CN107190093B - Functional molecular markers for screening late flowering traits in maize under long-day conditions and their applications - Google Patents

Abstract

Functional molecular markers for screening late flowering traits of maize under long-day light conditions and applications thereof, relating to the biological field. The functional molecular marker InDel-PZmCOL3 used for screening late flowering traits of maize under long-day conditions, the PCR primer sequences are: forward primer: 5'-AGCCTAAGCATCTCCAAGGG-3', reverse primer: 5'-CGGGTTTGCTCCTTTGTTCG-3'. The invention designs specific functional molecular markers according to the above-mentioned specific difference sequences, which is conducive to the introduction of tropical blood maize materials into the genetic breeding of temperate maize material production areas, and not only retains the excellent disease resistance alleles introduced into tropical blood maize materials, but also improves the The shortcomings of the late flowering of tropical blood maize materials cannot be directly applied to the production of temperate maize materials.

Description

Functional molecular markers for screening late flowering traits in maize under long-day conditions and their application

technical field

The invention relates to the field of biotechnology, in particular to a functional molecular marker for screening late flowering traits of maize under long-day illumination conditions and its application.

Background technique

Functional markers are developed based on polymorphic motifs within functional genes that cause variation in phenotypic traits. Once genetic effects are localized to specific functional motifs, functional markers developed based on this correlation can determine the presence or absence of target alleles in different genetic backgrounds without further validation. In application, functional markers can avoid selection errors caused by recombination and exchange, and are more effective in the detection of target genes in groups; because they come from within genes, they directly reflect the performance of target traits, and can accurately detect and track target genes. Its genetic effect value is universal and highly reliable, and it is effective for both artificial breeding populations and natural populations. When using backcross to transfer traits such as flowering regulation, the application of functional markers can better avoid chain burdens. Therefore, the development of functional markers is helpful to promote the application of molecular marker-assisted selection in breeding and the research based on candidate gene strategies in association analysis, and it is also beneficial to discover favorable genes in germplasm resources.

Corn originated in the tropical regions of Central and North America and is a typical short-day plant. The widespread cultivation of maize in temperate regions is the result of artificial domestication. The flowering of maize is obviously regulated by the photoperiod. After tropical and subtropical maize germplasms are introduced into temperate zones, most of them will have problems such as delayed flowering, uncoordinated male and female, and late maturity, which seriously limit the research and application of the above germplasm resources in temperate maize genetics and breeding. . Affected by the sensitivity of photoperiod, different maize inbred lines and varieties have different adaptability in different ecological regions, and many maize varieties cannot be promoted and planted across regions.

SUMMARY OF THE INVENTION

In order to solve the problem that breeders in the main spring maize producing areas cannot predict whether the maize germplasm resources can bloom normally before planting in temperate long-day sunshine areas, and it is impossible to estimate the appropriate interval for staggered flowering in hybridization and backcrossing. The technical problem is to overcome the disadvantage that tropical blood maize material blooms late and cannot be directly applied to temperate maize material production. The present invention provides a functional molecular marker for screening maize late flowering traits under long-day sunshine conditions and its application.

The technical scheme adopted by the present invention for solving the technical problem is as follows:

The functional molecular marker InDel-PZmCOL3 used to screen late flowering traits of maize under long-day conditions, and its PCR primer sequences are:

Forward primer: 5'-AGCCTAAGCATCTCCAAGGG-3',

Reverse primer: 5'-CGGGTTTGCTCCTTTGTTCG-3'.

As a preferred embodiment, the functional molecular marker InDel-PZmCOL3 is located in the promoter region of the ZmCOL3 gene that regulates the flowering stage of maize.

As a preferred embodiment, the maize flowering stage regulating gene ZmCOL3 belongs to the CCT class transcription factor and is located on the fifth chromosome of maize.

As a preferred embodiment, under long-day conditions, there is a specific difference sequence in the promoter region of the ZmCOL3 encoding gene of normal flowering maize and late flowering maize, the specific difference sequence is closely linked with the flowering time of maize, and the specific difference sequence It is located at the 261-811 bp upstream of the initiation codon of the ZmCOL3 coding gene.

As a preferred embodiment, under long-day conditions, in normal flowering maize, the specific difference sequence is as shown in SEQ ID NO: 1 in the sequence listing, and in late flowering maize, the specific difference sequence is as shown in the sequence listing SEQ ID NO: 2.

The present invention also provides a method for distinguishing normal flowering maize and late flowering maize by using the above-mentioned functional molecular marker InDel-PZmCOL3.

The present invention also provides the application of the above-mentioned functional molecular marker InDel-PZmCOL3 in the molecular marker-assisted selection breeding of normal flowering maize and late flowering maize new varieties.

The beneficial effects of the invention are as follows: under the condition of long sunshine, there are specific difference sequences in the ZmCOL3 coding gene promoter regions of normal flowering maize and late flowering maize, and the specific difference sequences are closely linked with the flowering time of maize, and the specific difference sequences are closely linked with the flowering time of maize. The sex difference sequence is located at the 261-811 bp upstream of the initiation codon (ATG) of the ZmCOL3 coding gene. The present invention designs a specific functional molecular marker for distinguishing normal-flowering and late-flowering maize under long-day conditions according to the above-mentioned specific difference sequence characteristics, and the functional molecular marker InDel-PZmCOL3 is beneficial to the introduction of tropical blood maize material into temperate maize material Genetic breeding in the production area. Spring maize and maize production area (long-day conditions) genetic breeding When introducing tropical blood maize germplasm resources, the functional molecular marker InDel-PZmCOL3 was used to purposely remove the late-flowering characteristic base sequence of this gene, which not only retained the introduction of tropical maize The excellent disease resistance alleles of the bloodline maize material also improved the defect that the tropical bloodline maize material bloomed late and could not be directly applied to the production of temperate maize material.

The application of the functional molecular marker of the present invention is beneficial to the temperate long-day sunshine areas, especially the breeding researchers in the main spring maize production areas to quickly predict the flowering period of the obtained maize germplasm resources. has important guiding significance. At the same time, it is also convenient for breeders to quickly distinguish between tropical blood materials and temperate blood materials (most tropical blood materials have good agronomic traits of resistance, but in temperate long-day areas, the biological characteristics of delayed flowering or normal flowering); this screening The popularization and application of the technology is of great significance for accelerating the application speed of the breeding of tropical blood maize germplasm resources in the main producing areas of temperate maize and improving the wide adaptability of maize varieties.

Description of drawings

Figure 1 is a sequence alignment analysis diagram.

Figure 2 is a genotyping diagram of a representative backbone inbred line that can normally flower in the long-day area. In the picture: M: DL2000Marker; 1: Zheng 58; 2: 7922; 3: Cheng 351; 4: Ji 853; 5: Ji V203; 6: B73; 7: PH6WC; 8: Four-144; 9: Four-287 ; 10: PH4CV; 11: Ji A001; 12: A6203; 13: Chang 7-2; 14: A188; 15: 8902; 16: Mo17; 17: H99; 18: Zhe 461; 19: Dan 598; 20: Law A.

Figure 3 is a genotyping diagram of commercial hybrids in the long-day area. In the picture: M: DL2000 Marker; 1: Xianyu 335; 2: Xianyu 696; 3: DK516; 4: DK517; 5: Zhengdan 958; 6: Jidan 27; 7: Jidan 519; 8: Demeiya 1; 9: Demeya 2; 10: Demeya 3.

Figure 4 is a genotyping map of representative inbred lines in Southwest China and Yunnan. In the picture: M: DL2000 Marker; 1: Chengzi 273; 2: 06S282; 3: Empty; 4: HF05-1; 5: K959; 6: WY8-1-2; 7: Q319; 8: C128; 9: C08; 10: Cross 51; 11: FS08H; 12: T497-2; 13: Empty; 14: HWZ03; 15: C319.

Detailed ways

A functional molecular marker InDel-PZmCOL3 for screening late flowering traits of maize under long-day illumination conditions of the present invention, and its PCR primer sequence is:

Forward primer (INDEL-F): 5'-AGCCTAAGCATCTCCAAGGG-3',

Reverse primer (INDEL-R): 5'-CGGGTTTGCTCCTTTGTTCG-3'.

The maize flowering stage regulatory gene ZmCOL3 belongs to the CCT class of transcription factors and is located on the fifth chromosome of maize. The functional molecular marker InDel-PZmCOL3 of the present invention is located in the promoter region of the ZmCOL3 gene regulating the flowering stage of maize.

Under long-day conditions, there are specific difference sequences in the promoter region of ZmCOL3 coding gene of normal flowering maize and late flowering maize. The specific difference sequence is closely linked with the flowering time of maize, and the specific difference sequence is located in the ZmCOL3 coding gene. The 261-811 bp upstream of the initiation codon (ATG).

Under long-day conditions, in normal flowering maize, the specific difference sequence is shown in SEQ ID NO: 1 in the sequence listing, and in late flowering maize, the specific difference sequence is replaced by a base sequence with a length of 217bp. Substitutions are shown in SEQ ID NO: 2 in the Sequence Listing.

The present invention also provides a method for distinguishing normal flowering maize and late flowering maize by using the above-mentioned functional molecular marker InDel-PZmCOL3.

The functional molecular marker InDel-PZmCOL3 of the present invention can also be applied to the molecular marker-assisted selection breeding of new varieties of normal flowering maize and late flowering maize. The development of functional molecular markers that control the specific transcription factor ZmCOL3 during maize flowering will help to accelerate the application of tropical blood maize germplasm resources in temperate maize genetics and breeding.

In the present invention, the normal flowering corn hybrid material refers to corn inbred lines or hybrids whose flowering period is not later than or close to the flowering period of the reference varieties Xianyu 335 and Zhengdan 958 in Jilin Province under long-day sunshine conditions. The late-flowering corn hybrid material refers to the corn material whose flowering time is delayed by more than 7 days relative to the reference variety, or even does not bloom. The inbred line material is based on the flowering date of the internationally sequenced variety B73.

Example 1 Acquisition of the ZmCOL3 promoter-specific differential sequence of the regulatory gene ZmCOL3 during maize flowering

1. Experimental materials

Normal flowering maize inbred line under long-day conditions: B73;

Late flowering maize inbred line under long-day conditions: CML432.

2. Genomic DNA extraction

The leaves were taken at the seedling stage of maize, and the genomic DNA of the normal flowering maize inbred line B73 and the late flowering maize inbred line CML432 were extracted by CTAB method respectively.

The specific steps are as follows: under the condition of liquid nitrogen, about 0.5-1 g of corn leaves are quickly ground into powder and then transferred to a 2 ml centrifuge tube, and about 800 μL of CTAB buffer preheated at 65°C is added, and fully mixed; Place in a water bath at 65°C for 15 minutes, then take out and add 10 μL RNAase (RNase, concentration of 10 mg/ml), continue the water bath for 1 to 1.5 hours, and mix gently for several times during the water bath; take out the centrifuge tube and add an equal volume of The mixed liquid of chloroform and isoamyl alcohol, in the mixed solution, the volume ratio of chloroform and isoamyl alcohol solution is V:V=24:1, after gentle mixing for 30min, centrifuge at 8000rpm for 20min; suck the supernatant and transfer it to another In a centrifuge tube, add equal volumes of mixed liquid of chloroform and isoamyl alcohol to each tube. In the mixed solution, the volume ratio of chloroform to isoamyl alcohol solution is V:V=24:1. After mixing, centrifuge at 8000 rpm and centrifuge. 20min (In order to prevent the protein between the two layers of liquid from being sucked, it should be noted that the supernatant should not be too much, just suck 2/3 of the volume of the supernatant. This step can be repeated once more); transfer the supernatant to another 1.5ml centrifuge tube, add an equal volume of pre-cooled isopropanol, mix gently, stand at -20°C for 20-30min, and then use Cut off the pipette tip to absorb DNA flocs, soak in 70% ethanol for 2 hours (the centrifuge tube may not be replaced, after sucking the DNA flocs, pour out the liquid in the centrifuge tube); rinse with 70% ethanol for 2 hours ～3 times, centrifuge briefly for 10 seconds in a microcentrifuge, pour out 70% ethanol and air dry; add 100-200 μL of l×TE, dissolve DNA at 56°C for half a day (the specific dissolution time depends on the situation); use NaNoDrop 2000 DNA concentration The analyzer detects the concentration and purity of DNA, and takes a small amount of samples to determine the quality of DNA by 0.8% Agarose gel electrophoresis.

3. PCR sequencing and sequencing analysis

Using the genome nucleotide sequences (MaizeGDB accession number: GRMZM2G021777) of the normal-flowering maize inbred line B73 under long-day conditions and the late-flowering maize inbred line CML432 under long-day conditions as templates, primers were designed to amplify the maize inbred line B73 by PCR. and the genomic DNA fragment of the maize inbred line CML432.

Primers were designed using software Primer5.0, synthesized by Sangon Bioengineering (Shanghai) Co., Ltd., diluted to 10 μmol/L with sterilized ddH ₂ O, and stored at -20°C for future use.

(1) The primer sequences are as follows:

A-551-type promoter primer sequence:

F:AGCCGAGTGTTTGGTACGAATGGTT;

R: GTTGGTGCGTGTCCGCCGTG.

P-217-type promoter primer sequence:

F: GCTGATGTGTTTGGTACGGGGTGG;

R: CCGGGCGGACTGGGTTGC.

(3) PCR amplification system (20 μL system) is as follows:

(4) The PCR amplification procedure is as follows:

The amplified product was detected by 1% agarose gel electrophoresis, and sequenced by Shanghai Bioengineering Co., Ltd. after purification by DNA purification kit.

Through sequence alignment analysis, it was found that there were specific sequence differences in the promoter region of the ZmCOL3 coding gene of the normal flowering maize inbred line B73 and the late flowering maize inbred line maize CML432 under long-day conditions, as shown in Figure 1. The promoter-specific difference sequence is located at the 261-811 bp upstream of the gene start codon (ATG). Under long-day conditions, in the normal flowering maize inbred line B73, the specific difference sequence is shown in SEQ ID NO: 1 in the sequence listing, and in the late flowering maize inbred line maize CML432, the specific difference sequence The sequence was replaced by a base sequence with a length of 217 bp, as shown in SEQ ID NO: 2 in the sequence listing.

Example 2 Development of a functional molecular marker InDel-PZmCOL3 for screening late flowering traits of maize under long-day conditions and its genotype analysis on tropical and temperate maize

1. Design primers

Select the sequences on both sides of the deletion segment in the ZmCOL3 gene of the normal flowering maize inbred line B73, and use the software Primer5. It was synthesized by Gongbi Engineering (Shanghai) Co., Ltd., diluted with sterilized ddH ₂ O to 10 μmol/L, and stored at -20 °C for later use.

The primer sequences are as follows:

Forward primer (INDEL-F): 5'-AGCCTAAGCATCTCCAAGGG-3' (SEQ ID NO: 3),

Reverse primer (INDEL-R): 5'-CGGGTTTGCTCCTTTGTTCG-3' (SEQ ID NO: 4).

2. Investigation of experimental materials and their flowering period

317 genetic populations for association analysis were used for genotyping and flowering phenotype testing.

It was found by PCR sequencing that 210 materials were A-551-type and 107 materials were P-217-type. The A-551-type promoter was closely linked to the flowering period of maize under two long-day conditions in Jilin Province and Beijing.

Table 1 shows the significance analysis between the two types of SNP-marked promoter differences and flowering period differences. p-value<0.05 means significant difference (0.0161, 0.0332, 0.0260, 0.0289), pvalue<0.01 means extremely significant difference (0.0094, 0.0016).

Table 1

551/217 DT DA DS 2014JL 0.0161 0.0094 0.0332 2012BJ 0.0260 0.0289 0.0016

Example 3 Genotype analysis and association analysis of flowering stage traits in F2 generation population using functional molecular marker InDel-PZmCOL3

In order to further clarify whether P-217-type and A-551-type are closely linked with maize flowering period in the genetic population, we used the tropical blood maize material CML189 (P-217-type) and the temperate blood maize material Mo17 (A-551- After hybridization, the F2 generation genetic population was constructed; they were planted in three different ecological regions of Hubei, Hainan and Jilin, and the related traits of flowering period such as tasseling, silk spinning and loose powder were investigated.

Genotype analysis of more than 1000 individual plants in the F2 generation genetic population was carried out using the functional molecular marker InDel-PZmCOL3. The study found that the genetic segregation ratio of homozygous P-217-type, heterozygous A-551/P-217-type and homozygous A-551-type was about 1:2:1. The results of association analysis showed that the P-217-type promoter was closely linked with late flowering traits under the condition of long sunshine in Jilin Province and Beijing. The functional molecular marker InDel-PZmCOL3 under long-day conditions (Jilin Province) can significantly distinguish flowering traits such as tasseling, silking and loose powder in maize, with P value <0.01, but not under short-day conditions. Table 2 shows the genotype and phenotype analysis of the F2 generation genetic population at three different locations.

Table 2

Example 4 Genotype detection of maize inbred lines and hybrids in spring maize regions in Southwest China, Huanghuaihai and Northeast China by InDel-PZmCOL3, a functional molecular marker

Using the functional molecular marker InDel-PZmCOL3 to analyze the marker genotypes of maize, two band types were amplified, one band type (P-217-type, size 363bp) in late flowering maize, and another in normal flowering maize. One band type (A-551-type, size 697bp).

Using the functional molecular marker InDel-PZmCOL3, the representative backbone inbred lines Zheng 58, Chang 7-2, PH6WC, PH4CV, Si-287, Si-287 and Si -144, A6202, Zhe 461, B73, Mo17, Ji 853 and other maize inbred lines were all detected as A-551-type genotypes (band size about 700bp), as shown in Figure 2.

Using functional molecular marker InDel-PZmCOL3 to identify representative hybrids of spring maize in Northeast China and Huanghuaihai region (temperate, long-day area) Zhengdan 958, Xianyu 335, Xianyu 696, Dika 516, Dika 517, Demeiya 1 No., Demeiya No. 2, Demeiya No. 3 and other maize inbred lines tested were all A-551-type genotypes (band size about 700bp), as shown in Figure 3.

Using the functional molecular marker InDel-PZmCOL3, the representative inbred lines in the southwest and Yunnan regions such as Chengzi 273, 698-3, HF05-1, K959, WY8-1-2, Jiao 51, C319, Q319 and other maize inbred lines were tested. Among them, the three maize inbred lines, Cross 51, C319 and Q319, showed a serious delay in flowering in Jilin Province, and the PCR amplification detection band size was about 350bp, which was identified as P-217-type, except for the above three maize The other inbred lines did not show serious delay in flowering in Jilin Province, and could bear fruit normally. The PCR amplification detection band size was about 700bp, and it was identified as A-551-type, as shown in Figure 4.

The above research results indicated that the P-217-type molecular marker was unique to late-flowering maize under long-day conditions, while the A-551-type molecular marker was unique to normal-flowering maize. Using the functional molecular marker InDel-PZmCOL3, the size of the electrophoretic bands can be used to distinguish whether the maize material has normal flowering or delayed flowering under long-day conditions. The application of the functional molecular marker InDel-PZmCOL3 is beneficial for the purposeful removal of delayed flowering gene loci when introducing the genetic background of tropical blood maize germplasm resources for temperate maize genetic breeding, and ensuring the excellent disease resistance of the introduction of tropical blood At the same time, the allele improves the shortcomings of the late flowering of tropical blood maize and cannot be directly applied to the production of temperate maize, and accelerates the application in maize production.

The above are only the preferred embodiments of the present invention. It should be pointed out that for those skilled in the art, without departing from the principles of the present invention, several improvements and modifications can be made. It should be regarded as the protection scope of the present invention.

SEQUENCE LISTING

<110> Jilin Academy of Agricultural Sciences

<120> Functional molecular marker for screening late flowering traits of maize under long-day conditions and its application

<160>4

<210>1

<211>551

<212> DNA

<213> Labor

<400>1

GGCTAGCTCCAACAAGGAGCTCTAAAGGGTCCTACACCCTAAATTTAGAGGATAAAGACA 60

TCCTCTACTCCCTCCAGCAGCGTCCTCTAAACGGTTCTCTAAATTTAGAGGACGTTGCTG 120

GATCCTCTATATATAGAGTTTCTCTAAACGGTTCTCTATCCATTTAAATACTTTAAACAA 180

CCGGTTTAGTAAAACTAAAATATGTACAATACATTCGAGAGTATGACAAATACGTATGTA 240

CAAAAAATTAAAAATAAAAAATGTCTTTAATATATGTATTTGCATATAGAGGACGTGATT 300

TAGAGGACGTTGTTGGAGAGGAAGGAGATATAGAGGATGAAATCTTTTAGAGAAGACTGT 360

AAAGGACGGATATAGAGGATGTTGCTGGAGACAGTCTACTCTATATGTAGCATCTCACTT 420

CAACAAACTTCTATCTAGTTTGGCTCTAGTGAGAGAGCTATTTTAGATACTCCAATAGCT 480

TGACAAGTTAGATAAATAGTCTGTTAAAGAATTATTTTGATGTTGAATGACTAAATAACT 540

AGTCTATGGGA 551

<210>2

<211>217

<212> DNA

<213> Labor

<400>2

ATATTATATATGTAGCATCTCACTCTAACAAACTATCTATATAGTTTGGCTAGTCAGGAT 60

AACTATGTAAGTTTCGTTAGTGAGAGAGCTAATTTAGATACTCAAATAGCTTGATGAGTT 120

AGATAACTAATCTATTAAAGAATTTATTTTTTTTATTGAATAGCTAAAATTTAGCTTTACG 180

AGTCGTTTACCTCGCCTTTCGGAGATGGTACAAGTAC 217

<210>3

<211>20

<212> DNA

<213> Labor

<400>3

INDEL-F: 5′-AGCCTAAGCATCTCCAAGGG-3′

<210>4

<211>20

<212> DNA

<213> Labor

<400>4

INDEL-R: 5′-CGGGTTTGCTCCTTTGTTCG-3′

Claims (7)

1. A functional molecular marker InDel-PZmCOL3 for screening maize late-flowering traits under long-day conditions is characterized in that a PCR primer sequence is as follows:

a forward primer: 5'-AGCCTAAGCATCTCCAAGGG-3' the flow of the air in the air conditioner,

reverse primer: 5'-CGGGTTTGCTCCTTTGTTCG-3' are provided.

2. The functional molecular marker InDel-PZmCOL3 as claimed in claim 1, wherein the functional molecular marker InDel-PZmCOL3 is located in maize flowering stage regulatory gene ZmCOL3 promoter region.

3. The functional molecular marker InDel-PZmCOL3 as claimed in claim 2, wherein the maize flowering phase regulatory gene ZmCOL3 belongs to CCT class transcription factor and is located on maize chromosome 5.

4. The functional molecular marker InDel-PZmCOL3 as claimed in claim 1, wherein under long-day conditions, there is a specific difference sequence in promoter region of ZmCOL3 encoding gene of normal flowering corn and late flowering corn, the specific difference sequence is closely linked with corn flowering time, and the specific difference sequence is located 261-811 bp upstream of the start code of ZmCOL3 encoding gene.

5. The functional molecular marker InDel-PZmCOL3 as claimed in claim 4, wherein, in normal flowering maize under long-day conditions, the specific difference sequence is as shown in SEQ ID NO: 1, in the late-flowering corn, the specific difference sequence is shown as SEQ ID NO: 2, respectively.

6. A method of distinguishing between normal and late-flowering maize using the functional molecular marker InDel-PZmCOL3 of any of claims 1 to 5.

7. Use of the functional molecular marker InDel-PZmCOL3 according to any one of claims 1 to 5 for molecular marker assisted selection breeding of new varieties of normal and late flowering maize.

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