Method for creating a waxy maize haploid induction line and applications thereof

Abstract

The application provides a waxy maize haploid induction line creation method and application, and belongs to the technical field of crop breeding. The application selects a high-frequency induction line CAU5, a waxy maize commercial hybrid Shenko Waxy 602 and its paternal parent SWL12 as parents, combines a haploid induction key gene molecular marker, uses hybridization, backcrossing and selfing means, and selects in the breeding process by combining single plant induction rate determination and kernel color marking, and selects a line with clear color marking, haploid induction key gene homozygosity and the highest induction rate as the final application induction line after good transformation. The waxy maize haploid induction line created by the application has the advantages of strong resistance to lodging, large pollen amount and long flowering period, the induced waxy maize haploid has clear color marking, and the scale application can be effectively expanded and the risk can be reduced when waxy maize haploid engineering breeding is carried out.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of crop breeding, and particularly relates to a method for creating a waxy maize haploid inducer line and application thereof. BACKGROUND

[0002] In maize breeding work, the preparation of pure lines is a key link. Conventional breeding methods mainly adopt selfing or backcrossing to purify the background of materials, and generally need more than 8 generations. In comparison, doubled haploid (DH) breeding technology only needs 2 generations to obtain a pure line. According to statistics, in major seed companies in Europe and the United States, most of the maize new variety parents contain one or more DH lines. The coverage area of maize hybrids bred by DH lines reaches 675 million acres. Doubled haploid breeding technology mainly consists of four aspects: induction, identification, doubling and DH line evaluation.

[0003] In normal production activities, haploids can be spontaneously generated, but the frequency is only 1 / 80000, which is far from the production demand. There are many methods for generating haploids in maize, such as anther in vitro culture, distant hybridization, ig gene induction, CenH3 modified material induction and maize parthenogenic haploid inducer line induction. At present, in maize doubled haploid breeding, maize parthenogenic haploid inducer line is mainly used for induction. Since Coe reported the first parthenogenic inducer line Stock6 in 1959, a large number of research works have been carried out on the improvement and breeding of inducer lines based on Stock6. A series of high-frequency inducer lines have been selected. This series of inducer lines all carry anthocyanin synthesis gene R1-nj. The gene is specifically expressed in the embryo and endosperm aleurone layer of maize. In identification, the endosperm aleurone layer of haploid embryo is purple, and the embryo is white. If the embryo and endosperm aleurone layer are both purple, it is a heterozygous diploid.

[0004] Although the doubled haploid breeding technology has been widely used in maize breeding at present. However, the research and utilization of doubled haploid breeding technology in waxy maize breeding is relatively lagging. At the same time, it is found in the work of waxy maize doubled haploid breeding that the expression of R1-nj gene in the embryo or endosperm aleurone layer of the inducer line is easily affected by the maternal material, which affects the accurate identification of haploid embryos. Therefore, a waxy maize haploid inducer line is urgently needed. SUMMARY

[0005] Therefore, the purpose of the present application is to provide a method for creating a waxy maize haploid inducer line and application thereof. The waxy maize haploid inducer line created has a higher induction rate of more than 12%.

[0006] In order to achieve the above-mentioned purpose of the application, the present application provides the following technical solutions.

[0007] A method for creating a waxy maize haploid induction line, comprising the following steps:

[0008] (1) Taking Shenke Nuo 602 as the female parent and the induction line CAU5 as the male parent, hybridization is performed to obtain F1, and F1 grains with obvious coloration of the embryo and endosperm aleurone are screened out;

[0009] (2) Single-seed sowing is performed on the F1 grains, and plants with obvious coloration of the embryo and endosperm aleurone are screened out as the female parent at the powder scattering stage, pollination is performed with SWL12 as the male parent, and (Shenke Nuo 602 x CAU5) x SWL12 F1 grains are obtained, 10 grains with obvious coloration of the embryo and endosperm aleurone are screened out from each plant;

[0010] (3) The (Shenke Nuo 602 x CAU5) x SWL12 F1 grains are sown according to the strain, and backcrossing is performed with the recurrent parent SWL12 for 5 generations, and during the period, single plants containing key sites of haploid induction genes are reserved by using molecular markers;

[0011] (4) After the backcrossing is completed, the single plants of the offspring are self-pollinated, single plants containing key sites of haploid induction genes are reserved by using molecular markers, and single plants with homozygous key sites of haploid induction genes are detected by one-generation sequencing;

[0012] (5) The single plants with homozygous key sites of haploid induction genes are tested for induction rate, and breeding is performed to obtain an excellent waxy maize haploid induction line.

[0013] Preferably, in step (1), 100 F1 grains with the most obvious purple coloration of the embryo and endosperm aleurone are screened out.

[0014] Preferably, in step (2), plants with late emergence and weak growth of F1 generation are eliminated, and 20 plants with strong root system, thick and strong stem, and large amount of pollen are selected as the female parent at the powder scattering stage.

[0015] Preferably, in step (2), 10 grains with the most obvious purple coloration of the embryo and endosperm aleurone are screened out from each plant.

[0016] Preferably, in step (3), (Shenke Nuo 602 x CAU5) x SWL12 F1 strains with late emergence and weak growth are eliminated, and strains with large amount of pollen and long flowering period are selected for backcrossing breeding.

[0017] Preferably, in step (3), 1 plant of each strain is selected to enter the next round of backcrossing in each round of backcrossing, and the selection criteria remain the same in each round of backcrossing.

[0018] Preferably, in steps (3) and (4), the molecular marker primer is GW48, the upstream primer sequence is shown in SEQ ID NO. 1, and the downstream primer sequence is shown in SEQ ID NO. 2.

[0019] Preferably, in the step (4), after backcrossing, 20 strains are planted in the field, and the screening with the molecular marker is continued, and the single plant containing the haploid induction gene key site is reserved, and the other single plants are eliminated.

[0020] Preferably, in the step (4), the self-pollination purification is performed on the reserved single plants, a fruit ear of each BC5F2 is reserved, and the sowing of the BC5F2 grains is continued, 20 plants are sown for each strain, sampling is performed at the seedling stage, the molecular marker screening and the first-generation sequencing detection are performed.

[0021] Another object of the present application is to provide the preparation method of the waxy maize haploid induction line or the application of the obtained waxy maize haploid induction line in corn breeding.

[0022] Compared with the prior art, the present application has the following beneficial effects:

[0023] The present application provides a preparation method of a waxy maize haploid induction line, in which the high-frequency induction line CAU has large pollen amount and long flowering period, the waxy maize hybrid Shenko Waxy 602 and its paternal parent SWL12 have developed root system, compact plant type, large pollen amount and long flowering period, and the prepared waxy maize haploid induction line has developed root system, thick and strong plant, large pollen amount and long flowering period, and has certain advantages in subsequent waxy maize haploid engineering breeding application.

[0024] The waxy maize haploid induction line prepared in the present application has the advantages of strong lodging resistance, large pollen amount and long flowering period, and the induced waxy maize haploid has clear color marker, which can effectively expand the scale application and reduce the risk in the waxy maize haploid engineering breeding. BRIEF DESCRIPTION OF DRAWINGS

[0025] Figure 1 GW48 primer molecular marker band pattern;

[0026] Figure 2 Preparation procedure of the waxy maize haploid induction line of the present application;

[0027] Figure 3 The figure is the first-generation sequencing peak value graph of the haploid induction gene key site, in which the upper graph is the non-homozygous site peak value graph, and the lower graph is the homozygous site peak value graph. DETAILED DESCRIPTION

[0028] The technical solutions provided by the present application are described in detail below in combination with the embodiments, but they should not be understood as limiting the scope of protection of the present application.

[0029] Example 1

[0030] Obtaining of the molecular marker

[0031] Obtaining of the molecular marker

[0032] The gene number of ZmPLA1 in the B73 V5 version of the corn genome database (http: / / www.maizegdb.org / ) is Zm00001eb019170. With the sequence as a reference, a pair of polymorphic primers (molecular markers) with clear band types are selected by using the NCBI online primer design tool after primer specificity alignment, and are named GW48. The primer sequences are as follows: GW48-F: AGGTTCCTGGTGC TGTCCAT (as shown in SEQ ID NO. 1); and GW48-R: TCACCGGTTCGTACCTCG (as shown in SEQ ID NO. 2). The genomic DNA of CAU5 is used as a template to obtain a PCR amplification product with a size of 350 bp.

[0033] Example 2

[0034] Application of the molecular marker

[0035] In the process of transformation, leaf samples are taken from single seedlings at the seedling stage, and the CTAB method is used to extract the genomic DNA of the single seedling. The obtained DNA is used as a template, and the GW48 primers obtained in Example 1 are used for PCR amplification to obtain a PCR amplification product.

[0036] The PCR amplification reaction system is as follows: 2 μl of template DNA, 0.5 μl of forward and reverse primers, 10 μl of PCR amplification reaction buffer Mix, and adding ultrapure water to 20 μl. A layer of liquid paraffin is covered on the top of the reaction solution.

[0037] The PCR amplification reaction conditions are as follows: 95 ℃ pre-denaturation for 4 min; 95 ℃ denaturation for 30 s, 60 ℃ annealing for 30 s, 72 ℃ extension for 30 s, 35 cycles; 72 ℃ extension for 10 min; and 12 ℃ preservation.

[0038] Gel electrophoresis: 1.5% agarose gel is prepared, and the above-mentioned PCR product is detected by electrophoresis and verified by sequencing. The electrophoresis voltage is 180 V, and the electrophoresis time is 15 min.

[0039] After detection, if the key site of the induced gene is contained in the single seedling of the transformation, the single seedling can amplify the target fragment; otherwise, the target fragment cannot be amplified, as shown in SEQ ID NO. 3. Figure 1

[0040] Therefore, the feature of the molecular marker of the present application is that in the process of creating the waxy corn haploid induction line, the target fragment can be amplified, the single seedling of the transformation induction line contains the key site of the haploid induction gene, and the single seedling is retained to enter the next round of selection; otherwise, the single seedling is eliminated. Through the molecular marker, the selection process of the waxy corn haploid induction line can be accelerated. ​

[0041] Example 3

[0042] A method for creating a waxy maize haploid induction line, the flowchart is shown as Figure 2 The steps are as follows:

[0043] (1) Single plant screening

[0044] Using waxy maize hybrid Shenkeno 602 as the female parent and high-frequency induction line CAU5 as the male parent, F1 was obtained by hybridization, and 100 F1 generation kernels with the most obvious coloration of embryo and endosperm aleurone were selected.

[0045] The selected F1 kernels of Shenkeno 602 x CAU5 were sown individually, and the male parent SWL12 of Shenkeno 602 was sown at the same time. The plants with late emergence and weak growth were eliminated, and 20 F1 plants of Shenkeno 602 x CAU5 with strong roots, thick stems, and large pollen were selected as the female parent at the anthesis stage. The pollen of SWL12 was applied, and 10 kernels of (Shenkeno 602 x CAU5) x SWL12 F1 generation with the most obvious coloration of embryo and endosperm aleurone were selected from each plant after harvesting the kernels, totaling 200 kernels.

[0046] (2) Backcross breeding

[0047] The 200 kernels in step (1) above were planted according to the line, with a total of 20 rows. The lines with late emergence and weak growth were eliminated, and the lines with large pollen and long flowering period were selected for backcross breeding. The recurrent parent SWL12 was planted at the same time, and the molecular markers designed in Example 1 were used for assistance. The molecular markers were used to retain single plants containing haploid induction gene key sites during the backcross breeding process. One plant from each line was selected for each backcross to enter the next round of backcross, and a total of 5 generations of backcross were performed. The selection criteria remained the same for each round of backcross.

[0048] (3) Self-purification

[0049] After completing 5 rounds of backcross, 20 lines were planted in the field, and molecular markers were used for selection to retain single plants containing haploid induction gene key sites, and the other single plants were eliminated. The remaining single plants were self-pollinated, and one ear of BC5F2 was retained from each line. BC5F2 kernels were continuously sown, with 20 plants per line. Sampling was performed at the seedling stage, and molecular markers were used to retain single plants containing haploid induction gene key sites. Then, the remaining single plants were further detected by one-generation sequencing, and single plants with non-homozygous haploid induction gene key sites were eliminated, as shown in Figure 3 .

[0050] The single plants with homozygous haploid induction gene key sites were tested for induction rate, and the two single plants with the highest induction rate were retained for breeding, and were named WXYD1 and WXYD2, respectively.

[0051] Example 4

[0052] Application of waxy maize haploid inducer line

[0053] Three waxy maize hybrids were used as female materials to detect the induction rate of WXYD1 and WXYD2, with high-frequency inducer line CAU5 as a control. At the stage of anthesis, the pollens of WXYD1, WXYD2 and CAU5 were applied to the three waxy maize hybrids before the female materials began to spin, with 10 plants in each combination. After the ears were harvested, haploid identification was performed.

[0054] After identification, the induction rates of WXYD1 and WXYD2 were 12.19% and 12.42% respectively, as shown in Table 1. Since the color marker of CAU5 was not clear, the data of CAU5 could not be accepted.

[0055] Table 1. Identification statistics of haploid induction rate

[0056]

[0057]

[0058] The above only describes the preferred embodiments of the present application, and it should be noted that for those skilled in the art, without departing from the principles of the present application, a number of improvements and refinements can be made, and these improvements and refinements should also be considered as the protection scope of the present application.

Claims

1. A method for creating a haploid inducible line of waxy maize, characterized in that, Includes the following steps: (1) Using Shenkenuo 602 as the female parent and CAU5 as the male parent, F1 was obtained by hybridization, and F1 grains with obvious coloration of embryo and endosperm aleurone layer were screened. (2) F1 generation seeds were sown individually, and the female parent was selected during the pollen shedding period. SWL12 was used as the male parent for pollination to obtain (Shenkenuo 602×CAU5)×SWL12 F1 generation seeds. Seeds with obvious coloring of embryo and endosperm aleurone layer were selected from each plant. (3) (Shenkeno 602×CAU5)×SWL12 F1 generation seeds were sown according to the lineage and backcrossed with the recurrent parent SWL12 for 5 generations. During this period, molecular markers were used to retain individual plants containing key sites of haploid induction genes. (4) After backcrossing, the progeny plants were self-pollinated and purified. Molecular markers were used to retain plants containing key sites of haploid induction genes. The plants that retained key sites of haploid induction genes were detected by first-generation sequencing. (5) The induction rate of homozygous single plants at key sites of haploid induction genes was tested, and seed was screened and propagated to obtain excellent waxy maize haploid induction lines.

2. The method for creating a haploid inducible line for waxy maize according to claim 1, characterized in that, In step (1), 100 F1 generation grains with the most obvious purple color in the embryo and endosperm aleurone layer are selected.

3. The method for creating a haploid inducible line of waxy maize according to claim 1, characterized in that, In step (2), plants with late emergence and weak growth in the F1 generation are eliminated, and 20 plants with strong roots, thick stems and large pollen production are selected as the mother plants during the pollen shedding period.

4. The method for creating a haploid inducible line of waxy maize according to claim 1, characterized in that, In step (2), 10 seeds from each plant are selected that show the most obvious purple color in the embryo and endosperm aleurone layer.

5. The method for creating a haploid inducible line for waxy maize according to claim 1, characterized in that, In step (3), the strains of (Shenkeno 602×CAU5)×SWL12 F1 generation that emerge late and grow weakly are eliminated, and strains with large pollen and long flowering period are selected for backcrossing and breeding.

6. The method for creating a haploid inducible line for waxy maize according to claim 1, characterized in that, In step (3), one strain from each line is selected for each backcross in each round of backcrossing, and the selection criteria remain consistent in each round of backcrossing.

7. The method for creating a haploid inducible line of waxy maize according to claim 1, characterized in that, In steps (3) and (4), the molecular marker primer is GW48; the upstream primer sequence is shown in SEQ ID NO.1; and the downstream primer sequence is shown in SEQ ID NO.

2.

8. The method for creating a haploid inducible line for waxy maize according to claim 1, characterized in that, In step (4), after backcrossing, 20 lines were planted in the field and molecular markers were used for further screening. Single plants containing key haploid induction gene sites were retained, while other single plants were eliminated.

9. The method for creating a haploid inducible line of waxy maize according to claim 1, characterized in that, In step (4), self-pollination purification includes self-pollination of the retained single plants, harvesting BC5F2 ears, retaining 1 ear for each line, continuing to sow BC5F2 grains, sowing 20 plants for each line, taking samples for testing during the seedling stage, and performing molecular marker screening and first-generation sequencing.

10. The method for creating the waxy maize haploid inducing line according to any one of claims 1-9, or the application of the obtained waxy maize haploid inducing line in maize breeding.

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