EST-SSR marker developed based on pennisetum sinese transcriptome sequence and application

Abstract

The invention belongs to the technical field of molecular biology, and particularly relates to an EST-SSR marker developed based on a pennisetum sinese transcriptome sequence and application. The EST-SSR marker provided by the invention can be used for germplasm resource identification, genetic diversity analysis, fingerprint construction, molecular marker-assisted breeding and the like of cenchrus fungus grass.

Description

Technical Field

[0001] The present invention belongs to the technical field of molecular biology, and in particular relates to an EST-SSR marker developed based on the transcriptome sequence of giant fungus Herba Lycopodii and its application. Background Art

[0002] Transcriptome sequencing (RNA-seq), a DNA sequencing technology, uses high-throughput deep sequencing of cDNA sequences generated by reverse transcription of mRNA, thereby comprehensively and rapidly obtaining information on all transcripts in a given species. Compared to other sequencing technologies, RNA-seq offers advantages such as low cost, high sensitivity, and high throughput. It has been applied to grain crops such as wheat, rice, and corn, as well as important forage grasses such as alfalfa, perennial ryegrass, and sheep fescue, primarily focusing on research on plant growth and development, resistance mechanisms, and molecular marker development. Pennisetum giganteum, a member of the genus Pennisetum in the Poaceae family, is characterized by rapid growth, high biomass, high crude protein and sugar content, and strong adaptability. Currently, Pennisetum giganteum is widely cultivated as a high-quality grass, forage, and ecological management material. It is a highly representative species within the Pennisetum genus, making it a viable choice for developing EST-SSR markers.

[0003] Currently, my country has a relatively rich collection of Juncao germplasm resources, but the types, quantity, and quality of available molecular markers are relatively limited, which restricts research in areas such as kinship identification, genetic diversity analysis, and variety protection and utilization. This also hinders the efficient discovery of superior germplasm materials and the creation of new germplasm. Therefore, this application proposes a method for developing EST-SSR markers based on the transcriptome sequence of Juncao to provide a basis for the rapid and accurate identification and utilization of Juncao germplasm. Summary of the Invention

[0004] In order to solve the above technical problems, the present invention provides an EST-SSR marker developed based on the transcriptome sequence of giant grass and its application.

[0005] The present invention is achieved by providing an EST-SSR marker developed based on the transcriptome sequence of giant grass, wherein the EST-SSR marker comprises a combination of the following 10 pairs of EST-SSR primers:

[0006] .

[0007] Provided is the application of the above-mentioned EST-SSR markers in the identification of Tribulus truncatula germplasm resources, genetic diversity analysis, fingerprint map construction and molecular marker-assisted breeding.

[0008] Preferably, the application of the EST-SSR markers in the identification and differentiation of Tribulus truncatula germplasm comprises the following steps:

[0009] 1) Extract DNA from different Juncao germplasms of the genus Tribulus to be identified;

[0010] 2) using the DNA obtained in step 1) as a template, and performing PCR amplification using the EST-SSR marker of claim 1 to obtain an amplified product;

[0011] 3) Performing electrophoresis on the amplified product obtained in step 2), reading the electrophoresis band data, and counting the band patterns of the samples. Different Juncao germplasms of the genus Tribulus are identified and differentiated by comparing the band patterns.

[0012] Provided is a kit for identifying and differentiating Tribulus truncatula germplasm, comprising the EST-SSR marker.

[0013] Compared with the prior art, the advantages of the present invention are:

[0014] The present invention provides an EST-SSR marker developed based on the transcriptome sequence of giant fungus grass and its application, which has the advantages of:

[0015] 1. Using Illumina high-throughput sequencing technology to perform RNA-seq on giant fungus grass, a large number of EST-SSR marker sites can be obtained, thereby enriching the number of molecular markers of the genus Tribulus forage grass; 2. Based on the Unigene sequence, Primer 5.0 was used for primer design to develop EST-SSR marker primer combinations, providing a new path for obtaining new primers; 3. Using polyacrylamide gel electrophoresis technology, suitable EST-SSR primers were screened to obtain highly polymorphic EST-SSR primer combinations, providing a reliable and convenient reference for subsequent research on the diversity of the genus Tribulus forage grass at the molecular level; 4. The newly developed EST-SSR markers were used to construct a fingerprint map, providing a molecular basis for the protection and utilization of fungus grass germplasm resources. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 Annotate species distribution for Nr function;

[0017] Figure 2 GO functional classification for Unigene;

[0018] Figure 3 Classify KOG annotations for Unigene;

[0019] Figure 4Figure 3 is the electrophoresis diagram of PCR amplification of 20 Juncao materials using primer SSR-JJC11 (1-20 represent elephant grass, Nanmu No. 2, hybrid elephant grass No. 1, hybrid elephant grass No. 2, Zhangzhou elephant grass, giant jungle grass No. 1, thick-stem elephant grass, king grass, purple elephant grass, Nanmu No. 1, dwarf elephant grass, giant jungle grass, elephant grass N51, elephant grass N85, sweet elephant grass, Minmu No. 6, Mote dwarf elephant grass, elephant grass-LY, elephant grass-XM, and giant jungle grass-BZ, respectively). DETAILED DESCRIPTION

[0020] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention will be further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0021] Example 1: Development of EST-SSR marker primers for giant fungus grass

[0022] 1. Test materials

[0023] The material was Giant Juncao. Three tissue samples of roots, stems, and leaves were collected from the seedling stage of Giant Juncao. All samples were mixed, with three replicates for each tissue sample. After quick freezing in liquid nitrogen, they were stored in a -80°C ultra-low temperature freezer until use.

[0024] 2. RNA and DNA extraction and detection

[0025] Total RNA from the giant herb was extracted using the Trizol method using a plant genomic RNA extraction kit. RNA integrity was assessed using an Agilent 2100, and concentration and purity were determined using a NanoDrop 2000 spectrophotometer. RNA that passed these tests was used for cDNA library construction.

[0026] DNA was extracted from G. gigantea using a plant genomics kit, and its quality and concentration were determined. DNA purity was assessed using a UV gel imaging system and a NanoDrop ND 2000 spectrophotometer. Sample DNA was diluted to 50 ng / μL and used for PCR amplification using EST-SSR marker primers.

[0027] 3. cDNA library construction and quality control

[0028] RNA from each tissue sample was diluted to 80 ng / µL. Using a pipette, equal volumes of RNA from each tissue sample were transferred to the same 2 mL centrifuge tube and mixed thoroughly. The RNA from the giant herb was then mixed for cDNA library construction. Initial quantification of the constructed library was performed using a Qubit 3.0 fluorometer, requiring a concentration greater than 1 ng / µL. Inserts were then analyzed using a Qsep 400 high-throughput analysis system. Once the inserts met expectations, the effective concentration of the library was accurately quantified by q-PCR (>2 nM) to ensure library quality.

[0029] 4. Transcriptome sequencing and data assembly

[0030] Transcriptome sequencing (RNA-seq) was performed using the high-throughput sequencing platform PE150. The sequencing raw reads were broken into short fragments (K-mer) using Trinity software and then extended into long fragments (Contig). Finally, high-quality CleanData was assembled to obtain transcript sequences (Transcript), and Unigene was statistically analyzed.

[0031] RNA-seq was performed on P. gigantea using the PE150 sequencing platform, and a total of 21 462 224 reads were obtained, with 6.36 Gb of high-quality bases, a GC content of 53.89%, an error rate of 0.04%, and Q20 and Q30 values ​​of 97.95% and 94.73%, respectively (Table 1). This indicates that the sequencing data of P. gigantea are of high quality and fully meet the requirements of subsequent bioinformatics analysis.

[0032] Table 1. Statistics of the evaluation of the sequencing data of giant grass

[0033] 5. Unigene functional annotation

[0034] The Unigene sequences were compared with the NR, Swiss-Prot, COG, KOG, eggNOG, and KEGG databases using DIAMOND software, the GO results of the new genes were analyzed using the InterPro integrated database, and the HMMER software was used to compare with the Pfam database to obtain the Unigene annotation information.

[0035] The assembled unigenes were compared and annotated against nine functional databases using BLAST and HMMER, respectively, with F-values ​​set to e ≤ 10⁻⁵ and e ≤ 10⁻⁵. The results are shown in Table 2. A total of 4700 unigenes were successfully annotated for P. gigantea. TrEMBL and Nr had the largest number of annotated unigenes, accounting for 4650 (44.2%) and 4404 (41.9%) of the total unigenes, respectively. GO had 2894 unigenes (27.5%), eggNOG had 2848 (27.1%), Pfam had 2262 (21.5%), KEGG had 2027 (19.3%), and Swissprot had 1950 (18.5%). KOG has 1,534 entries (accounting for 14.6% of all Unigenes), while GOG has the fewest Unigenes annotated, with only 704 entries (accounting for 6.7% of all Unigenes).

[0036] Table 2 Unigene annotation statistics of Giant Juncus

[0037] 5.1 Nr functional annotation

[0038] Depend on Figure 1 As shown in the Nr database, a total of 4404 Unigenes of Giant Juncus were found to be homologous sequences. Among them, the species with the largest number of annotated genes was Setaria italica, with 1740, accounting for 39.51% of the highest homology; followed by Setaria viridis, Panicum millaceum, Zea mays, Panicum hallii, Digitaria exilis, and Sorghum bicolor, with 784, 329, 266, 253, 179, and 161 Unigenes, accounting for 17.80%, 7.47%, 6.04%, 5.74%, 4.06%, and 3.66%, respectively; and Eragrostis curvula, Dichanthelium oligosanthes, and Panicum The homology of the three plants of halliivar is relatively low, with 125, 119 and 115 unigenes respectively, accounting for 2.84%, 2.70% and 2.61% respectively, which is greater than 2.00%; in addition, 333 unigenes match other species, accounting for 7.56%.

[0039] 5.2 GO functional annotation classification

[0040] The unigenes of giant grass were functionally classified in the GO database and annotated into three categories: biological process, molecular function and cellular component, including 41 subcategories ( Figure 2 Among them, there are 65,716 unigenes in the 15 subcategories annotated to molecular functions, with the highest proportion being the molecular binding subcategory and the catalytic activity subcategory, with 33,062 and 25,101 annotated genes, respectively. There are relatively few genes expressing small molecule sensor activity, molecular carrier activity, and toxin activity. There are 48,375 unigenes in the three subcategories annotated to cellular components, with the highest proportion being the cellular anatomical activity and intracellular subcategories, with 29,649 and 14,449 annotated genes, respectively. The protein-containing complex subcategory has the least proportion, with 4,277 genes. There are 78,282 unigenes in the 23 subcategories annotated to biological processes, with the cellular process, metabolic process, biological regulation, and response subcategories. The subcategories of stimulation and localization accounted for the highest proportion, with 26 605, 24 980, 8 190, 5 631 and 4 389 annotations, respectively. However, the expression of genes related to carbon utilization, biological adhesion, nitrogen utilization and pigmentation was relatively low.

[0041] 5.3 KEGG metabolic pathway analysis

[0042] As shown in Table 3, KEGGG metabolic pathway enrichment analysis of unigenes obtained from RNA-sep analysis of P. gigantea revealed a total of 2,027 unigenes, accounting for 19.3% of the total unigenes. A total of 115 metabolic pathways were involved, with 24 significantly enriched pathways, and the number of unigenes ranged from 139 to 20. The most abundant pathways were ribosomes, plant-pathogen interactions, endocytosis, oxidative phosphorylation, plant hormone signaling, and ubiquitin-mediated proteolysis, with 139, 110, 59, 53, and 49 unigenes, respectively. Amino sugar and nucleotide sugar metabolism and the MAPK signaling pathway each had 40 unigenes involved. Benzoxazine biosynthesis and amino acid biosynthesis had the fewest unigenes, with 21 unigenes each.

[0043] Table 3 Some KEGG metabolic pathways of the Unigene of the transcriptome of Giant Juncus

[0044] 5.4 KOG Annotation Classification

[0045] The KOG functional annotation classification of the Unigenes of Giant Juncus was performed, and a total of 1534 Unigenes were annotated, accounting for 14.6% of the total Unigenes, and were divided into 25 functional categories ( Figure 3Among them, the general function prediction showed that the number of genes in the three categories of post-translational modification, protein turnover, molecular chaperone and translation, and ribosome structure and biogenesis was the largest, with 271 (17.67%), 227 (14.8%), and 160 (10.43%) Unigenes, respectively. The next categories were unknown function (121, 7.89%), signal transduction mechanism (119, 7.76%), intracellular transport, secretion and vesicle transport (107, 6.98%), energy production and conversion (95, 6. 19%), transcription (92, 6%), carbohydrate transport and metabolism (60, 3.91%), biosynthesis, transport, and catabolism of secondary metabolites (59, 3.85%), RNA processing and modification (53, 3.46%), inorganic ion transport and metabolism (49, 3.19%), replication, recombination, and repair (44, 2.87%), amino acid transport and metabolism (43, 2.8%), and cell cycle control, cell division, and chromosome division (37, 2.41%). Cytoskeleton, lipid transport and metabolism, nucleotide transport and metabolism, defense mechanisms, a very small number of chromosome structure and dynamics, and cell wall / membrane biogenesis were represented in relatively small numbers (all less than 2%). Only nuclear structure, coenzyme transport and metabolism, extracellular structure, and cell motility were less than 0.8%. Therefore, the genes in giant fungus grass are the most numerous for function prediction, post-translational modification, protein turnover, molecular chaperones and translation, ribosome structure and biology, followed by genes involved in signal transduction mechanism, intracellular transport, secretion and vesicle transport, energy production and conversion, transcription, etc., and the genes for defense mechanism, cell movement, etc. are the least numerous.

[0046] 6. Development of SSR molecular markers and screening of polymorphic primers

[0047] Unigene sequences were aligned using STAR software, and SSR loci were mined using MISA software and the obtained Unigene sequences. Using DNA from five Juncao accessions as templates, the initially obtained EST-SSR primers were screened for polymorphisms, and complete primer sequences were synthesized. EST-SSR PCR systems were established, gene fragments were amplified, and products were analyzed by gel electrophoresis according to existing methods.

[0048] According to the SSR detection results, the front or back sequence of the SSR site was removed and imported into Primer5.0 primer design software to obtain the primer sequences for EST-SSR markers, from which 42 pairs of suitable primers with rich polymorphisms and clear amplification bands were screened. The primers were named and numbered (Table 4). Primers ranged in length from 18 to 20 bp, and the expected product fragments were all larger than 200 bp. Primers SSR-JJC17 and SSR-JJC35 were the largest, each with a length of 500 bp. SSR-JJC36, SSR-JJC3, SSR-JJC4, SSR-JJC18, SSR-JJC5, SSR-JJC19, and SSR-JJC20 were the next largest, with fragments ranging from 591 to 480 bp. SSR-JJC33, SSR-JJC15, SSR-JJC34, and SSR-JJC16 were the smallest, with fragments of 283, 278, 275, and 233 bp, respectively. The development of these SSR marker primers will lay the foundation for molecular identification of G. gigantea germplasm resources, genetic diversity, and fingerprint construction.

[0049] Table 4 EST-SSR primer information for polymorphic strains of Glechoma longituba

[0050]

[0051]

[0052] Example 2: Screening of highly polymorphic primers for identification, differentiation, and fingerprint construction of different Juncao germplasms of the genus Tribulus

[0053] PCR amplification was performed on 20 Juncao germplasm materials of the genus Tribulus (Elephant Grass, Nanmu No. 2, Hybrid Elephant Grass No. 1, Hybrid Elephant Grass No. 2, Zhangzhou Elephant Grass, Giant Juncao No. 1, Thick Stem Elephant Grass, King Grass, Purple Elephant Grass, Nanmu No. 1, Dwarf Elephant Grass, Giant Juncao, Elephant Grass N51, Elephant Grass N85, Sweet Elephant Grass, Minmu No. 6, Mote Dwarf Elephant Grass, Elephant Grass-LY, Elephant Grass-XM, and Giant Juncao-BZ) using the EST-SSR primers in Example 1. Ultimately, 10 pairs of primers with clear bands, easy identification, and stable amplification were obtained (Table 5). The number of polymorphic sites ranged from 2 to 7, the polymorphism ratios ranged from 30.00% to 58.33, and the average polymorphism ratio was 46.59%, indicating that these 10 pairs of EST-SSR marker primers had good polymorphism and strong reliability. A fingerprint map containing 15 marker sites was constructed for the 20 materials mentioned above using primer SSR-JC11, which could clearly distinguish the 20 materials and played a good identification role (reference Figure 4 , Table 6).

[0054] Table 5 Amplification results of 10 pairs of EST-SSR polymorphism primers

[0055]

[0056] Table 6 Digital fingerprints of 20 Juncao materials

[0057] .

Claims

1. EST-SSR markers developed based on the transcriptome sequence of giant grass are characterized by: The EST-SSR markers include the following 10 pairs of EST-SSR primers: 。 2. Application of the EST-SSR marker according to claim 1 in identification of Juncao germplasm resources of the genus Tribulus, genetic diversity analysis, fingerprint construction and molecular marker-assisted breeding.

3. The use of the EST-SSR marker according to claim 2 in the identification and differentiation of Tribulus truncatula germplasm, characterized in that: The steps include: 1) Extract DNA from different Juncao germplasms of the genus Tribulus to be identified; 2) using the DNA obtained in step 1) as a template, and performing PCR amplification using the EST-SSR marker of claim 1 to obtain an amplified product; 3) Performing electrophoresis on the amplified product obtained in step 2), reading the electrophoresis band data, and counting the band patterns of the samples. Different Juncao germplasms of the genus Tribulus are identified and differentiated by comparing the band patterns.

4. A kit for identifying and differentiating Tribulus terrestris germplasm, characterized in that: Including the EST-SSR marker according to claim 1.

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