Molecular marker of wheat TaACL5-4A gene and application of molecular marker

Abstract

The invention discloses a molecular marker of a wheat TaACL5-4A gene and application of the molecular marker, and belongs to the technical field of crop seed selection and cultivation. The molecular marker is a KASP marker and is obtained by amplifying a primer group as shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, the 5'ends of two front primers are respectively marked with HEX and FAM, a PCR amplification product is detected by using a microplate reader, and genetic typing is carried out by using KlusterCaller software. The KASP molecular marker has the beneficial effects that by applying the developed KASP molecular marker, the grain number per ear, the ear length, the spikelet number and other characters of wheat can be predicted, the cost is saved, the selection efficiency is greatly improved, the breeding process can be accelerated, and new possibilities are provided for efficient screening of excellent allelic genes of the TaACL5-4A gene and cultivation of high-yield wheat varieties.

Description

Technical Field

[0001] The present invention relates to a molecular marker and an application thereof, in particular to a molecular marker of wheat TaACL5-4A gene and an application thereof in assisted breeding, belonging to the technical field of crop seed selection and breeding. Background Art

[0002] Wheat (Triticum aestivum L.) is the world's most widely distributed and cultivated grain crop, making increasing wheat yield crucial. Wheat ear traits are highly correlated with yield and serve as key selection criteria for wheat yield breeding. These traits primarily include kernel number per ear, ear length, and the number of spikelets per ear. As one of the three key yield factors, kernel number per ear is a crucial trait affecting final wheat yield. Improving kernel number per ear is an effective way to increase yield.

[0003] The wheat TaACL5-4A gene was previously mapped for grain number per spike using a recombinant inbred line population constructed from Kenong 9204 and Jing 411. A major stable QTL controlling grain number per spike, qKnps-4A, was located on chromosome 4A. Fine mapping of this QTL pinpointed qKnps-4A within a ~2.19 Mb interval (673.55 Mb-675.74 Mb). Candidate gene analysis identified TraesCS4A02G398300 as a potential candidate gene for qKnps-4A. Functional annotation revealed that TraesCS4A02G398300 encodes a gene encoding thermospermine synthase, which is homologous to the ACL5 (ACAULIS5) gene in Arabidopsis thaliana. Therefore, the gene was named TaACL5-4A. The ACL5 gene product not only regulates plant organ elongation but also plays a key role in maintaining the proliferation activity of the floral meristem. However, the impact of natural variation in the wheat TaACL5-4A gene on wheat yield and the associated molecular markers have not been reported. Therefore, identifying haplotype molecular markers for the TaACL5-4A gene and applying them is of great significance for achieving high and stable wheat yields. Summary of the Invention

[0004] The present invention provides a molecular marker of the wheat TaACL5-4A gene and its application in identifying wheat haplotypes, aiming to provide effective gene resources and molecular markers for crop genetic improvement.

[0005] In order to achieve the above objectives, the present invention adopts the following technical solutions: A molecular marker of the wheat TaACL5-4A gene, comprising two haplotypes, TaACL5-4A-Hap I and TaACL5-4A-Hap II, wherein the allelic variant bases at positions 675186426, 675186459, 675186707, 675186835, 675187781, and 675187814 of TaACL5-4A-Hap I in the Chinese spring wheat reference genome sequence RefSeq v2.1 are A, C, A, G, C, and T, respectively; and the allelic variant bases at positions 675186426, 675186459, 675186707, 675186835, 675187781, and 675187814 of TaACL5-4A-Hap II in the Chinese spring wheat reference genome sequence RefSeq v2.1 are A, C, A, G, C, and T, respectively. The allelic variant bases at positions 675186426, 675186459, 675186707, 675186835, 675187781, and 675187814 in v2.1 are G, G, G, A, G, and C, respectively. The molecular marker is a KASP marker, which is amplified by two forward primers represented by SEQ ID NO: 1 and SEQ ID NO: 2 and one backward primer represented by SEQ ID NO: 3. The 5' end of the forward primer represented by SEQ ID NO: 1 is labeled with a HEX fluorescent group, and the 5' end of the forward primer represented by SEQ ID NO: 2 is labeled with a FAM fluorescent group. The PCR amplification products are detected using a microplate reader and genotyped using KlusterCaller software. When the KlusterCaller result is red, the haplotype of the wheat TaACL5-4A gene is TaACL5-4A-Hap I, When the result of KlusterCaller is blue, the haplotype of the wheat TaACL5-4A gene is TaACL5-4A-Hap II.

[0006] The molecular markers of the aforementioned wheat TaACL5-4A gene were used to breed the wheat TaACL5-4A-Hap II haplotype. Compared with the TaACL5-4A-Hap I haplotype, the TaACL5-4A-Hap II haplotype has a larger number of grains and spikelets per spike and a longer spike length.

[0007] The present invention is beneficial in that: (1) The present invention develops a new KASP molecular marker. The KASP molecular marker developed by the present invention is derived from the genetic variation analysis of the TaACL5-4A gene in natural wheat populations, providing unique genetic information and a new tool for wheat breeding; (2) By applying the KASP molecular marker developed by the present invention, wheat traits such as grain number per spike, spike length, and spikelet number can be predicted, which not only saves costs but also greatly improves selection efficiency, accelerates the breeding process, and provides new possibilities for efficiently screening superior alleles of the TaACL5-4A gene and cultivating high-yield wheat varieties. BRIEF DESCRIPTION OF THE DRAWINGS

[0008] Figure 1 This is the SNP variation site and haplotype typing results of the wheat TaACL5-4A gene; Figure 2 This is the KASP marker genotyping result diagram of some wheat TaACL5-4A genes, where blue represents GG, red represents AA, green represents heterozygous, purple represents signal but no clear typing, and pink represents no signal or weak signal (invalid result). DETAILED DESCRIPTION

[0009] The present invention will be described in detail below with reference to the accompanying drawings and embodiments.

[0010] 1. Determination of polymorphic sites and haplotypes of the wheat TaACL5-4A gene 1. Acquisition of polymorphic sites of the wheat TaACL5-4A gene The experiment used 48 common hexaploid wheat varieties, the specific varieties are shown in Table 1.

[0011] Table 1 48 common hexaploid wheat varieties

[0012] The first-generation sequencing results of 48 common hexaploid wheat varieties listed in Table 1 were comprehensively analyzed for multiple sequence alignment.

[0013] The analysis results showed that there were 6 SNP variations in the wheat TaACL5-4A gene (SEQ ID NO: 4), including 4 SNP variations in the exon region and 2 SNP variations in the intron region. The physical positions of these 6 SNP variations in the Chinese spring wheat reference genome sequence RefSeq v2.1 were 675186426, 675186459, 675186707, 675186835, 675187781 and 675187814, respectively.

[0014] 2. Acquisition of TaACL5-4A gene haplotype The sequence variation of 48 common hexaploid wheat varieties listed in Table 1 was summarized and analyzed. The results showed that there were two haplotypes in the TaACL5-4A gene ( Figure 1): TaACL5-4A-Hap I, TaACL5-4A-Hap II, wherein the allelic variant bases of TaACL5-4A-Hap I at positions 675186426, 675186459, 675186707, 675186835, 675187781 and 675187814 in the Chinese spring wheat reference genome sequence RefSeq v2.1 are A, C, A, G, C and T, respectively, and TaACL5-4A-Hap II II The allelic variant bases at positions 675186426, 675186459, 675186707, 675186835, 675187781 and 675187814 in the Chinese spring wheat reference genome sequence RefSeqv2.1 are G, G, G, A, G and C, respectively.

[0015] 2. Identification of wheat TaACL5-4A gene haplotypes Competitive allele-specific PCR (KASP) molecular marker-assisted selection breeding can select for target traits at the DNA level, which not only stabilizes the results and reduces the cost of phenotypic evaluation, but also improves the efficiency of wheat breeding.

[0016] To validate and utilize the identified SNP variants and further develop KASP markers, SNP 3 was selected from SNPs 1, 2, 3, 4, 5, and 6. Primer sequences were designed and evaluated using WheatOmics (http: / / wheatomics.sdau.edu.cn / PrimerServer / ) and validated in 260 accessions.

[0017] For SNP 3, the designed primers consist of two front primers (Primer Allele X, Primer Allele Y) and one back primer (Common). The sequences of the two front primers are shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively, and the sequence of the back primer is shown in SEQ ID NO: 3.

[0018] In order to detect the PCR amplification product using a microplate reader, the 5' end of the forward primer (PrimerAllele X) shown in SEQ ID NO: 1 was labeled with a HEX fluorescent group, and the labeled primer was recorded as AlleleHEX. The 5' end of the forward primer (Primer Allele Y) shown in SEQ ID NO: 2 was labeled with a FAM fluorescent group, and the labeled primer was recorded as AlleleFAM.

[0019] PCR amplification was performed using the two labeled forward primers (AlleleHEX, AlleleFAM) and one backward primer (Common) on the DNA of 260 common hexaploid wheat varieties (seedlings) listed in Tables 2-1, 2-2, 2-3, 2-4, and 2-5. The PCR reaction system and amplification procedure are as follows: (1) The PCR reaction system consists of 2 μL DNA template (30 ng / μL), 2.5 μL KASP Master Mix (LGCGenomics, KBS-1016-002), and 0.08 μL primer working solution, which is made up to 5 μL with sterile ultrapure water. The primer working solution is prepared as follows: 12 μL of 100 μM AlleleHEX, 12 μL of 100 μM AlleleFAM, and 30 μL of 100 μM Common are mixed, and the volume is made up to 100 μL with ddH2O. (2) The amplification procedure was as follows: pre-denaturation at 95°C for 15 min; denaturation at 95°C for 20 s, annealing and extension at 65-57°C for 60 s, 9 cycles, with the temperature decreasing by 1°C each cycle; denaturation at 95°C for 20 s, annealing at 57°C for 60 s, 32 cycles; and storage at 10°C.

[0020] The PCR products were sequenced and the TaACL5-4A gene was typed based on the sequencing results. Specifically: When the allelic variant bases of SNP 1 (located at position 675186426 of the Chinese spring wheat reference genome sequence RefSeq v2.1), SNP 2 (located at position 675186459 of the Chinese spring wheat reference genome sequence RefSeq v2.1), SNP 3 (located at position 675186707 of the Chinese spring wheat reference genome sequence RefSeq v2.1), SNP 4 (located at position 675186835 of the Chinese spring wheat reference genome sequence RefSeq v2.1), SNP 5 (located at position 675187781 of the Chinese spring wheat reference genome sequence RefSeq v2.1), and SNP 6 (located at position 675187814 of the Chinese spring wheat reference genome sequence RefSeq v2.1) are A, C, A, G, C, and T, respectively, the typing result of the wheat TaACL5-4A gene is TaACL5-4A-HapI; When the allelic variant bases of SNP 1 (located at position 675186426 of the Chinese spring wheat reference genome sequence RefSeq v2.1), SNP 2 (located at position 675186459 of the Chinese spring wheat reference genome sequence RefSeq v2.1), SNP 3 (located at position 675186707 of the Chinese spring wheat reference genome sequence RefSeq v2.1), SNP 4 (located at position 675186835 of the Chinese spring wheat reference genome sequence RefSeq v2.1), SNP 5 (located at position 675187781 of the Chinese spring wheat reference genome sequence RefSeq v2.1), and SNP 6 (located at position 675187814 of the Chinese spring wheat reference genome sequence RefSeq v2.1) are G, G, G, A, G, and C, respectively, the typing result of the wheat TaACL5-4A gene is TaACL5-4A-HapII.

[0021] The typing results of the TaACL5-4A gene in 260 natural wheat populations are shown in Tables 2-1, 2-2, 2-3, 2-4 and 2-5.

[0022] Table 2-1 Typing results of wheat TaACL5-4A gene from the 1st to the 48th

[0023]

[0024] Table 2-2 Typing results of wheat TaACL5-4A gene from accessions 49 to 108

[0025] Table 2-3 Typing results of wheat TaACL5-4A gene from accessions 109 to 168

[0026] Table 2-4 Typing results of wheat TaACL5-4A gene from accessions 169 to 228

[0027] Table 2-5 Typing results of wheat TaACL5-4A gene from accessions 229 to 260

[0028] KASP marker genotyping was performed on 260 common hexaploid wheat varieties listed in Tables 2-1, 2-2, 2-3, 2-4, and 2-5. The specific steps are as follows: (1) PCR amplification Using genomic DNA of 260 wheat samples to be tested as templates, PCR amplification reaction was performed using the two labeled front primers (AlleleHEX, AlleleFAM) and one back primer (Common). The PCR reaction system and amplification procedure were the same as before and will not be repeated here.

[0029] The experiment also set up a blank control (NTC) in which no DNA template was added to the PCR reaction system, and set up one or more blank controls on each plate.

[0030] PCR amplification products were scanned using a multifunctional microplate reader (PHERAstar Plus, BMG LABTECH, Germany) with an excitation wavelength of 485 nm and an emission wavelength of 520 nm for FAM, an excitation wavelength of 535 nm and an emission wavelength of 556 nm for HEX, and an excitation wavelength of 575 nm and an emission wavelength of 610 nm for the system reference fluorescence ROX.

[0031] KlusterCaller software (LGC Genomics, Beverly, USA) was used to analyze the scanning data of the multifunctional microplate reader, and genotype analysis was performed based on the fluorescence signal. Specifically: When the result of KlusterCaller is red, the genotype of wheat TaACL5-4A gene at SNP 3 is AA, and the haplotype is TaACL5-4A-Hap I; When the KlusterCaller result is blue, the genotype of the wheat TaACL5-4A gene at SNP 3 is GG, and the haplotype is TaACL5-4A-Hap II; When the result of KlusterCaller is green, the genotype of wheat TaACL5-4A gene at SNP 3 is heterozygous; When the KlusterCaller result is purple or pink, the detection result is invalid.

[0032] The KASP genotyping results of TaACL5-4A gene of some wheat varieties among 260 common hexaploid wheat varieties are shown in Figure 2 .

[0033] The genotyping results of the KASP marker showed that the typing results of the KASP marker were completely consistent with the haplotype prediction results based on network data and laboratory sequencing results of 48 wheat samples, and the typing results of the KASP marker were good.

[0034] This shows that the KASP marker has been successfully developed and can be further used in breeding material testing.

[0035] 3. Association analysis between wheat TaACL5-4A gene haplotypes and yield traits The natural population phenotypic data of wheat grain number per ear, ear length, and spikelet number were collected from six environments (E1: Qixia Experimental Base in 2018; E2: College of Horticulture, Ludong University, Yantai Experimental Base in 2020; E3: Shijiazhuang Experimental Base in 2020; E4: Laishan Pula Valley Experimental Base in Yantai in 2021; E5: Muyu Village Experimental Base in Yantai in 2022; E6: Laishan Pula Valley Experimental Base in Yantai in 2022) over four years, and the best linear unbiased estimator (BLUE) of each trait in multiple environments was calculated using the R package lme4.

[0036] The data were statistically analyzed using Excel software, and Student's t-test was used for significance analysis. The results of the association analysis of the agronomic traits (grain number per spike, spike length, and spikelet number) of the wheat TaACL5-4A gene in multiple environments are shown in Tables 3-1, 3-2, and 3-3.

[0037] Table 3-1 Results of association analysis of wheat TaACL5-4A gene grain number per spike in multiple environments

[0038] Table 3-2 Results of association analysis of wheat TaACL5-4A gene in multiple environments for ear length

[0039] Table 3-3 Results of association analysis of wheat TaACL5-4A gene spikelet number in multiple environments

[0040] Note: NS means P >0.05, * indicates P <0.05, ** indicates P <0.01, *** indicatesP <0.001.

[0041] In summary, the haplotype analysis results showed that: (1) Compared with the TaACL5-4A-Hap I type, the number of grains per spike in the TaACL5-4A-Hap II strain increased significantly by 5.14-12.24%; (2) Compared with the TaACL5-4A-Hap I type, the spike length of TaACL5-4A-Hap II increased significantly by 6.70-21.74%; (3) Compared with the TaACL5-4A-Hap I type, the spikelet number of TaACL5-4A-Hap II increased significantly by 0.42-6.85%.

[0042] These results indicate that the two haplotypes of the wheat TaACL5-4A gene significantly regulate traits such as grain number per spike, spike length, and spikelet number. A higher number of grains per spike and spikelet number directly increases total grain yield per unit area by increasing the number of seeds set per spike, making it a key agronomic trait for increasing yield. Based on the performance of various traits, the TaACL5-4A-Hap II haplotype exhibits excellent agronomic traits and is a superior haplotype for wheat, with potential value in the development of high-yielding varieties.

[0043] In summary, the two haplotypes of the wheat TaACL5-4A gene identified in the present invention are significantly associated with wheat grain number per spike, spike length, and spikelet number, and have important application value for improving wheat yield traits.

[0044] It should be noted that the above embodiments are merely examples for the purpose of clearly illustrating the present invention and are not intended to limit the embodiments of the present invention. Those skilled in the art will appreciate that other variations or modifications may be made based on the above description. It is not possible to enumerate all embodiments here. Any obvious variations or modifications arising from the technical solution of the present invention remain within the scope of protection of the present invention.

Claims

1. A molecular marker for the wheat TaACL5-4A gene, characterized in that: The TaACL5-4A gene comprises two haplotypes, TaACL5-4A-Hap I and TaACL5-4A-Hap II, wherein the allelic variant bases of TaACL5-4A-Hap I at positions 675186426, 675186459, 675186707, 675186835, 675187781 and 675187814 in the Chinese spring wheat reference genome sequence RefSeq v2.1 are A, C, A, G, C and T, respectively; and the allelic variant bases of TaACL5-4A-Hap II at positions 675186426, 675186459, 675186707, 675186835, 675187781 and 675187814 in the Chinese spring wheat reference genome sequence RefSeq v2.1 are A, C, A, G, C and T, respectively. The allelic variant bases at positions 675186426, 675186459, 675186707, 675186835, 675187781, and 675187814 in v2.1 are G, G, G, A, G, and C, respectively. The molecular marker is a KASP marker, which is amplified by two forward primers represented by SEQ ID NO: 1 and SEQ ID NO: 2 and one backward primer represented by SEQ ID NO:

3. The 5' end of the forward primer represented by SEQ ID NO: 1 is labeled with a HEX fluorescent group, and the 5' end of the forward primer represented by SEQ ID NO: 2 is labeled with a FAM fluorescent group. The PCR amplification products are detected using a microplate reader and genotyped using KlusterCaller software. When the KlusterCaller result is red, the haplotype of the wheat TaACL5-4A gene is TaACL5-4A-Hap I, When the result of KlusterCaller is blue, the haplotype of the wheat TaACL5-4A gene is TaACL5-4A-Hap II.

2. Use of the molecular marker of the wheat TaACL5-4A gene according to claim 1 in breeding the wheat TaACL5-4A-Hap II haplotype, wherein the TaACL5-4A-Hap II haplotype has a larger number of grains and spikelets per spike and a longer spike length than the TaACL5-4A-Hap I haplotype.

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