A molecular marker closely related to flowering time of cabbage and its application

Abstract

The present invention belongs to the field of molecular biology, and in particular to a molecular marker related to the early or late flowering of cabbage. The nucleotide sequence of the molecular marker is shown in SEQ ID No.1. The present invention cloned the gene sequence of BoTTG1 and its promoter sequence from cabbage with extremely early and extremely late flowering, and found that the early / late flowering BoTTG1 gene sequences were consistent, while there was an insertion / deletion of 341bp in the promoter region. The InDel-TTG1 marker was designed based on this insertion / deletion sequence, and the marker was verified in 89 natural cabbages, and it was found that 40 cabbages contained early flowering markers and 49 cabbages contained late flowering markers. According to the identification of flowering period for three consecutive years, it was determined that the InDel-TTG1 marker was closely related to the early or late flowering period of cabbage. The marker can be directly applied to the field of cabbage bolting resistance breeding, and early identification and auxiliary selection of bolting resistance can be performed to improve breeding efficiency.

Description

Technical Field

[0001] The invention belongs to the field of molecular biology, and in particular relates to a molecular marker closely related to the early or late flowering of cabbage and an application thereof. Background Art

[0002] Cabbage Brassica oleracea var.capitata ), hereinafter referred to as cabbage, is a biennial vegetable crop of the genus Brassica in the family Cruciferae, and its main edible organ is the cabbage head. The bolting and flowering of cabbage are mainly affected by the vernalization pathway, the photoperiod pathway, the circadian rhythm pathway, the gibberellin pathway and the autonomous pathway. These pathways complement each other and jointly regulate the expression of flowering integration factors, affecting bolting and flowering. In the actual production process of cabbage, if the cabbage head is subjected to a long period of low temperature before maturity, it is easy to cause the plant to switch from vegetative growth to reproductive growth, causing premature bolting, resulting in a decrease in cabbage yield and quality, causing serious economic losses. Therefore, cultivating cabbage varieties that are resistant to bolting is an effective way to solve this problem, and the prerequisite for achieving this breeding goal is to select and breed late-flowering cabbage parent materials that are resistant to bolting.

[0003] TTG1 It has multiple effects and is involved in regulating the accumulation of anthocyanins, seed coloring, seed coat mucilage, epidermal hairs, and root hair formation (Tian and Wang 2020). In recent years, scientists have discovered in Arabidopsis that TTG1 Mutants flower early, and overexpression delays flowering (Paffendorf et al. 2020)(Seo et al. 2011). However, there are few reports on BoTTG1 Related to the early or late flowering. Summary of the invention

[0004] To solve the above problems, the inventors cloned the BoTTG1 The gene sequence and its promoter sequence of early / late flowering BoTTG1 The gene sequences are consistent, but there is an insertion / deletion of 341bp in the promoter region. Based on this insertion / deletion sequence, the InDel-TTG1 marker was designed. The marker was verified in 89 natural cabbages, and it was found that 40 cabbages contained early flowering markers and 49 cabbages contained late flowering markers. According to the identification of flowering period for three consecutive years, it was determined that the InDel-TTG1 marker is closely related to the early and late flowering period of cabbage. This marker can be directly applied to the field of cabbage bolting resistance breeding, and early identification and auxiliary selection of bolting resistance can improve breeding efficiency.

[0005] The first object of the present invention is to provide a molecular marker closely related to the early or late flowering of cabbage, which can be used for molecular marker-assisted selection of cabbage breeding materials and varieties that are resistant to bolting and late flowering. The nucleotide sequence of the molecular marker is shown in SEQ ID No. 1.

[0006] The second object of the present invention is to provide a primer pair for amplifying a gene containing the molecular marker.

[0007] In a specific embodiment of the present invention, the forward primer sequence of the primer pair is shown as SEQ ID No. 2, and the reverse primer sequence is shown as SEQ ID No. 3.

[0008] The invention also provides application of the molecular marker in selecting early and late flowering period markers of cabbage.

[0009] In a specific embodiment of the present invention, cabbage genomic DNA is used as a template, PCR amplification is performed using the primer pair, and the product is detected by electrophoresis. If a 136 bp target fragment appears, it is an early-flowering cabbage material; if a 477 bp target fragment appears, it is a late-flowering cabbage material.

[0010] The present invention also provides a method for molecular marker-assisted selection of early or late flowering period of cabbage. The cabbage genomic DNA is used as a template, and a fragment containing the molecular marker is obtained by PCR amplification. If the PCR amplified fragment contains the sequence shown in SEQ ID No. 1, it is a late-flowering cabbage material. If the PCR amplified fragment does not contain the sequence shown in SEQ ID No. 1, it is an early-flowering cabbage material.

[0011] In a specific embodiment of the present invention, PCR amplification is performed using the cabbage genomic DNA as a template using the primer pair, and the product is detected by electrophoresis. If a 136 bp target fragment appears, it is an early-flowering cabbage material; if a 477 bp target fragment appears, it is a late-flowering cabbage material.

[0012] The present invention clones the extremely late-flowering and extremely early-flowering cabbages respectively. BoTTG1 The coding region and promoter sequence of the gene were sequenced and compared, and it was found that the early and late flowering BoTTG1The coding region sequences are consistent, while there is an insertion / deletion of 341bp in the promoter region. Based on this insertion / deletion sequence, the InDel-TTG1 marker was designed. The PCR product of this marker in extremely early-flowering broccoli is 136bp (early-flowering marker), and the PCR product in extremely late-flowering broccoli is 477bp (late-flowering marker). This marker was used in 89 cabbage inbred line populations, and 40 cabbages containing early-flowering markers and 49 cabbages containing late-flowering markers were screened out. The flowering period was counted for three consecutive years and it was found that the average flowering period of cabbage containing early-flowering markers was significantly earlier than that of cabbage containing late-flowering markers (early-flowering 2019 = 201.20 ± 7.76 days, late flowering 2019 = 205.45 ± 8.73 days, P <0.05; early flowering 2020 = 201.13 ± 7.69 days, late flowering 2020 = 204.29 ± 7.37 days, P <0.05; early flowering 2021 = 189.9 ± 6.06 days, late flowering 2021 = 193.82 ± 6.89 days, P <0 .05); and late flowering markers were significantly positively correlated with flowering period r 2019 =0.249, P <0.05; r 2020 = 0.207, P = 0.052; r 2021 = 0.289, P <0 .01), and early flowering markers were significantly negatively correlated with flowering period r 2019 = -0.249, P <0.05; r 2020 = -0.207, P = 0.052; r 2021 = -0.289, P <0 .01). It was confirmed that the molecular marker was closely related to the early or late flowering period. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 In Example 1, the primer pair (BoTTG1-F1 + BoTTG1-R1) was used for PCR amplification. BoTTG1 Gene coding region sequence product, primer pair (BoTTG1-P-F1 + BoTTG1-P-R1) PCR amplification BoTTG1Gene promoter region sequence product.

[0014] Figure 2 Example 1 involves primers in BoTTG1 The position of the gene promoter sequence.

[0015] Figure 3 This is a sequence alignment diagram of the difference fragments between early-flowering and late-flowering cabbages in Example 1, and the dotted line portion is the 341 bp fragment missing from the early-flowering cabbage.

[0016] Figure 4 is the difference segment position involved in Example 1, where the black area indicates BoTTG1 Gene coding region.

[0017] Figure 5 Example 3 InDel-TTG1 marker primer pair PCR amplification BoTTG1 The sequence product of the gene promoter deletion fragment region; wherein, M represents DL5000DNAMarker, E and Early represent early flowering cabbage, and L and Late represent late flowering cabbage.

[0018] Figure 6 This is the relationship between the InDel-TTG1 marker in Example 3 and the flowering period of a natural population of cabbage. DETAILED DESCRIPTION

[0019] The following examples are used to illustrate the present invention but are not intended to limit the scope of the present invention.

[0020] Example 1 BoTTG1 Anchoring of genetically differential fragments

[0021] (1) Based on BoTTG1 Primers were designed using Oligo7 online primer design software based on the genome sequence. BoTTG1 gene primers (Forward primer: 5′-CGCCATGGACAACTCAGCTCCG-3′, Reversed primer: 5′-CGCGGATCCTCAAACTCTGAGGAGCT-3′), 20 μl PCR reaction system (ddH2O: 4.2 μl; 2×PhantaMax buffer: 10 μl; dNTP Mix (10 mM each): 0.4 μl; Primer F (10 μM): 1 μl; Primer R (10 μM): 1 μl; Phanta max super-fidelity DNA Polymerase (1 U / μl): 0.4 μl; DNA (100 ng / μl): 3 μl), and PCR reaction program (95°C 5 min, {95°C 15 s, 55°C 30 s, 72°C 1 min}×34 cycles, 72°C The PCR product was amplified from genomic DNA of extremely early-flowering (F416) and late-flowering (P1) cabbage under the conditions of 10 min and 16°C storage. BoTTG1 Gene sequence, 1% agarose gel electrophoresis imaging, the results are shown in Figure 1 The size of the amplified fragments in the coding region of early-flowering and late-flowering cabbage was about 1.0 kb, with no difference in size; BGI sequencing found that the sequences were completely consistent.

[0022] (2) Based on BoTTG1 The primers were designed with reference to the promoter region sequence of the genome using Oligo7 online primer design software. BoTTG1 Promoter primers (Forward primer: 5'-TGAGGGGTTCAAATCACGGG-3', Reversed primer: 5'-GAAGGACATCGCGTAGAGGG-3') (position see Figure 2The PCR product was amplified from the genomic DNA of extremely early-flowering (F416) and late-flowering (P1) cabbage under the conditions of 20 μl reaction system (ddH2O: 4.2 μl; 2×PhantaMax buffer: 10 μl; dNTPMix (10 mM each): 0.4 μl; Primer F (10 μM): 1 μl; Primer R (10 μM): 1 μl; Phanta max super-fidelity DNA Polymerase (1 U / μl): 0.4 μl; DNA (100 ng / μl): 3 μl) and PCR reaction program (98℃ 5 min, {98℃ 15 s, 65℃ 30 s -1℃ / c, 72℃ 2 min}×10 cycles, {98℃15 s, 55℃ 30 s, 72℃ 2 min}×20 cycles, 72℃ 10 min, stored at 16℃). BoTTG1 Gene promoter region, 1% agarose gel electrophoresis imaging, the results are shown in Figure 1 The size of the amplified fragment in the promoter region of early-flowering and late-flowering cabbage was not significantly different, and the amplified fragment size was about 2700bp; the promoter sequence was cloned and sent to BGI for sequencing. The sequencing results showed that the early-flowering cabbage material lacked 341bp compared with the late-flowering cabbage. The results are shown in Figure 3-4 It was preliminarily speculated that the 341bp-sized differential fragment might be related to the flowering period of cabbage.

[0023] Example 2 BoTTG1 Development of promoter BoTTG1-indel markers

[0024] According to extreme early flowering and extreme late flowering BoTTG1The promoter has a 341bp difference, and the InDel-TTG1 marker primer pair was designed at both ends (Forward primer: 5'-GAAATACTATTCCCTCCAAATTCACCT-3', reversed primer: 5'-ACGATGGAAAGTTCCACGGTCAGT-3'). The PCR reaction system of this primer pair is: 20μl system (ddH2O: 12.2μl; 10×Taq buffer: 2μl; dNTP Mix (10mM each): 0.4μl; Primer F (10μM): 1μl; Primer R (10μM): 1μl; Taq DNA Polymerase (1U / μl): 0.4μl; DNA (100ng / μl): 3μl), PCR reaction program is: 95℃5min, (95℃ 15s, 55℃ 30s, 72℃ 30s)×28cycles, 72℃ 10min, stored at 16℃), the PCR product of this marker in early-flowering broccoli was 136bp (early-flowering marker), and the PCR product in late-flowering broccoli was 477bp (late-flowering marker).

[0025] Example 3 BoTTG1 Correlation between promoter InDel-TTG1 marker and flowering time of cabbage

[0026] 89 cabbage samples were randomly selected from the cabbage inbred lines, and the flowering period for three consecutive years (2019, 2020 and 2021) was counted. PCR amplification was performed in these 89 cabbage samples using the InDel-TTG1 marker, and electrophoresis was performed to verify that the amplified bands of 40 cabbage samples were 136 bp and the amplified bands of 49 cabbage samples were 477 bp ( Figure 5 ). Regarding the flowering period in 2019, the average flowering period of cabbage with early flowering markers was significantly earlier than that of cabbage with late flowering markers (early flowering 2019 =201.20±7.76 days, late flowering 2019 =205.45±8.73 days, P <0.05); and late-flowering markers were significantly positively correlated with flowering period ( r 2019 =0.249, P <0 .05), and early flowering markers were significantly negatively correlated with flowering time ( r 2019 =-0.249, P <0 .05). Regarding the flowering period in 2020, the average flowering period of cabbage with early flowering markers was significantly earlier than that of cabbage with late flowering markers (early flowering 2020 =201.13±7.69 days, late flowering 2020=204.29±7.37 days, P <0.05); and late-flowering markers were significantly positively correlated with flowering period ( r 2020 =0.207, P =0.052), and the early flowering marker was significantly negatively correlated with the flowering period ( r 2020 =-0.207, P =0 .052). Regarding the flowering period in 2021, the average flowering period of cabbage with early flowering markers was significantly earlier than that of cabbage with late flowering markers (early flowering 2021 =189.9±6.06 days, late flowering 2021 =193.82±6.89 days, P <0 .05); and the late-flowering marker was significantly positively correlated with the flowering period ( r 2021 =0.289, P <0 .01), and early flowering markers were significantly negatively correlated with flowering time ( r 2021 =-0.289, P <0.01) ( Figure 6 ). The results proved that the InDel-TTG1 marker was closely related to the early or late flowering period of cabbage.

[0027] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the technical principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A molecular marker associated with the early or late flowering period of cabbage. It is characterized in that Its nucleotide sequence is shown in SEQ ID No.

1.

2. Amplifying a primer pair containing the molecular marker according to claim 1, It is characterized in that The forward primer sequence is shown in SEQ ID No. 2, and the reverse primer sequence is shown in SEQ ID No.

3.

3. Use of the molecular marker described in claim 1 in selecting early or late flowering period markers for cabbage.

4. The use according to claim 3, It is characterized in that The following steps are involved: The cabbage genomic DNA is used as a template and the primer pair of claim 2 is used for PCR amplification. The product is detected by electrophoresis. If a 136 bp target fragment appears, it is an early-flowering cabbage material; if a 477 bp target fragment appears, it is a late-flowering cabbage material.

5. A molecular marker-assisted selection method for early or late flowering period of cabbage, It is characterized in that Using cabbage genomic DNA as a template, a fragment containing the molecular marker according to claim 1 is obtained by PCR amplification. If the PCR amplified fragment contains the sequence shown in SEQ ID No. 1, it is a late-flowering cabbage material. If the PCR amplified fragment does not contain the sequence shown in SEQ ID No. 1, it is an early-flowering cabbage material.

6. The molecular marker-assisted selection method for early or late flowering period of cabbage according to claim 5, It is characterized in that The cabbage genomic DNA is used as a template and the primer pair of claim 2 is used for PCR amplification. The product is detected by electrophoresis. If a 136 bp target fragment appears, it is an early-flowering cabbage material; if a 477 bp target fragment appears, it is a late-flowering cabbage material.

Images