1) Collect fresh fresh pollen from triploid male poplar clones and hybridize them with fertile female poplars to obtain aneuploid offspring populations; (2) Using the 19 pairs of SSR primers of claim 1 to identify or determine the chromosome number of individuals in the aneuploid progeny population, and screen out suspected poplar primary trisomy plant offspring.

5. The application according to claim 4, characterized in that, The triploid poplar male clones mentioned above include Beilin Male Poplar No. 1, Beilin Male Poplar No. 2, or Sanmao Poplar No.

8.

6. The application according to claim 4, characterized in that, The female poplar tree mentioned is the silver gland poplar.

7. The application according to claim 4, characterized in that, The step (2) described in which the chromosome number of individuals in the aneuploid progeny population is identified or determined using the 19 pairs of SSR primers of claim 1 includes: using the genomic DNA of the aneuploid progeny population as a template, performing PCR amplification using the 19 pairs of SSR primers of claim 1, performing capillary electrophoresis on the PCR amplification products, analyzing them using GeneMarker V2.2.0 software, screening them using CERVUS 3.0 software, determining the chromosome number of hybrid progeny based on the position and peak size of the capillary electrophoresis bands, and screening out suspected poplar primary trisomy plant progeny.

8. The application according to claim 4, characterized in that, Also includes: Chromosome number counting was used to identify suspected poplar primary trisomy plants to determine whether they were true poplar primary trisomy.

9. A PCR kit for identifying chromosomes in aneuploid poplar progeny plants, comprising: SSR primers, PCR Mix, fluorescent primers, and ddH2O; characterized in that the SSR primers are the 19 pairs of SSR primers as described in claim 1.

10. The PCR kit according to claim 9, characterized in that, The poplar trees mentioned are selected from white poplar (Populus tomentosa). Populus tomentosa ) or Silver Gland Poplar ( P. alba × P. glandulosa ).

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