Detection primer pair of EST-SSR (expressed sequence tag-simple sequence repeat) marker for identifying leafy character of medicago sativa and application of detection primer pair

Abstract

The invention discloses a detection primer pair of an EST-SSR (expressed sequence tag-simple sequence repeat) marker for identifying the leafy character of medicago sativa and application of the detection primer pair. The EST-SSR marker comprises at least one of MS.gene047479 or MS.gene36334; the nucleotide sequences of the primer pair for detecting the MS.gene047479 are shown as SEQ ID NO.1-2, and the nucleotide sequences of the primer pair for detecting the MS.gene36334 are shown as SEQ ID NO.3-4. The invention further discloses a kit for detecting the MS.gene36334. According to the application, two EST-SSR molecular markers highly associated with the multi-leaf character of medicago sativa are screened, so that medicago sativa can be accurately distinguished from medicago sativa, and the EST-SSR molecular markers have the characteristics that amplification products are stable and strips are easy to identify. Reliable technical support is provided for researching the multileaf medicago sativa in the future, and the molecular marker-assisted breeding process can be accelerated.

Description

TECHNICAL FIELD

[0001] The application relates to a detection primer pair of an EST-SSR marker for identifying a multi-leaf trait of Medicago sativa and application thereof, and belongs to the technical field of biotechnology / molecular breeding. BACKGROUND

[0002] Medicago sativa L. is the most widely planted and economically valuable forage grass in the world, and plays a key role in forage industrialization, ecological environment construction and adjustment of the structure of agriculture and animal husbandry. However, Medicago sativa is a homologous tetraploid, a perennial legume with allogamy, and its genetic background is complex, which makes it difficult to improve traits and to some extent restricts the efficiency and process of breeding new varieties of alfalfa.

[0003] Leaves are the core organs of forage nutrients and biomass, and the size, shape and number of leaves directly affect photosynthesis, forage quality and biomass. As one of the forage grasses with the highest digestible protein, Medicago sativa is rich in protein (about 70% of the whole plant), and the cellulose content is only 1 / 3 of that of the stem. Studies have shown that the contribution rate of leaves to forage yield of alfalfa can reach 37-50%. In addition, the extract of alfalfa leaves has been proven to have the effects of lowering cholesterol, lowering blood lipids, protecting nerves, antioxidant and antibacterial. Therefore, accelerating the breeding of multi-leaf alfalfa varieties is of great significance to improve the quality and yield of alfalfa and ensure national food security.

[0004] Simple sequence repeat (SSR) is a heritable co-dominant molecular marker, which is widely used in genetic diversity analysis, population structure analysis, fingerprinting and germplasm identification of alfalfa due to its advantages of large number, simple development, high polymorphism and good stability. Among them, EST-SSR is a molecular marker based on EST (Expressed Sequence Tags), which has higher universality and accuracy compared with genomic SSR markers because its polymorphism may be directly related to gene function expression. However, the existing technology still has significant limitations in the research and application of multi-leaf traits of Medicago sativa: ①It is difficult to identify the multi-leaf trait of Medicago sativa at an early stage, and the expression of the multi-leaf trait is highly related to the growth period. The multi-leaf rate is the lowest at the seedling stage, and traditional morphological identification is time-consuming and easily affected by the environment. ②The existing SSR markers are mostly based on random regions of the genome, and lack molecular markers directly related to key genes of compound leaf development. ③The characteristics of high heterozygosity and cross-pollination of Medicago sativa lead to complex population genetic structure, and it is difficult to accurately distinguish the hybrid offspring.

[0005] In view of this, the present application provides an EST-SSR molecular marker for identifying the multi-leaf trait of alfalfa in the early breeding stage and a primer combination for detecting the molecular marker. SUMMARY

[0006] The present application aims to provide a primer pair for identifying the multi-leaf trait of alfalfa and the application thereof, which has high specificity and stability.

[0007] The primer pair for identifying the multi-leaf trait of alfalfa according to the present application comprises at least one of MS.gene047479 or MS.gene36334; the primer pair for detecting MS.gene047479 has the nucleotide sequence shown as SEQ ID NO. 1-2, and the primer pair for detecting MS.gene36334 has the nucleotide sequence shown as SEQ ID NO. 3-4.

[0008] Further, the primer pair of SEQ ID NO. 1-2 is used for identifying multi-leaf and trifoliate alfalfa; when the amplification result is a 237bp band, the alfalfa to be tested is multi-leaf alfalfa; and when the amplification result is a 237bp band and a 264-287bp band, the alfalfa to be tested is trifoliate alfalfa.

[0009] Further, the primer pair of SEQ ID NO. 3-4 is used for identifying the hybrid F1 generation trait of multi-leaf and trifoliate alfalfa; when the amplification result is consistent with the maternal parent, the alfalfa to be tested is a self-crossed species; and when the amplification result is a specific band type between the parent and maternal parent bands in addition to the band consistent with the maternal parent, the alfalfa to be tested is a true hybrid.

[0010] Further, the maternal parent is S2, and the paternal parent is Huaiyin alfalfa.

[0011] Further, the reagent or kit further comprises 2xReaction Mix, Golden DNA Polymerase and ddH2O.

[0012] The primer pair, reagent or kit according to the present application is used for early identification of the multi-leaf trait of alfalfa or identification of the hybrid offspring of multi-leaf and trifoliate alfalfa; the primer pair for detecting MS.gene047479 is used for identifying multi-leaf and trifoliate alfalfa; and the primer pair for detecting MS.gene36334 is used for identifying the hybrid F1 generation trait of multi-leaf and trifoliate alfalfa.

[0013] Further, the PCR amplification procedure of the application in detection is: 94℃ 3min; 94℃ 30s, 57-59℃ 30s, 72℃ 1min, 30 cycles; 72℃ 5min.

[0014] Beneficial effects: Compared with the prior art, the application has the following remarkable advantages: two EST-SSR molecular markers highly related to the leafiness of alfalfa are screened, which can accurately distinguish the multi-leaf alfalfa from the trifoliate alfalfa, and have the characteristics of stable amplification products and easy identification of bands. It provides reliable technical support for future research on multi-leaf alfalfa, and is beneficial to accelerate the process of molecular marker-assisted breeding. BRIEF DESCRIPTION OF DRAWINGS

[0015] Figure 1 The compound leaf types of 20 alfalfa materials for testing;

[0016] Figure 2 The amplification results of the primer of MS.gene047479 in multi-leaf and trifoliate alfalfa are detected;

[0017] Figure 3 The application of the primer of MS.gene36334 in identifying the true or false hybrid of the F1 population of multi-leaf alfalfa and trifoliate alfalfa. DETAILED DESCRIPTION

[0018] The technical solutions of the application will be further described below in combination with the drawings.

[0019] Example 1 Development of molecular markers

[0020] (1) Using "Leaf blade development", "Compound leaf development", "Leaf morphogenesis" as keywords, 283 candidate genes involved in plant leaf development were obtained after searching and screening in Pubmed by TBtools software, including 13 types of functional genes such as ARP, KNOX1, MKNOX, LFY, SLM1, AUXIN, HD-ZIPIII, C2H2, BEL1, CUC / NAM, MYB, TCP and WOX. The above genes play an important role in the processes of initial development of leaf primordium, establishment of leaf polarity, and determination of initial position and number of leaflets. The CDS sequences of the functional genes in NCBI were downloaded in batches by using the Bulk NCBI Sequence Download tool of TBtools. Then, the CDS sequences of the leaf development genes were used as a data set. Local BLAST program (E-value≤10 -5) The Medicago sativa cv. Zhongsi 4 genome database (genome sequence downloaded from the National Center for Biotechnology Information (accession numbers: CRA005190 and CRA003659)) was detected, and non-redundant homologous genes were obtained by the CD-HIT (c=0.95) program. Finally, 749 homologous genes were identified. For the above identified homologous genes, the EST-SSR primer was designed using the SSRminer plug-in of TBtools, and the primer specificity was detected by the Primer Check plug-in. After the primer was synthesized by Shenguo Bioengineering (Shanghai) Co., Ltd., it was used for subsequent experiments.

[0021] (2) 71 SSR sites were searched in 39 gene sequences of Medicago sativa, and 48 pairs of primers were successfully designed to detect some of the SSR sites. PCR amplification was performed using DNA from 3 multifoliate alfalfa (Zhongsi 1, S2, and multifoliate in the BC2 segregating population) and 2 trifoliate alfalfa (Huaying alfalfa and trifoliate in the BC2 segregating population) as templates (Nos. 1, 3, 7, 11, and 17 in Table 1, respectively).

[0022] Among them, the BC2 segregating population is: Huaying alfalfa as the female parent and PH34HQ as the male parent to obtain hybrid F1, and the multifoliate material is selected as the female parent, and PH34HQ is used as the male parent to construct BC1, and the multifoliate material is selected as the female parent, and PH34HQ is used as the male parent to construct the BC2 population. In the whole construction of the BC population, PH34HQ is used as the recurrent parent.

[0023] (2.1) Extraction and dilution of genomic DNA: Fresh alfalfa leaves were quickly frozen in liquid nitrogen and ground into powder, and 20 portions of alfalfa leaf genomic DNA were extracted by SDS method. The DNA quality and concentration were determined by NanoDrop ND1000 spectrophotometer and agarose gel electrophoresis, and the qualified DNA was diluted to 25 ng / μL, and then stored at -20°C in the refrigerator.

[0024] (2.2) The PCR reaction system and amplification program are as follows:

[0025] ① The PCR reaction system is 10 μl

[0026]

[0027] Note: The amount of enzyme in the system can not be included in the total amount

[0028] ② Amplification program

[0029]

[0030] The obtained PCR products were subjected to polyacrylamide gel electrophoresis and developed and stained.

[0031] 48Of the 36 pairs of primers, 9 pairs of primers successfully amplified a single clear band with the expected size. These 9 pairs of primers were further tested in 20 alfalfa materials.

[0032] (3) The DNA of 10 strains of multi-leaf alfalfa (multi-leaf rate: 18.05%-70.85%) and 10 strains of trifoliate alfalfa with different genetic backgrounds was used as a template for PCR amplification with the above-mentioned 9 pairs of primers (the information of sample's compound leaf type and multi-leaf rate is shown in Table 1), and the DNA extraction, PCR amplification system and procedure were the same as in step (2). From them, one pair of primers (MS.gene047479) that can be used to accurately distinguish multi-leaf type and trifoliate type was screened out (Table 2). The compound leaf types of the 20 samples of Medicago sativa L. are shown in Figure 1 , and the amplification results of primer MS.gene047479 are shown in Figure 2 , in which the amplification results of multi-leaf alfalfa are a 237 bp band, and the amplification results of trifoliate alfalfa are a 237 bp band and a 264-287 bp band. Figure 2 , in which the multi-leaf alfalfa materials include the excellent multi-leaf single plants of Hangmao No. 1 (Nos. 1 and 2), the self-crossed single plants of South Australia multi-leaf alfalfa PH34HQ (Nos. 3-5), the different strain single plants of BC1 (Nos. 6, 9 and 10) and BC2 (Nos. 7 and 8) with Huaying alfalfa and PH34HQ as parents. The trifoliate alfalfa materials include 8 varieties: Huaying, Feli, Qiangjing, Sandeli, WL525, Klaudi (Nos. 11-16), and the different strain single plants of BC1 (No. 18) and BC2 (Nos. 17, 19 and 20) with Huaying alfalfa and PH34HQ as parents.

[0033] Table 1 Information of compound leaf type and multi-leaf rate of 20 samples

[0034]

[0035] (4) S2 self-crossed single plant with 7 leaves as the main part and a multi-leaf rate of 65.45% was used as the female parent, and the trifoliate alfalfa with an upright plant type and large plant height and leaves was used as the male parent for artificial pollination. The hybridization experiment was carried out in the morning from 9:00 to 11:00 on a sunny and windless day. After pollination, the whole inflorescence was wrapped with two layers of paper towels to prevent external pollen from affecting the hybridization results, and mature seeds were harvested after one month. The information of parents is shown in Table 3.

[0036] The harvested F1 hybrid was sowed after germination, and the first three compound leaves were fully expanded. The leaf blades were taken to extract DNA, and the genomic DNA of 15 F1 single plants was amplified by the above 9 pairs of primers to obtain primer pair MS.gene36334 (Table 2) which can be used to accurately identify the multi-leaf alfalfa (S2) and the trifoliate alfalfa (Huaying alfalfa) and their hybrid offspring. The amplification results of primer MS.gene36334 are shown in Figure 3 , the maternal amplification band is 161 bp, the paternal amplification band is 161 bp+208 bp; 15 single plants in the hybrid F1 population were amplified to obtain two band types, which were marked with the numbers "1" and "2", respectively. Among them, band type "1" amplified a specific band type (about 185 bp) between the parent and parent main bands in addition to the 161 bp band which the parent did not have, which can be determined as true hybrid, and band type "2" has the same amplification result as the maternal parent, which is self-crossed.

[0037] Table 2 Two pairs of EST-SSR markers related to the multi-leaf trait of purple alfalfa

[0038]

[0039]

[0040] Table 3 Hybrid parent information of multi-leaf alfalfa and trifoliate alfalfa

[0041]

[0042] Reference genome "Zhongmu No. 4" purple alfalfa MS.gene047479 (SEQ ID NO. 5) and MS.gene36334 (SEQ ID NO. 6) gene CDS sequence, wherein the bold is the forward and reverse primer sequence, and the underline is the SSR repeat sequence.

[0043] SEQ ID NO. 5:

[0044]

[0045] SEQ ID NO. 6:

[0046] Based on the above results, the two EST-SSR markers provided in the application can be used to identify the multi-leaf trait of purple alfalfa.

Claims

1. A primer pair for detecting EST-SSR markers used to identify the multi-leaved trait of alfalfa, characterized in that, The EST-SSR marker includes at least one of MS.gene047479 or MS.gene36334; the primer pair nucleotide sequences for detecting MS.gene047479 are shown in SEQ ID NO.1-2, and the primer pair nucleotide sequences for detecting MS.gene36334 are shown in SEQ ID NO.3-4.

2. The detection primer pair for the EST-SSR marker used to identify the multi-leaf trait of alfalfa according to claim 1, characterized in that, Primer pairs SEQ ID NO.1-2 are used to identify alfalfa and clover. When the amplification result is a 237bp band, the alfalfa to be tested is alfalfa. When the amplification result is a 237bp band and a 264-287bp band, the alfalfa to be tested is clover.

3. The detection primer pair for the EST-SSR marker used to identify the multi-leaf trait of alfalfa according to claim 1, characterized in that, Primer pairs SEQ ID NO.3-4 are used to identify the F1 generation traits of hybrid alfalfa and trifoliate alfalfa. If the amplification result is consistent with the maternal parent, the alfalfa to be tested is a self-pollinated species. If the amplification result, in addition to the band consistent with the maternal parent, also amplifies a specific band pattern that is not present in the parents and is between the main bands of the parents, the alfalfa to be tested is a true hybrid.

4. The detection primer pair for the EST-SSR marker used to identify the multi-leaf trait of alfalfa according to claim 3, characterized in that, The maternal parent is S2, and the paternal parent is Huaiyin alfalfa.

5. A reagent or kit containing the primer pair as described in any one of claims 1 to 4.

6. The reagent or kit according to claim 5, characterized in that, The reagents or kits also include 2×Reaction Mix, Golden DNA Polymerase, and ddH2O.

7. The application of the detection primer pair according to any one of claims 1 to 4, and the reagent or kit according to claims 5 to 6, in the early identification of the multi-leaved trait of alfalfa or in the identification of hybrid offspring of multi-leaved and trifoliate alfalfa, characterized in that, The primer pair for detecting MS.gene047479 was used to identify alfalfa and clover; the primer pair for detecting MS.gene36334 was used to identify the F1 generation traits of hybrids of alfalfa and clover.

8. The application according to claim 7, characterized in that, The PCR amplification program used in the detection process is as follows: 94℃ for 3 min; 94℃ for 30 s, 57~59℃ for 30 s, 72℃ for 1 min, 30 cycles; 72℃ for 5 min.

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