SSR Molecular Markers Linked to Thermo-photosensitive Genic Male Sterile Lines in Wheat and Their Application

Abstract

The present invention discloses an SSR molecular marker linked to a temperature-sensitive male nuclear sterile line of wheat and its application. The primers are SSR marker 2B-TPSGMS located on chromosome 2B of wheat and SSR marker 6B-TPSGMS located on chromosome 6B of wheat. The two SSR primers in the present invention are used to carry out molecular marker-assisted selection of wheat male nuclear sterile lines, which can double the efficiency of wheat male nuclear sterile line breeding; in addition, the two markers of the present invention are verified by molecular marker-assisted selection, and are indeed closely linked to the sterile gene of the wheat male nuclear sterile line, which helps breeders understand the genetic law of sterility of the temperature-sensitive male nuclear sterile line of wheat, and lays a foundation for the precise positioning and cloning of sterile genes.

Description

Technical Field

[0001] The invention relates to the field of wheat molecular markers, in particular to SSR primers tightly linked to wheat sterility and applications thereof. Background Art

[0002] The annual planting area of ​​wheat in the world is about 3.3 billion mu, which is an important staple food crop for mankind. The utilization of wheat hybrid advantage is considered to be one of the effective ways to further improve the yield level, stress resistance and stable yield, and enrich the genetic basis of wheat varieties. It is also a scientific and technological problem that has been tried to solve in wheat breeding for more than half a century. The "two-line" method of temperature-sensitive nuclear sterility is one of the new methods of utilizing wheat hybrid advantage created by my country. The temperature-sensitive sterile line is characterized by low-temperature short-day sterility and long-day high-temperature fertility. It does not require a maintenance line, has a wide recovery source, and has a simple seed production procedure. So far, there has been no report of the discovery or breeding of such sterile lines abroad. In recent years, my country's hybrid wheat research has made significant progress and has begun to move towards large-scale production and application. At the same time, it has also achieved good demonstration results in South Asia and Southeast Asia. For example, the "Jingza: series" and "Yunza" series of hybrid wheat in Pakistan and Vietnam have increased yields by more than 30% compared with local controls.

[0003] The expression of sterility in thermo-photosensitive genic male sterile materials is controlled by both genetics and environmental temperature and light conditions. It takes 5-8 years to breed stable sterile lines through traditional breeding methods. The use of molecular marker-assisted selection can effectively reduce the breeding years and improve the breeding accuracy, thereby greatly improving the breeding efficiency. At present, molecular marker technology has been used to preliminarily locate some sterility genes of the thermo-photosensitive genic male sterile lines BNY-S, ES-10, 337S, BS20, BS210, and 291S on chromosomes 1BS, 2A, 2B, 3A, 5B, 5D, 6B, 7B, and 7D, but there are few reports on the use of molecular markers for assisted selection of thermo-photosensitive male genic male sterile lines in wheat.

[0004] The low efficiency of breeding of superior sterile lines and the small number of superior sterile lines for testing and matching strong superior combinations are the main problems affecting the efficiency of screening strong superior combinations of hybrid wheat by the light-temperature-sensitive two-line method. The small number of stable molecular markers that can be used for molecular marker-assisted selection of thermo-photosensitive male nuclear sterile lines of wheat is one of the main reasons for the low efficiency of breeding of superior sterile lines. In addition, the thermo-photosensitive male nuclear sterile lines of wheat are unique to my country and have not yet been protected by relevant intellectual property rights. Therefore, obtaining molecular markers linked to sterile genes will help reveal the genetic characteristics of thermo-photosensitive male nuclear sterile wheat. The use of these molecular marker technologies can improve the efficiency of breeding of superior sterile lines, and at the same time lay the foundation for the next step of cloning sterile genes and effectively protecting my country's unique thermo-sensitive nuclear sterile wheat gene resources through gene patents. Summary of the invention

[0005] To solve the above problems, the present invention aims to provide SSR molecular markers that can be used for assisted selection of temperature-photosensitive male sterile lines in wheat, which is specifically achieved through the following technical solutions:

[0006] An SSR molecular marker linked to a temperature-photosensitive male sterile line of wheat, wherein the primers of the molecular marker are SSR marker 2B-TPSGMS located on wheat chromosome 2B and SSR marker 6B-TPSGMS on chromosome 6B, the upstream nucleotide sequence of 2B-TPSGMS is shown as SEQIDNO.1, and the downstream nucleotide sequence is shown as SEQIDNO.2; the upstream nucleotide sequence of 6B-TPSGMS is shown as SEQIDNO.3, and the downstream nucleotide sequence is shown as SEQIDNO.4.

[0007] Furthermore, the primers 2B-TPSGMS and 6B-TPSGMS are primers linked to the fertility gene of the temperature-photosensitive male sterile line of wheat, obtained by screening polymorphic primers present in two parents and two extreme separation pools and verified by double haploid populations.

[0008] Application of SSR molecular markers in detecting and selecting individual plants of temperature-photosensitive male sterile lines in wheat.

[0009] The beneficial effects of the present invention are as follows: the present invention obtains for the first time SSR primer sequences 2B-TPSGMS and 6B-TPSGMS that are closely linked to the temperature-photosensitive male sterile line of wheat, and the primers can be used to detect the breeding individual plants of the temperature-photosensitive male sterile line of wheat. By utilizing the characteristics of the above two markers being closely linked to fertility, the detection can be performed at the seedling stage, and the individual plants carrying the two markers can be bagged and fertilized and watered in advance in order to obtain more seeds. By combining marker detection with mutual verification of fruiting rate, the difficulty of sterile line breeding caused by environmental impact can be reduced, and the efficiency and accuracy of sterile line breeding can be improved. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 The results of polyacrylamide gel electrophoresis of primer 6B-TPSGMS in fertile and sterile plants. The bands in the upper part are fertile, and the bands in the lower part are sterile.

[0011] Figure 2 This is the result of polyacrylamide gel electrophoresis detection of fertile and sterile plants using primer 2B-TPSGMS. The band at the top is the fertile band type, and the band at the bottom is the sterile band type. DETAILED DESCRIPTION

[0012] The present invention is described in detail below in conjunction with some implementation examples, but the present invention is not limited to these specific embodiments.

[0013] The sources of the biological materials involved in the embodiments are as follows:

[0014] The fertile parent 1069, the sterile parents K78S and K456S and their hybrid progenies were all provided by the Institute of Food Crops, Yunnan Academy of Agricultural Sciences.

[0015] The enzymes, reagents and kits involved in the examples are all common commercially available products.

[0016] 1. Positioning Group Construction

[0017] The fertile parent 1069 was used as the male parent and the sterile parent K78S was used as the female parent. The F1 generation used the wheat-maize hybridization to induce wheat haploid technology to construct a double haploid (DH) population with a total of 468 plants.

[0018] 1. Primer acquisition

[0019] 1.1 Polymorphic primer screening

[0020] Using the 2000 pairs of SSR primers already in this application, genotype detection was performed on the fertile parent 1069, the sterile parent K78S, the fertile pool (a mixture of 20 completely fertile plants in the DH population), and the sterile pool (a mixture of 20 completely sterile plants in the DH population). Primers that were polymorphic between the parents and the two pools were screened as alternative primers, totaling 6 pairs, with 2 pairs and 4 pairs on chromosomes 2B and 6B, respectively.

[0021] 1.2 Primer determination

[0022] The genotypes of 468 plants in the DH population were detected using 6 alternative pairs of primers. The genotypes were combined with the phenotypes (fertility). The results showed that there was one pair of primers on chromosomes 2B and 6B that were closely linked to the sterility gene, named 2B-TPSGMS and 6B-TPSGMS. The upstream nucleotide sequence of 2B-TPSGMS was 5-GAACATCCCTCACCTGCAAT-3, and the downstream nucleotide sequence was 3-GTACGTTGCACCTCAAGCCT-5; the upstream nucleotide sequence of 6B-TPSGMS was 5-CTGATCGTCCGTCTCTGTCC-3, and the downstream nucleotide sequence was 3-GGGTGAAACAGATAGTGGCG-5.

[0023] The obtained SSR primers were used for molecular marker-assisted selection breeding of temperature-photosensitive male sterile wheat lines. The specific examples are as follows:

[0024] Example 1

[0025] Breeding of thermo-photosensitive male sterile wheat lines in 2018 / 2019

[0026] The offspring of the sterile line selected this year came from the hybrid offspring with K78S as the female parent, totaling 232 plants. K78S is a sterile line for large-scale seed production. It has been identified for many years in Baoshan, Wenshan, Lijiang, Qujing, Dali and other places in Yunnan Province, with a sterility of >99%, a hybrid wheat seed purity of >85%, and an average seed yield of more than 200 kg / mu. It is an ideal sterile line in hybrid wheat production. The hybrid offspring were sown on November 15, 2018, and the leaves of individual plants were taken for testing on February 20, 2019. The steps are as follows:

[0027] 1. DNA Extraction

[0028] (1) Weigh 1.5 g of leaves, place in a pre-cooled mortar, pour in liquid nitrogen, and grind the leaves as quickly as possible.

[0029] (2) Transfer the powder into a 2.0 ml centrifuge tube, add 800 ul 2×CTAB extraction buffer and 20 ul β-mercaptoethanol, and preheat to 65°C.

[0030] (3) After thorough mixing, place in a 65°C water bath for 30 min.

[0031] (4) Remove the centrifuge tube, cool it to room temperature, add 800 μl of chloroform:isoamyl alcohol (volume ratio 24:1), and shake at room temperature for 30 min.

[0032] (5) Centrifuge at 3000 rpm for 5 min at room temperature, take the supernatant into a 96-well deep-well plate, add 600 μl of isopropanol, and place in a -20°C refrigerator overnight.

[0033] (6) Centrifuge at 5000 rpm for 1 min at room temperature, discard the supernatant and keep the precipitate, wash the precipitate twice with 70% ethanol, air-dry the precipitate, and add 200 ul TE solution for later use.

[0034] 2. PCR Amplification

[0035] (1) PCR reaction system 10ul, including Tiangen PCR Mix 4ul, about 10ng DNA template 4ul, forward primers 0.5ul each, and sterile double distilled water 1ul.

[0036] (2) The PCR reaction procedure is:

[0037] Pre-denaturation at 94°C for 5 min; denaturation at 94°C for 1 min, annealing at 55°C for 1 min, extension at 72°C for 1 min, 35 cycles; extension at 72°C for 5 min; storage at 4°C.

[0038] 3. Polyacrylamide gel electrophoresis

[0039] (1) Preparation of polyacrylamide gel

[0040] ① 380g acrylamide and 20g N,N'-methylenebisacrylamide, add water to make up to 1000ml to prepare 40% acrylamide mother solution; take 200ml of the mother solution, add water to make up to 1000ml to prepare 8% acrylamide.

[0041] ② Take 100ml of 8% acrylamide solution, add 1ml of 10% ammonium persulfate and 100μl of TEMED, stir well and immediately pour into the glass plate sealed with agarose gel, fill it up and lay it flat so that it is at an angle of about 10° to the table. Insert a comb and let it stand for 1 to 2 hours to allow the gel to solidify.

[0042] (2) Sample loading

[0043] After the gel solidifies, add electrophoresis buffer to the bottom of the vertical electrophoresis apparatus, fix the glass plate on the vertical electrophoresis tank, clamp the glass plate, add electrophoresis buffer between the two glass plates, remove the comb, take the PCR amplification product, and use a microinjector to take 4μl and inject it into the spotting hole.

[0044] (3) Electrophoresis and staining

[0045] Electrophoresis was performed at 160V for 60 minutes. The gel was removed and placed in a fixative solution for 12 minutes. Then, the gel was placed in a 1‰ silver nitrate solution for 10 minutes. The gel was washed with distilled water for 20 seconds. The gel was transferred to distilled water and a color developing solution was added. The color was developed for 1-2 minutes. The gel was washed with clean water and placed in a plastic wrap. The band pattern of each individual strain was read. Figure 1 and Figure 2 The band pattern was recorded, with the fertile plant band pattern being recorded as "1", the sterile plant band pattern being recorded as "2", the heterozygous plant band pattern being recorded as "3", and the absence of a band being recorded as "0".

[0046] 121 plants were detected with both 2B-TPSGMS and 6B-TPSGMS banding types of "2", indicating that these 121 plants carried sterility genes located on chromosomes 2B and 6B at the same time. Combined with the fertility expression characteristics of the sterile line, these 121 plants were bagged in advance and water and fertilizer management was carried out. On April 10, 2019, all plants were identified in the field. The results showed that the 121 plants carrying two sterility genes showed sterility when the main ear opened, and the tillering ears were bagged in advance. In addition, there were more tillering ears due to the early water and fertilizer management. All 121 sterile plants received seeds, and the number of seeds was >30, laying a good foundation for the next step of reproduction and stage identification. Compared with the case without the use of molecular markers, the efficiency was increased by more than 2 times. In the past, there was no molecular marker assistance and it was entirely dependent on personal experience. Although the fertility expression of the temperature-light-sensitive male nuclear sterile line of wheat is controlled by nuclear genes, it is also affected by the environment. It is fertile at high temperatures and sterile at low temperatures. The selection of sterile lines requires that the main ear exhibits sterility characteristics, such as opening of the lemma and exposure of the stigma. At the same time, the tillering ears must be fertile or partially fertile so that seeds can be left for reproduction and stage identification. However, the selection of sterile lines is carried out in the field environment, and the temperature during the fertility sensitive period is not controlled. Many sterile lines are managed with water and fertilizer after expressing sterility characteristics. The treatment measures are taken too late, resulting in too few tillering ears, very little seeds are received, and some even no seeds are received, resulting in natural elimination of the sterile line. Some sterile lines encounter high temperatures during the fertility sensitive period, making the main ear fertile but not selected.

[0047] Example 2: Breeding of temperature-light-sensitive male sterile wheat lines in 2017 / 2018

[0048] The offspring of the sterile line bred this year came from the hybrid offspring with K456S as the female parent, totaling 186 plants. K456S is a sterile line for large-scale seed production, and was bred from the hybrid offspring of K78S. The hybrid offspring was sown on November 15, 2017, and individual leaves were taken for testing on February 27, 2018 to determine which plants carried the sterility-controlling genes (the above-mentioned 2B and 6B SSR markers) (the detection steps are the same as in Example 1). 95 plants were detected to carry both genes at the same time. Combined with the fertility expression characteristics of the sterile line, these 95 plants were bagged in advance and water and fertilizer management was carried out. On April 10, 2018, all plants were identified in the field. The results showed that 95 plants carrying two sterility genes showed sterility in the main ear opening, the tillering ears were bagged in advance, and the tillering ears were more due to the early water and fertilizer management. All 95 sterile plants received seeds, and the number of seeds was >20. The harvested seeds were planted into rows for propagation on June 2, 2018, and all rows were harvested on October 2, 2018. The harvested row seeds were sown in three phases on October 20, October 30 and November 9, 2018 for identification and selection. The results showed that the 95 selected plants all showed high sterility (average sterility in three phases>95.4%). Combined with the characteristics of plant height, stress resistance (disease resistance, drought resistance) and outcrossing fruiting rate, 22 excellent sterile lines were finally selected for the screening of hybrid wheat superior combinations. Using traditional breeding methods, this project has only selected 2 sterile lines for the screening of hybrid wheat superior combinations in the past 10 years.

[0049] Experimental conclusion: It can be seen from Examples 1 and 2 that the use of the two SSR primers in the present invention for molecular marker-assisted selection of wheat male sterile lines can double the breeding efficiency of wheat male sterile lines; in addition, the two markers of the present invention have been verified by molecular marker-assisted selection and are indeed closely linked to the sterility gene of the wheat male sterile line, which helps breeders to better understand the genetic laws of sterility of wheat male sterile lines and lays a foundation for the precise positioning and cloning of sterility genes.

[0050] The above is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any changes or substitutions that are not conceived through creative work should be included in the protection scope of the present invention. Therefore, the protection scope of the present invention should be based on the protection scope defined in the claims. Sequence Listing <110> Institute of Food Crops, Yunnan Academy of Agricultural Sciences <120> SSR Molecular Markers Linked to Thermo-photosensitive Genic Male Sterile Lines in Wheat and Their Application <160> 4 <170> SIPOSequenceListing 1.0 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <400> 1 gaacatccct cacctgcaat 20 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <400> 2 gtacgttgca cctcaagcct 20 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <400> 3 ctgatcgtcc gtctctgtcc 20 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <400> 4 gggtgaaaca gatagtggcg 20

Claims

1. Application of primers of SSR molecular markers in detecting and selecting individual plants of thermophotosensitive male sterile lines of wheat, the primers of the SSR molecular markers are 2B-TPSGMS and 6B-TPSGMS, the nucleotide sequence of the upstream primer of the 2B-TPSGMS is shown in SEQ ID NO.1, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO.2; the nucleotide sequence of the upstream primer of the 6B-TPSGMS is shown in SEQ ID NO.3, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO.4, the 2B-TPSGMS and 6B-TPSGMS are primers linked to the fertility gene of the thermophotosensitive male sterile line of wheat obtained by screening polymorphic primers existing in two parents and two extreme separation pools, and verifying them through double haploid populations.

Images