Novel elytrigia minor translocation line detection method and application thereof

Abstract

The invention discloses a novel thinopyrum triticum translocation line detection method and application thereof. According to the method, specific markers CINAU1181 (SEQ ID NO.17-18) and CINAU1381 (SEQ ID NO.19-20) of thinopyrum triticum are expanded through PCR (Polymerase Chain Reaction), and a translocation line is judged according to a 253bp / 254bp band. The marker and the leaf rust resistant gene LrSN21044 are co-separated (the co-separation rate is greater than or equal to 98%). The method can be applied to wheat genetic improvement, and is especially suitable for rapid breeding of wheat leaf rust resistant varieties.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of crop molecular marker assisted breeding, and particularly relates to a method for detecting a small einkorn wheat translocation line and application thereof. BACKGROUND

[0002] China is the largest wheat producer and consumer in the world, accounting for 17% of the world's total wheat production and 16% of the total consumption (Liu et al., 2018). Although China's grain production is showing a good trend of steady growth, it still faces problems such as continuous reduction of arable land, invasion of various pests and diseases, and lack of original and breakthrough germplasm and varieties. Therefore, cultivating high-yield and disease-resistant varieties and strengthening germplasm innovation are important ways to maintain the sustainable development of wheat breeding and production (Singh et al., 2016). With the emergence of new pathogens, the resistance of existing disease resistance genes is gradually lost. For example, Pm8 located on the short arm of rye 1R chromosome has been widely used, but it has now lost resistance to new powdery mildew strains (Ren et al., 2017). In addition, due to the influence of artificial selection, the genetic diversity of modern wheat breeding is reduced, and the shortage of genetic resources is not conducive to the cultivation and promotion of new disease-resistant and stress-tolerant varieties, and it is urgent to explore new disease resistance genes and create new disease-resistant germplasm to broaden the resources of wheat breeding.

[0003] There are many wild relatives of wheat with rich genetic diversity, and more than 20 wild relatives of wheat have been successfully hybridized with wheat (Dong, 2000). The allohexaploid Thinopyrum ponticum (2n = 10x = 70, StStStStE e E e E b E b E x E x or JJJJJJJ S J S J S J S) is a perennial herb, which shows excellent resistance to wheat powdery mildew, stripe rust, stem rust, scab, yellow dwarf and other diseases, and is a valuable resource for wheat genetic improvement. So far, multiple disease resistance genes have been found in the octoploid longspike wildrye, including the scab resistance gene Fhb7 (Wang et al., 2020), the powdery mildew resistance gene Pm51 (Zhan et al., 2014), the wheat stripe virus resistance gene Cmc2 (Li et al., 2009), the leaf rust resistance genes Lr19, Lr24, Lr29 (McIntosh et al., 2020), the stripe rust resistance gene Yr69 (Hou et al., 2016), and the stem rust resistance genes Sr24, Sr25, Sr26, Sr43 (Cui et al., 2015; Li et al., 2023), which play a crucial role in wheat disease resistance breeding.

[0004] The existing translocation line identification relies on chromosome in situ hybridization (GISH-FISH), which is complex to operate and has a long cycle (3-5 days are required), and is difficult to screen in a high-throughput manner; there is a lack of molecular markers closely linked to disease resistance genes, resulting in low efficiency of resistance phenotype tracking in breeding (field inoculation identification is required by traditional methods). SUMMARY

[0005] To solve the problems of few original breakthrough disease-resistant germplasm and singleization of wheat breeding parents, the present application provides a novel translocation line SN21044 of stripe rust and leaf rust resistant small wildrye.

[0006] The present application is realized by the following technical solutions:

[0007] SNTE20 is an octoploid small wildrye created by crossing the octoploid longspike wildrye with Yanong 15 and Shan Nong Fa 63, which is immune to common wheat diseases such as rust and powdery mildew. SNTE20 is crossed with line SN637 to solve the problems of high stem (118 cm) and late maturity of SNTE20, and a novel T3DS·3DL-Tp translocation line SN21044 is created.

[0008] The novel translocation line SN21044 of stripe rust and leaf rust resistant small wildrye provided by the present application is a T3DS·3DL-Tp small fragment translocation line, which has 42 chromosome numbers, including 40 normal wheat chromosomes and 2 wheat-octoploid longspike wildrye translocation chromosomes involving 3D.

[0009] SN21044 has multiple excellent traits: dwarf (66 cm), more grains (74 grains), heat tolerance, high resistance to wheat stripe rust and leaf rust. The ideal agronomic traits and excellent resistance of SN20144 make it of great value in wheat disease resistance breeding.

[0010] The present application develops intron targeting (IT) markers based on translocation fragments, and screens two specific markers: primer CINAU1181 of the third partial homology group and primer CINAU1381 of the fifth partial homology group

[0011] CINAU1181, amplified fragment 253bp;

[0012] CINAU1381, amplified fragment 254bp.

[0013] Mycelial resistance identification in the seedling stage shows that SN21044 is nearly immune to stripe rust and leaf rust (as shown in Table 1), and pedigree analysis shows that the leaf rust resistance is derived from the decaploid Elytrigia repens. Figure 1 The sequential GISH-FISH identification result shows that SN21044 is a T3DS·3DL-Tp double translocation line (as shown in Table 2), and during the formation process, the chromosomes such as 4D, 5B, 5D and 6A have obvious structural variation (as shown in Table 3). Figure 2 The intron targeting (IT) markers are used for amplification of SN21044 and its parents, and six decaploid Elytrigia repens specific markers are screened, of which four are derived from the first partial homology group, and three can simultaneously amplify specific bands in decaploid Elytrigia repens and Elytrigia trachycaulum (as shown in Table 4). Figure 3 The leaf rust resistant and susceptible separation ratio in the F2 population of SN21044 and Huixuanhong is consistent with 3:1 (χ 2 =0.466, P>0.05), and CINAU1181 and CINAU1381 are linked to the leaf rust resistance phenotype. It is inferred that the leaf rust resistance in SN21044 is controlled by a pair of dominant genes, which is located in the exogenous chromosome fragment and is temporarily named as LrSN21044. In addition, the spike grain number (74 grains) and the thousand-grain weight (51.11g) of SN21044 are increased by 13.85% and 10.13% respectively compared with its common wheat parent SN637. Figure 4 The present application provides a method for detecting small wheat translocation line SN21044, which comprises the following steps:

[0014] (1) extracting the genomic DNA of the wheat strain to be tested;

[0015] (2) using primer combination CINAU1181 / CINAU1381 for PCR amplification, and the primer sequences are shown in SEQ ID NO. 17-20;

[0016]

[0017] ​(3) Detection of amplification products: if specific bands of long-awned Aegilops tauschii appear, CINAU1181 specific band 253bp; CINAU1381 specific band 254bp, then it is determined that the SN21044 translocation line carries T3DS·3DL-Tp translocation chromosome.

[0018] Preferably, the molecular marker combination of the detection method comprises:

[0019] Primer CINAU1181: forward sequence SEQ ID NO. 17, reverse sequence SEQ ID NO. 18;

[0020] Primer CINAU1381: forward sequence SEQ ID NO. 19, reverse sequence SEQ ID NO. 20;

[0021] The marker combination is co-segregated with the anti-leaf rust gene LrSN21044, and the co-segregation rate is ≥98%.

[0022] Preferably, the PCR amplification procedure is: 94℃ pre-denaturation for 5min; 35 cycles (94℃ 30s, 58℃ 30s, 72℃ 30s); 72℃ extension for 5min.

[0023] Preferably, the translocation chromosome is verified by genomic in situ hybridization, using the genome DNA of the decaploid long-awned Aegilops tauschii as a probe and the genome DNA of Yan Nong 15 as a blocking agent, and the blocking ratio is ≥50:1.

[0024] The application provides a method for breeding anti-leaf rust wheat by using a molecular marker combination, comprising:

[0025] (1) crossing SN21044 with a susceptible wheat to obtain an F2 population;

[0026] (2) extracting single plant DNA and screening positive plants using CINAU1181 / CINAU1381;

[0027] (3) identifying the positive plants by leaf rust inoculation, and retaining the disease-resistant plant lines with IT≤1.

[0028] The application has the beneficial effects that the novel anti-stripe rust and anti-leaf rust Aegilops tauschii translocation line provided by the application has excellent resistance and ideal agronomic traits, and has important utilization value in wheat genetic improvement. In wheat breeding, shortage of germplasm resources and single breeding parents are one of the problems to be solved. The novel anti-stripe rust and anti-leaf rust Aegilops tauschii translocation line provided by the application provides a new germplasm resource for wheat disease-resistant breeding. BRIEF DESCRIPTION OF DRAWINGS

[0029] Figure 1Identification of SN21044 and its parents for seedling stripe and leaf rust resistance; wherein A is seedling stripe rust resistance identification, B is seedling leaf rust resistance identification, 1-7 are respectively Elytrigia elongata, SNTE20, Yannong 15, Shannong 63, SN637, SN21044, Huixianhong.

[0030] Figure 2 Identification of SN21044 by in situ hybridization; wherein A-C are respectively genomic in situ hybridization (GISH) identification, fluorescence in situ hybridization (FISH) identification, FISH banding pattern of SN21044. Arrow shows translocation chromosome.

[0031] Figure 3 FISH banding pattern comparison of SN21044 and its parents; a-c are respectively SNTE20, SN637, SN21044. Arrow shows FISH banding pattern difference site.

[0032] Figure 4 Specific bands of marker CINAU1181 / CINAU1381 in translocation line; wherein lane figure M, 1-9 are respectively Marker (DL2000 DNA Marker), Elytrigia elongata, diploid Elytrigia elongata, Roegneria procera, Psathyrostachys juncea, SNTE20, Yannong 15, Shannong 63, SN637, SN21044; A-F are respectively CINAU942, CINAU947, CINAU955, CINAU956, CINAU1181, CINAU1381. Arrow shows specific band, size is respectively 175bp, 300bp, 252bp, 269bp, 253bp, 254bp.

[0033] Figure 5 Amplification results of Elytrigia elongata specific IT marker in SN21044 in SN21044 / Huixianhong F2 population; wherein lane figure M, 1-9 are respectively Marker (DL2000 DNA Marker), Elytrigia elongata, diploid Elytrigia elongata, Roegneria procera, Psathyrostachys juncea, SNTE20, Yannong 15, Shannong 63, SN637, SN21044; A-F are respectively CINAU942, CINAU947, CINAU955, CINAU956, CINAU1181, CINAU1381. Arrow shows specific band, size is respectively 175bp, 300bp, 252bp, 269bp, 253bp, 254bp. Figure 1 Elytrigia elongata is 2, SN21044 is 3, GSTR425 is 4, Huixianhong is 5-88; A is CINAU1181, B is CINAU1381; M is DL2000 DNA Marker. R shows resistance, S shows susceptibility. Arrow shows specific band 253 / 254bp.

[0034] Figure 6 Plant, ear and grain morphology of SN21044; wherein A-C are respectively plant morphology, ear morphology, grain morphology of SN21044. DETAILED DESCRIPTION

[0035] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. Unless otherwise specified, the examples are based on conventional experimental conditions.

[0036] Example Detection Method and Molecular Marker Screening of the Stripe Rust and Leaf Rust Resistance Translocation Line SN21044

[0037] 1 Test materials

[0038] Decaploid Elytrigia longissima; diploid Elytrigia longissima, Elytrigia pseudo-goosegrass, and Elytrigia bessa are the original parents of decaploid Elytrigia longissima; octoploid Elytrigia spp. SNTE20 is the hybrid offspring of decaploid Elytrigia longissima and common wheat Yannong 15 and Shannong Fu 63; Elytrigia spp. translocation line SN21044 ( Figure 6 ), which is the hybrid offspring of SNTE20 and SN637; common wheat Huixianhong, which is the susceptible control.

[0039] 2 Test methods

[0040] 2.1 Disease resistance identification at the seedling stage

[0041] SN21044, its parents, and the susceptible control Huixianhong were planted in small plastic pots. When they had two leaves and one heart, they were independently inoculated with the leaf rust race (PHQS) and the stripe rust race (CYR34), respectively. The response type was evaluated after the susceptible control was fully diseased.

[0042] The seedling resistance identification method uses the 0-4 level identification standard (Roelfs et al., 1992) with the following reference standards, where the reaction level 0-2 represents resistance and the level 3-4 represents susceptibility.

[0043] 0—immune, no spores and infection points;

[0044] 0;——Nearly immune, no spore piles, obvious necrosis;

[0045] 1 - Highly resistant, small spore piles with necrotic spots around them;

[0046] 2 - medium resistance, small to medium spore piles with necrosis or chlorosis around them;

[0047] 3 - moderate, with medium spore mass and no surrounding necrotic spots;

[0048] 4 - Highly susceptible, large spore mass, no chlorosis.

[0049] 2.2 Cytological identification

[0050] (1) Observation of root tip cell (RTC) chromosomes

[0051] The seeds were soaked for 12 h at room temperature according to the method of Kato et al. (2004), and then the excess water was discarded and the seeds were placed in a petri dish with wet filter paper. The roots were taken when the root length was 1.5-2.0 cm. The suspension was formed by APM fixation, nitrous oxide treatment, 90% acetic acid fixation, cellulose pectinase digestion, 70% alcohol washing, and crushing with a dissecting needle. Then the suspension was centrifuged, the supernatant was discarded, and 90% acetic acid was added to dissolve the sample. The sample was dropped on a slide and observed under a microscope.

[0052] (2) Genomic in situ hybridization (GISH)

[0053] The method of Fu (2012) was used with slight modifications.

[0054] Probe preparation: The high-quality genomic DNA of the test material was labeled with the nick translation method, and all the fluorescein was stored in the dark. The probe system contained ddH2O, 2 mM dNTP, DNA pol I, template DNA, DNase I (100 Mu / ul), TEXAS RED-5-dCTP (or 488-5-dUTP), etc. After mixing, the prepared system was incubated at 15°C for 2 h, 8 volumes of single-stranded DNA blocking agent (ssDNA) and 2.5 volumes of pre-cooled precipitation solution (containing 90% anhydrous ethanol solution and 10% 3M sodium acetate solution mixture) were added, and the mixture was precipitated in a -20°C refrigerator for 12 h. Then, the mixture was centrifuged at 12000 rpm and 4°C for 30 min, washed with 70% ethanol solution for 2-3 times, and dried. Then, 60 μL of 1×TE and 2×SSC mixed solution was added to dissolve the sample, and the sample was stored at 4°C for later use.

[0055] Hybridization: The prepared hybridization solution was mixed and added dropwise to the prepared glass slide, covered with a cover glass, denatured in a boiling water bath for 5 min, and then placed in a 55°C constant temperature box for hybridization for more than 6 h.

[0056] The GISH hybridization solution contains probes, blocking agents, 1×TE+2×SSC, etc., and the probe-blocking ratio is 1:200.

[0057] Microscopic examination: DAPI reagent was added dropwise under light-proof conditions to the dividing phase, and microscopic examination was performed by fluorescence microscopy (Nikon (ECLIPSE Ni-U type)), and image acquisition was performed by a microscope camera (Nikon DS-Ri1, Japan).

[0058] (3) Fluorescence in situ hybridization (FISH)

[0059] Preparation of the slide: The same as genomic in situ hybridization.

[0060] Hybridization: Refer to the method of Huang et al. (2018) with slight modifications. For in situ hybridization, mix the hybridization solution, denature at 105°C for 13 min, and then place it in -20°C for more than 8 min. Place the prepared glass slide in 0.15 mol / L denaturing solution (70% ethanol + 1 mol / L NaOH) and denature at 44°C for 5 min. After dehydration with anhydrous ethanol, blow dry. Drop the hybridization solution onto the split phase and place it in the black box. Hybridize at 37°C for more than 6 h. The hybridization solution contains DFA, 20×SSC, salmon sperm DNA (ssDNA), 8 oligonucleotide probes, 50% dextran sulfate (DS), and other reagents.

[0061] 8 oligonucleotide probe sequences:

[0062] Table 1. Oligonucleotide probes

[0063] Signal detection: Take out the glass slide, knock off the cover glass, and soak in 2×SSC at 44°C for 10 min. Then rinse with deionized water and blow dry.

[0064] Microscopy: The same as genomic in situ hybridization.

[0065] 2.3 Extraction of wheat genomic DNA using the CTAB method

[0066] (1) Take 2 g of fresh wheat leaves and grind them into powder in a 2 ml centrifuge tube containing liquid nitrogen;

[0067] (2) Add 800 μL of CTAB and incubate at 65°C for 45 min. Shake well 2-3 times during the water bath;

[0068] (3) Add 800 μL of chloroform-isoamyl alcohol (24:1) and shake well. Centrifuge at 12000 rpm for 10 min at 4°C. Transfer the supernatant to a new 2 ml centrifuge tube for the next round of extraction;

[0069] (4) Use phenol-chloroform-isoamyl alcohol (25:24:1) or phenol-chloroform (1:1) for extraction;

[0070] (5) Take the supernatant to a new 2 ml centrifuge tube and add 480 μL of isopropanol (-20°C pre-cooled isopropanol). Shake well until white flocculent appears, then transfer to a -20°C refrigerator;

[0071] (6) After 20 min, centrifuge at 12000 rpm for 10 min and discard the supernatant. Rinse the precipitate with 70% ethanol for 2-3 times, then air dry;

[0072] (7) After air drying, add 50 μL of ddH2O and store at 4°C.

[0073] 2.4 PCR amplification

[0074] (1) PCR amplification system was 10 μL: template DNA 50 ng, 7 μL of 2x Power Taq PCR MasterMix, 1 μL of forward and reverse primers (2.5 μmol / L) respectively, and ddH2O to 10 μL.

[0075] (2) PCR amplification program was 95℃ for 3 minutes; 95℃ for 15 seconds, 57℃ for 15 seconds, 72℃ for 1 minute, 36 cycles; 72℃ for 10 minutes.

[0076] After PCR amplification, the IT marker amplification product was detected by 8% polyacrylamide non-denaturing gel (PAGE) electrophoresis, and photographed and observed by Tanon Gis-2010 gel imaging system.

[0077] Table 2 IT marker sequence

[0078] 2.5 Agronomic trait identification

[0079] According to the "Wheat Germplasm Resource Description Specification and Data Standard" (Li Lihui and Li Xiushuan, 2006), agronomic traits such as flowering period, heading period, plant height, ear length, ear type, spikelet number, grain number per ear, and thousand kernel weight were investigated at the agronomy experiment station of Shandong Agricultural University. Among them, plant height, ear length, ear type, spikelet number, and grain number per ear were investigated on 10 plants and averaged, thousand kernel weight was obtained by scanning 500-1000 wheat kernels with a Wanshen SC-G automatic seed analysis instrument and thousand kernel weight system software, and the test was repeated three times.

[0080] 3 Results and analysis

[0081] 3.1 Seedling stage disease resistance identification

[0082] The seedling stage resistance to stripe rust identification results (as shown in Figure 1 A), the decaploid long-eared wildrye showed immunity to PHQS (IT=0), the octaploid small wildrye SNTE20 showed near immunity (IT=0;), Shannong 63, Yannong 15, and SN637 showed moderate susceptibility (IT=3); the new germplasm SN21044 of small wildrye showed near immunity (IT=0;). Figure 1CYR34 (IT=0), and SNTE20 (IT=0) showed near-immunity. Shanmai 63, Yanmai 15 and SN637 showed high resistance (IT=1). SN21044 showed near-immunity to leaf rust (IT=0).

[0083] 3.2 GISH-FISH identification

[0084] GISH identification using the genomic DNA of E. elongatum as a probe and the genomic DNA of Yanmai 15 as a block showed that SN21044 contained 20 normal wheat chromosomes and 1 translocation chromosome (as shown in Fig. Figure 2 A).

[0085] After removing the GISH signal, FISH analysis using 8 oligonucleotide probes showed that SN21044 contained a complete set of wheat chromosomes (1A-7A, 1B-7B, 1D-7D), in which the long arm of 3D chromosome carried a pair of small fragments of E. elongatum chromosomes, indicating that SN21044 was a T3DS·3DL-Tp small fragment translocation line (as shown in Fig. Figure 2 B). Figure 3 As shown in Fig.

[0086] 3.3 Molecular marker analysis

[0087] Molecular marker detection of SN21044 using 841 IT primers showed that primers CINAU942, CINAU947, CINAU955, CINAU956 from the first homoeologous group, primer CINAU1181 from the third homoeologous group, and primer CINAU1381 from the fifth homoeologous group could all amplify E. elongatum-specific bands in SN21044 (175 bp, 300 bp, 252 bp, 269 bp, 253 bp, and 254 bp, respectively), among which CINAU942, CINAU947, and CINAU1381 could also amplify obvious specific bands in the original parent E. elongatum (as shown in Fig. Figure 4 ).

[0088] 3.4 Chromosomal localization of leaf rust resistance gene LrSN21044

[0089] SN21044 was crossed with susceptible material, Huixianhong, and F1 was selfed to obtain F2 segregating population. 121 F2 single plants were randomly selected and inoculated with leaf rust pathogen physiological race PHQS at seedling stage, among which 94 plants showed resistance and 27 plants showed susceptibility, which was in accordance with the expected segregation ratio of 3:1 (χ 2 =0.466, P>0.05). Scanning of F2 population with 6 specific IT markers of E. elongatus showed that CINAU1181 (the third homoeologous group) and CINAU1381 (the fifth homoeologous group) were linked to the resistance phenotype (as shown in Table 2), and the segregation rate was ≥98%. It was inferred that the leaf rust resistance in SN21044 was controlled by a pair of dominant genes, which was located in the E. elongatus chromosomal fragment at the long arm end of 3D chromosome, and was temporarily named as LrSN21044. Figure 5

[0090] 3.5 Identification of agronomic traits

[0091] Ten SN21044 plants were randomly selected to investigate the main agronomic traits such as heading stage, flowering stage, spike length, and spikelet number, and the results were as follows: the plant height of SN21044 was lower (66.10 cm) than that of the parent SN637 (68.93 cm); the spike length was 11.65 cm, which was lower than that of SNTE20, but increased by 28.02% compared with the parent SN637 (9.10 cm); the spikelet number was 23, which was higher than that of SN637 (22). The spike grain number of SN21044 was as high as 74, which increased by 13.85% compared with the common wheat parent SN637; the grains were full, the thousand-grain weight was 51.11 g, which was 10.13% higher than that of SN637; the grain length was 0.78 cm, and the grain width was 0.39 cm, both of which were longer than those of the parent SNTE20.

[0092] In summary, the excellent resistance and ideal agronomic traits of SN21044 make it have important utilization value in wheat genetic improvement.

[0093] The above only describes the preferred embodiments of the patent, and it should be noted that for ordinary skilled persons in the technical field, several improvements and replacements can be made without departing from the technical principles of the patent, and these improvements and replacements should also be considered as the protection scope of the patent.​

Claims

1. A method for detecting the translocation line SN21044 of Triticum aestivum, characterized in that The following steps are involved: (1) Extract genomic DNA from the wheat lines to be tested; (2) PCR amplification was performed using the primer combination CINAU1181 / CINAU1381, wherein the primer sequences are shown in SEQ ID NOs. 17-20; (3) Detection of amplification products: If a specific band of Elytrigia longissima appears, the specific band of CINAU1181 is 253 bp; the specific band of CINAU1381 is 254 bp, then it is determined to be the SN21044 translocation line carrying the T3DS·3DL-Tp translocation chromosome.

2. A molecular marker combination for use in the detection method of claim 1, comprising: Primer CINAU1181: forward sequence SEQ ID NO.17, reverse sequence SEQ ID NO.18; Primer CINAU1381: forward sequence SEQ ID NO.19, reverse sequence SEQ ID NO.20; The marker combination co-segregates with the leaf rust resistance gene LrSN21044, and the co-segregation rate is ≥98%.

3. The method according to claim 1, wherein: The PCR amplification program was as follows: pre-denaturation at 94°C for 5 min; 35 cycles (94°C for 30 s, 58°C for 30 s, 72°C for 30 s); and extension at 72°C for 5 min.

4. The method according to claim 1, characterized in that Also includes: The translocation chromosome was verified by genomic in situ hybridization, using genomic DNA of deploid Thinopsis elongata as probe and genomic DNA of Yannong 15 as blocking agent, with a blocking ratio of ≥50:

1.

5. A method for breeding leaf rust-resistant wheat using the molecular marker combination of claim 2, comprising: (1) Hybridizing SN21044 with susceptible wheat to obtain an F2 population; (2) Extract DNA from individual plants and screen positive plants using CINAU1181 / CINAU1381; (3) Inoculate and identify the positive plants for leaf rust, and retain the disease-resistant strains with IT≤1.

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