Waxy maize wx gene natural mutant, molecular marker and application thereof

Abstract

The present application belongs to the field of genetic engineering, and particularly relates to a natural mutant of a maize wx gene, which has a 12bp insertion at the 301 nucleotide position of the 1st exon relative to the wild type gene, causes a change in the nucleotide sequence, and is a new wx gene mutation type.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of plant molecular biology, and particularly relates to a natural mutant of a maize wx gene, a molecular marker thereof and application thereof. BACKGROUND

[0002] Maize (Zea mays L.) is an important food, economic and forage crop in the world, and its planting area ranks third in the world. Maize starch is widely used in human life and industrial production, and the amylose and amylopectin contained therein have very high economic value. Waxy maize Origin In China, it is also called waxy maize or sticky maize. Waxy maize belongs to a subspecies of maize. About 95% to 100% of the endosperm of waxy maize is amylopectin, and almost no amylose (Luo Gaoling et al., 2003), so it has the characteristics of stickiness, softness, fineness and flexibility, unique flavor, excellent eating quality and rich nutrition, and is favored by consumers.

[0003] The waxy property of waxy maize is controlled by a pair of recessive genes (wxwx). Sprague et al. located the wx gene of waxy maize on chromosome 9 through research (Sprague G F et al., 1997). The wild type Wx gene locus is located on the short arm of chromosome 9, and the full length of the gene is 3718 bp, which is composed of 14 exons and 13 introns, and encodes a 60 kd granule-bound starch synthase (GBSSI). The enzyme mainly controls the synthesis of amylose in maize endosperm and pollen. In the wx mutant, the GBSSI enzyme activity is reduced, the synthesis of amylose is blocked, the content of amylose in the endosperm and pollen of maize seeds is reduced, and thus the amylose is converted into amylopectin, so that the maize has waxy property (Li Fangfang et al., 2018; Li Xiangdong et al., 2015; Tan Caixia et al., 2008).

[0004] At present, a number of mutants have been found and isolated, and the mutation positions are distributed throughout the wx gene. A large part of them are caused by the insertion of transposons or retrotransposons of different sizes into different positions of the gene, which are wx-m1, wx-m6, wx-m7, wx-m9, wx-B4, etc. formed by the insertion of transposon AC-Ds; wx-m8 formed by the insertion of transposon of Spm control family and wx-Stonor, wx-G, wx-65, wx-Cin4, wx-Reina, wx-I, wx-K, wx-B5, etc. formed by the insertion of retrotransposon. Some stable wx mutants are caused by deletion or insertion mutation at the key position of the wx gene, such as wx-D7 and wx-D10, which are caused by base deletion of the 7th exon and the 10th exon of waxy1 respectively (Fan, 2008; Tian Mengliang, 2008).

[0005] Waxy maize germplasm resources are rich, in addition to the above-mentioned mutation types, there may be some new unknown wx gene mutation types, these rare wx gene mutations are easy to be ignored and lost in the process of waxy maize germplasm selection and breeding. Therefore, exploring new wx gene mutants and developing molecular markers thereof can not only expand the germplasm resources of waxy maize and provide material basis for special maize new variety breeding, but also promote the application of molecular marker assisted breeding of waxy maize. SUMMARY

[0006] The present application provides a natural mutant of corn wx gene, which has an insertion of 12bp at the 301st nucleotide position of the 1st exon relative to the wild type gene, causing changes in nucleotide sequence and amino acid sequence. It is a new wx gene mutation type, which expands the germplasm resources of waxy maize and provides material basis for special maize new variety breeding. The present application also provides a molecular marker of the wx gene mutant, which promotes the application of molecular marker assisted breeding of waxy maize and realizes the seedling selection of waxy maize, thereby shortening the breeding cycle.

[0007] To solve the above problems, the technical scheme of the present application is as follows.

[0008] A waxy maize inbred line 4031HN1 is obtained by natural mutation in the process of inbred line selection. The inbred line has good combining ability and excellent waxy taste. In order to identify the type of wx gene mutation of the inbred line, the DNA of 18 core maize inbred lines collected by the research group and commonly used in the market is extracted (CTAB method), and the wx gene is amplified and sequenced from the first exon to the 14th exon by using three pairs of primers. The results show that the inbred line 4031HN1 is a new wx gene allelic mutant. The E1F / E1R primer pair is used for PCR amplification and sequencing of the first to third exons of 18 core waxy maize inbred lines including 4031HN1 inbred line. The PCR amplification conditions are as follows: 94℃, 2min, then 94℃, 30s, 53℃, 30s, 68℃, 1min30s for 36 cycles, and finally 68℃ extension for 10min. It is found that there are more than 10 different sites in the genome of different waxy maize. Among them, the second exon of the wx gene of the 4031HN1 inbred line has a 12bp insertion at the 301 nucleotide position, which is different from the other 17 waxy maize. We name it wx-301. Compared with other reported wx alleles, the mutation is a new type of wx gene mutation.

[0009] The present application has the following beneficial effects:

[0010] The present application provides a natural mutant of maize wx gene, which has a 12bp insertion at the 301 nucleotide position of the first exon relative to the wild type gene, causing changes in nucleotide sequence and amino acid sequence. It is a new type of wx gene mutation, which expands the germplasm resources of waxy maize and provides material basis for the breeding of new varieties of special maize. The present application also provides a molecular marker of wx gene mutant, which promotes the application of molecular marker assisted breeding of waxy maize and realizes the seedling selection of waxy maize, thereby shortening the breeding cycle. BRIEF DESCRIPTION OF DRAWINGS

[0011] Figure 1 Multiple sequence alignment information: E1F / E1R, the rest of the sequences are the same as 1-1

[0012] Figure 2 wx gene transcript schematic diagram and amplification position of three pairs of PCR primers

[0013] Figure 34031HN1 and its wild type (normal maize) wx gene fragment amplification electrophoresis detection results. The first, second lane is the use of E1F and E1R primer respectively on waxy corn 4031HN1 and its wild type (normal maize) wx gene of the first segment amplification electrophoresis detection; The third, fourth lane is the use of E2F and 2R primer respectively on waxy corn 4031HN1 and its wild type (normal maize) wx gene of the second segment amplification electrophoresis detection; The fifth, sixth lane is the use of 3F and E3R primer respectively on waxy corn 4031HN1 and its wild type (normal maize) wx gene of the third segment amplification electrophoresis detection. PCR products were detected by 1% agarose gel electrophoresis.

[0014] Figure 4 Wx gene sequence alignment of waxy corn mutant 4031HN1 and its wild type (normal maize).

[0015] Figure 5 Comparison of the nucleotide sequence of wx gene mutant wx-301 with the sequence of the cDNA start codon to the 3rd exon of the wild type and reference genome Wx gene.

[0016] Figure 6 Analysis of the amino acid sequence of the cDNA start codon to the 3rd exon of the Wx gene.

[0017] Figure 7 Analysis of the amino acid sequence of the encoded protein.

[0018] Figure 8 Detection results of Indel-301 functional molecular marker. The waxy corn inbred lines and normal maize we collected were amplified with Indel301-F1 and Indel301-R1, and the PCR products were detected by polyacrylamide gel electrophoresis. M is DNA marker, 1-18 lanes are 18 waxy corn inbred lines used in this study, of which lane 12 is waxy corn 4031HN1, and lanes 19-20 are normal maize.

[0019] Figure 9 Detection of Indel-301 functional molecular marker on wx-301 type waxy corn and normal maize F2 generation

[0020] PCR products were detected by polyacrylamide gel electrophoresis. M is a DNA marker; the first lane is the 4031HN1 waxy inbred line, the second lane is the H521 inbred line, the third, seventh, eighth and twelfth lanes are homozygous wx-301 type waxy corn in the hybrid F2 generation; the sixth, thirteenth and fifteenth lanes are homozygous normal corn type in the hybrid F2 generation; the fourth, fifth, ninth, tenth, eleventh, fourteenth and sixteenth lanes are heterozygous types. Detailed implementation method

[0021] Example 1 sequence alignment

[0022] A waxy inbred line 4031HN1 was obtained by natural mutation during inbred line breeding. The inbred line has good combining ability and excellent waxy taste. In order to identify which type of wx gene mutation the inbred line is, the DNA of 18 core inbred lines commonly used in the market and bred by the research group was extracted (CTAB method), and the wx gene was amplified and sequenced from the first exon to the 14th exon using three pairs of primers. The results show that the inbred line 4031HN1 is a new wx gene mutant. The 18 core waxy inbred lines including the 4031HN1 inbred line were amplified and sequenced by PCR using the E1F / E1R primer pair containing the first to third exons. The PCR amplification conditions are: 94°C for 2 min, then 94°C for 30 s, 53°C for 30 s, 68°C for 1 min 30 s for 36 cycles, and finally 68°C for 10 min. As shown in Figure 1 Figure 1, more than 10 differences were found in the genome of different waxy corn. Among them, the 4031HN1 inbred line wx gene has a 12 bp insertion at the 301 nucleotide position of the second exon, which is different from the other 17 waxy corn. We named it wx-301. Compared with other reported wx alleles, this mutation is a new type of wx gene mutation.

[0023] Example 2 verification of mutation

[0024] In order to verify whether it is the mutation at this position that causes the normal corn to become waxy corn, the wx gene of 4031HN1 and its wild type (normal corn) was amplified by the three pairs of primers (Table 1) described above, and the amplification products were 900 bp, 1.5 Kb and 1.6 Kb (Figure 2), respectively. The difference is not obvious by agarose gel electrophoresis detection. Further sequencing and comparison of PCR products found that the waxy mutant and the wild type normal corn only have a 12 bp difference in the second exon (Figure 3). Figures 2-3 ), and the difference is not obvious by agarose gel electrophoresis detection. Further sequencing and comparison of PCR products found that the waxy mutant and the wild type normal corn only have a 12 bp difference in the second exon (Figure 3).Figure 4 , Figure 5 The inserted sequence, ACGTCCTCGGCG, is a repeat of the nucleotide sequence at positions 289-301 of the gene, further proving that the 12bp insertion is the reason why the maize inbred line 4031HN1 becomes waxy.

[0025]

[0026] Table 1

[0027]

[0028]

[0029] Table 2

[0030] Example 3: Amino acid sequence analysis of the maize wx gene mutant wx-301

[0031] The nucleotide sequence of the wx gene mutant wx-301 was compared with the cDNA start codon to exon 3 sequence of the wild-type and reference genome Wx gene and translated into amino acid sequences. Figure 6 Analysis revealed that the mutant wx-301 had four extra amino acid sequences compared to the wild type. Further analysis showed that the difference in these amino acid sequences was located in the conserved catalytic domain of starch synthase. This result further confirmed that the loss of function of the wx gene in waxy maize of the wx-301 type is related to the insertion of 12 bp in the second exon of the wx gene at the 301 nucleotide position.

[0032] Example 4: Development and application of specific functional molecular markers for the maize wx gene mutant wx-301

[0033] Based on the 12bp difference at the 301bp position of the first exon in wx-301 type waxy corn compared to ordinary corn, a site-specific Indel molecular marker, Indel-301, was developed. Primer sequences are shown in Table 3. PCR amplification conditions were: 94℃ for 5 min, followed by 34 cycles of 94℃ for 30 s, 55℃ for 30 s, and 72℃ for 1 min, with a final extension at 72℃ for 10 min. PCR products were detected by polyacrylamide gel electrophoresis. The primer pair Indel301-F1 and Indel301-R1 ​​amplified 132bp and 120bp target fragments, respectively, in waxy corn 4031HN1, other types of waxy corn, and ordinary corn. Figure 8 ).

[0034] The developed Indel-301 functional molecular marker is used for genotyping detection of the wx-301 type waxy corn inbred line 4031HN1, its wild type and its hybrid F2 generation with common corn H521. The PCR amplification results show that the wx-301 type waxy corn inbred line 4031HN1 only amplifies a 132 bp fragment, the common corn inbred line H521 only amplifies a 120 bp fragment, and in the hybrid F2 generation, 132 bp and 120 bp fragments are amplified at the same time. Figure 9 The above results show that the PCR functional molecular marker can distinguish the corn materials carrying wx-301 type homozygous mutant type, heterozygous type and wild type wx gene.

[0035]

[0036] Table 3.

Claims

1. A molecular marker of natural mutant of waxy gene of waxy corn, characterized in that, The primer for amplifying the molecular marker consists of primer Indel301-F1 and primer Indel301-R1, and the wx gene natural mutant molecular marker is different from common corn inbred line H521 in that there is a 12bp insertion at the position of 301bp of the first exon, the insertion sequence is ACGTCCTCGGCG, Indel301-F1: GCCGTGGAGCAAGACCG, and Indel301-R1: CGGCAAGAGCACGAAGAAGA.

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