Molecular markers tightly linked to the major QTL-qSHL-3B for coleoptile length under drought stress in wheat and their application

Abstract

The present invention discloses a molecular marker tightly linked to the major effect QTL‑qSHL‑3B for coleoptile length under drought stress in wheat and its application, belonging to the field of wheat molecular biotechnology and breeding technology. Wherein, the molecular marker is KNchr3B‑195, which is tightly linked to the major effect QTL‑qSHL‑3B for coleoptile length under drought stress located on wheat chromosome 3B, and the nucleotide sequence is shown in SEQ ID NO: 3, with a sequence length of 475bp, which can be amplified by the upstream primer shown in SEQ ID NO: 1 and the downstream primer shown in SEQ ID NO: 2. The molecular marker provided by the present invention can quickly and accurately determine whether a wheat variety (line) under drought stress has the major effect QTL‑qSHL‑3B for coleoptile length, and provides excellent gene resources and selection tools for molecular breeding of wheat yield traits.

Description

Technical Field

[0001] The present invention relates to a molecular marker and an application thereof, in particular to a molecular marker tightly linked to a major effect QTL-qSHL-3B for coleoptile length under wheat drought stress and an application thereof in wheat assisted breeding and genetic improvement, belonging to the field of wheat molecular biotechnology and breeding technology. Background Art

[0002] Drought affects the growth and quality of wheat to a great extent, and affects wheat yield. The bud stage is a critical period for wheat growth. The drought environment also affects the morphology of the wheat root system to a certain extent, but its drought resistance identification cannot be completed based on a single indicator. Wheat drought resistance is a quantitative trait controlled by multiple genes, and the drought resistance mechanism is complex. Discovering new drought-resistant genetic loci in wheat and its closely related species and applying them to breeding work has become the most economical, effective and safe method for breeding drought-resistant wheat varieties.

[0003] The coleoptile is a sheath-like structure unique to monocotyledons, and its tip contains auxin. Its length and elongation rate are of great significance to the emergence of seedlings. In the early stages of seedling growth, it helps to protect wheat seedlings from deep sowing and successfully drill out of the soil. Studies have shown that the length of wheat coleoptiles varies among different drought-resistant groups under drought stress, and the length of wheat coleoptiles at the bud stage can be used as one of the indicators for identifying drought resistance at the bud stage. Therefore, exploring excellent alleles that control sheath length and developing functional molecular markers can greatly accelerate the breeding process of new high-yield wheat varieties and provide excellent gene resources and selection tools for molecular breeding of wheat yield traits.

[0004] Wheat sheath length is a quantitative trait controlled by multiple genes, with a relatively complex genetic background and a great influence from the environment. Therefore, genetic research on wheat sheath length is difficult. Generally, physical and chemical methods are used to reflect it by calculating the mean and variance, but they cannot accurately show the location of the gene and its specific role. With the development of molecular biology and biostatistics, quantitative trait loci (QTL) positioning can determine the correlation between unknown QTL and known genomic DNA markers, providing an effective technical means for studying the inheritance of quantitative traits. Therefore, it is extremely important in breeding to obtain genes related to wheat sheath length by developing molecular markers in the target segment.

[0005] At present, there have been a large number of research reports on the growth and physiological response mechanism of coleoptiles. Li Zhuokun et al. used 168 lines and parents of the DH population constructed by wheat varieties Huapei No. 3 and Yumai 57 as materials to locate 8 additive QTLs controlling coleoptile length on chromosomes 2A, 4B and 4D. Wang Jin et al. pointed out that under osmotic stress, the coleoptiles of wheat varieties with strong drought resistance have stronger growth potential than those with weak drought resistance. Rebetzke et al. used a double haploid (DH) population constructed by wheat Cranbrook×Halberd to detect multiple QTLs controlling coleoptile length on chromosomes 2B, 2D, 4A, 4B, 5D and 6B under different temperature and planting depth treatment conditions. At present, there are few reports on the positioning of wheat coleoptile length QTL under polyethylene glycol (PEG) simulated water stress conditions, and there are relatively few reports on sheath length QTL on chromosome 3B. Summary of the invention

[0006] The purpose of the present invention is to provide a molecular marker tightly linked to the major effect QTL-qSHL-3B of wheat coleoptile length under drought stress and its application, and to detect whether wheat varieties (lines) have QTL sites for increasing coleoptile length by obtaining molecular markers tightly linked to the major effect QTL of wheat coleoptile length under drought stress, so as to accelerate the breeding process of new high-yield wheat varieties.

[0007] In order to achieve the above object, the present invention adopts the following technical solution:

[0008] A molecular marker tightly linked to a major QTL-qSHL-3B for coleoptile length under drought stress in wheat, the molecular marker being KNchr3B-195, tightly linked to a major QTL-qSHL-3B for coleoptile length under drought stress located on wheat chromosome 3B, the nucleotide sequence being shown in SEQ ID NO: 3, the sequence length being 475 bp, and being amplified by an upstream primer shown in SEQ ID NO: 1 and a downstream primer shown in SEQ ID NO: 2, wherein both the upstream primer and the downstream primer are single-stranded DNA molecules.

[0009] The aforementioned molecular marker KNchr3B-195 tightly linked to the major effect QTL-qSHL-3B for coleoptile length under drought stress in wheat is used for identifying the coleoptile length trait under drought stress in wheat, and for breeding wheat with the major effect QTL-qSHL-3B for coleoptile length.

[0010] The present invention is beneficial in that:

[0011] (1) The molecular marker KNchr3B-195 provided by the present invention fully reflects the major effect QTL-qSHL-3B of coleoptile length under drought stress in wheat varieties (lines). Using the genomic DNA of the wheat variety (line) to be tested as a template, PCR amplification of the wheat variety (line) is performed using the upstream primer shown in SEQ ID NO: 1 and the downstream primer shown in SEQ ID NO: 2. It can quickly and accurately determine whether the wheat variety (line) has the major effect QTL-qSHL-3B of coleoptile length under drought stress, providing an excellent gene resource and selection tool for molecular breeding of wheat yield traits;

[0012] (2) The molecular marker KNchr3B-195 provided by the present invention, which is tightly linked to the major effect QTL-qSHL-3B for coleoptile length under wheat drought stress, is used in wheat molecular breeding, which can greatly reduce the workload of phenotypic identification and can be used in the wheat bud stage, thereby saving breeding costs and improving breeding efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 This is the confidence interval diagram of the major effect QTL-qSHL-3B of coleoptile length under wheat drought stress on chromosome 3B, where the hollow rectangle represents the chromosome, the right side of the hollow rectangle is marked with the name of the molecular marker, and the left side is marked with the position of the molecular marker on the chromosome (unit is Mb); there are two molecular markers (InDel molecular marker and SNP molecular marker) in the figure, KNchr3B-195 is an InDel molecular marker, and the rest are SNP molecular markers; the three solid rectangles on the right side of the hollow rectangle represent the confidence interval of the major effect QTL-qSHL-3B of sheath length under three environments (three replicates) of D1, D2, and D3, respectively, and D1, D2, and D3 represent three replicates under drought stress;

[0014] Figure 2 It is a graph of the PCR amplification results of the molecular marker KNchr3B-195 in some families of the RIL population, wherein M represents 2000 bp DNA Marker, K represents the amplification result of the wheat variety Kenong 9204, J represents the amplification result of the wheat variety Jing 411, and numbers 1-21 represent the amplification results of some families of the RIL population;

[0015] Figure 3 This is the result of single marker analysis of coleoptile length of 188 families in RIL populations under different environments based on molecular marker KNchr3B-195, where white bars are alleles from Kenong 9204, black bars are alleles from Jing 411, * indicates significant difference (P<0.05), ** indicates extremely significant difference (P<0.01). DETAILED DESCRIPTION

[0016] The present invention is described in detail below with reference to the accompanying drawings and embodiments.

[0017] 1. Get the RIL group

[0018] Wheat varieties Kenong 9204 (longer coleoptile length) and Jing 411 (shorter coleoptile length) were selected for the experiment. Kenong 9204 was used as the female parent and Jing 411 as the male parent. Hybrid F1 was obtained by hybridization, and F1 was self-pollinated to obtain F2. F2 was self-pollinated generation by generation to form an F6 generation RIL population containing 188 families.

[0019] 2. Hydroponic culture and phenotypic identification of RIL populations in different experimental environments

[0020] Thirty seeds with full grains were selected from each family of the RIL population and placed evenly. They were cultured hydroponically under drought treatment (10wt% PEG-6000) and control conditions (deionized water), respectively. The culture room temperature was 23°C, and the light / dark period was 20h / 4h. The coleoptile length (SHL) was measured on the 8th day. This was repeated three times.

[0021] Method for measuring coleoptile length: Use a ruler with an accuracy of 0.01 cm to measure the length of wheat seeds from the base of the coleoptile to the top of the coleoptile, and measure 10 plants in each family and take the average value.

[0022] 3. Results of major QTL detection for coleoptile length

[0023] The physical map was constructed using IciMapping v4.1 software. The six groups of coleoptile length phenotype values ​​obtained under control conditions and drought treatment were sorted according to the BIP module format requirements of IciMapping v4.1, and then additive QTL positioning was performed. With 1Mb as the step interval, 1000 permutation tests were performed to detect the LOD peak under different environments, and the LOD threshold was set to 2.0.

[0024] QTL analysis was performed by combining the high-density genetic linkage map of 188 families in the RIL population with the phenotypic data of coleoptile length in six environments (three replicates under control conditions and three replicates under drought treatment). The results showed that under control conditions, no major QTL for sheath length was detected on wheat chromosomes; under drought treatment, a major QTL for sheath length, qSHL-3B, was detected on wheat chromosome 3B (Table 1, Figure 1 ), which can be stably detected in three environments (D1, D2, and D3) (i.e., three replicates of drought treatment), with LOD values ​​of 2.21-4.12, which can explain 7.92-14.31% of the phenotypic variation in sheath length. The allele from Kenong 9204 increases the sheath length by 0.03-0.12 cm.

[0025] Table 1 Results of major QTL detection for sheath length in 188 families in the RIL population

[0026] environment LocationMb Left Mark Right Mark LOD value PVE / % Additive effect / cm D1 187.1675 AX-111672319 AX-109913603 2.21 7.92 0.09 D2 195.1675 AX-109913603 AX-110073730 3.10 14.31 0.12 D3 188.1675 AX-111672319 AX-109913603 4.12 12.04 0.03

[0027] 4. Development of molecular markers and design of corresponding primer pairs

[0028] An InDel molecular marker with polymorphism between parents was developed near the LOD prediction peak of the major QTL-qSHL-3B section of sheath length, and was recorded as KNchr3B-195. The corresponding primer pair was designed based on the molecular marker KNchr3B-195, wherein the nucleotide sequence of the upstream primer was: CCAGCACGTCAAGACCCAA (SEQ ID NO: 1), and the nucleotide sequence of the downstream primer was: CTCCTGGGTCACTTGAGGC (SEQ ID NO: 2), and both the upstream primer and the downstream primer were single-stranded DNA molecules.

[0029] 5. Genotyping of RIL population using molecular marker KNchr3B-195

[0030] 1. Extract DNA of each strain of RIL population using the improved CTAB method

[0031] The method for extracting DNA from each strain of the RIL population using the modified CTAB (cetyltrimethylammonium bromide) method is as follows:

[0032] (1) Take a steel ball and 0.2 g of fresh wheat leaves and put them into a 2.0 mL centrifuge tube. Then quickly put the centrifuge tube into liquid nitrogen and shake the centrifuge tube to grind the wheat leaves into fine powder.

[0033] (2) Continue to add 0.8 mL of CTAB extract to the centrifuge tube, shake well, and incubate at 65°C in a water bath for 60 min, inverting 4 times from time to time to shake well;

[0034] (3) Cool the centrifuge tube at room temperature, add an equal volume of chloroform-isoamyl alcohol (volume ratio 24:1), shake vigorously for 1 min to mix, and centrifuge at 8000 rpm for 10 min;

[0035] (4) Aspirate 600 μL of supernatant into another 1.5 mL centrifuge tube, add 0.8 times the volume of pre-cooled isopropanol (-20° pre-cooled) to precipitate DNA, and centrifuge at 12000 rpm for 6 min;

[0036] (5) Pour off the supernatant and add an appropriate amount of 70% ethanol to wash the precipitate twice. Place the centrifuge tube at an angle in a fume hood to air dry. After the alcohol smell is gone, add 400 μL TE to dissolve it and store it in a -20°C refrigerator for long-term storage.

[0037] 2. Genotyping of RIL population based on wheat genomic DNA using molecular marker KNchr3B-195

[0038] (1) PCR amplification of DNA from RIL population

[0039] Upstream primer: CCAGCACGTCAAGACCCAA (SEQ ID NO: 1)

[0040] Downstream primer: CTCCTGGGTCACTTGAGGC (SEQ ID NO: 2)

[0041] The PCR amplification system is 10 μL, specifically including: 2 μL DNA template, 0.5 μL upstream primer, 0.5 μL downstream primer, 5 μL 2×Taq PCR premix reagent, and 2 μL ddH2O.

[0042] The PCR amplification program used a common program, specifically: pre-denaturation at 95°C for 5 min; denaturation at 95°C for 30 s, annealing at 65°C for 30 s, extension at 72°C for 40 s, 34 cycles; extension at 72°C for 5 min; end amplification, and store at 12°C.

[0043] (2) Electrophoresis of PCR amplification products

[0044] The PCR amplification products were electrophoresed using a 6.0% non-denaturing polyacrylamide gel (preparation method: dissolve 5.85g acrylamide and 0.15g methylene acrylamide in 100mL distilled pure water), the electrophoresis buffer was 1×TBE, and the electrophoresis was performed at a constant voltage of 147V for 2h.

[0045] The PCR amplification results of some families in the RIL population are shown in Figure 2 .

[0046] (3) Analysis of electrophoresis results

[0047] The size of the PCR amplified fragment of wheat variety Kenong 9204 was 475 bp (SEQ ID NO: 3), and the size of the PCR amplified fragment of wheat variety Jing 411 was 415 bp (SEQ ID NO: 4). Among the 188 families in the RIL population, 96 had the same banding pattern as Kenong 9204, 66 had the same banding pattern as Jing 411, 9 were heterozygous, and 17 were missing.

[0048] VI. Analysis of the association between molecular marker KNchr3B-195 and sheath length

[0049] In three replicates under drought treatment, the molecular marker KNchr3B-195 was used to genotype 188 families in the RIL population, and the difference in sheath length between different genotypes was analyzed. The results of single marker analysis of sheath length of 188 families based on molecular marker KNchr3B-195 in three replicates are shown in Figure 3 .

[0050] The results showed that the allele from Kenong 9204 increased sheath length compared with the allele from Jing 411 which reduced sheath length, and this result was verified in three replicates under drought treatment.

[0051] The above results prove that the molecular marker KNchr3B-195 is a molecular marker tightly linked to wheat sheath length and can be effectively used in the molecular marker-assisted selection breeding program for wheat sheath length.

[0052] It should be noted that the above embodiments are merely examples for clearly illustrating the present invention, and are not intended to limit the embodiments of the present invention. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is impossible to list all the embodiments here. Any obvious changes or modifications derived from the technical solution of the present invention are still within the scope of protection of the present invention.

Claims

1. A molecular marker tightly linked to the major QTL-qSHL-3B for coleoptile length under drought stress in wheat, characterized in that: The molecular marker is KNchr3B-195, which is closely linked to the major effect QTL-qSHL-3B of coleoptile length located on wheat chromosome 3B. The nucleotide sequence is shown in SEQ ID NO: 3, the sequence length is 475bp, and can be amplified by the upstream primer shown in SEQ ID NO: 1 and the downstream primer shown in SEQ ID NO:

2. Both the upstream primer and the downstream primer are single-stranded DNA molecules.

2. Use of the molecular marker KNchr3B-195 tightly linked to the major QTL-qSHL-3B for coleoptile length under drought stress in wheat according to claim 1 in identifying the trait of coleoptile length in wheat.

3. Use of the molecular marker KNchr3B-195 tightly linked to the major effect QTL-qSHL-3B for coleoptile length under drought stress in wheat according to claim 1 in breeding wheat with the major effect QTL-qSHL-3B for coleoptile length.

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