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CN110129342B - Molecular marker of corn high-lysine gene ZmcytMdh4 and application thereof - Google Patents

Molecular marker of corn high-lysine gene ZmcytMdh4 and application thereof Download PDF

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CN110129342B
CN110129342B CN201910413352.2A CN201910413352A CN110129342B CN 110129342 B CN110129342 B CN 110129342B CN 201910413352 A CN201910413352 A CN 201910413352A CN 110129342 B CN110129342 B CN 110129342B
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付志远
陈永强
汤继华
郭战勇
张雪海
陈晓阳
李卫华
丁冬
王洪秋
薛亚东
李浩川
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Abstract

本发明公开了一种玉米高赖氨酸基因ZmcytMdh4的分子标记及其应用，旨在解决现有高赖氨酸突变体苗期生长弱、为粉质胚乳、产量较低、易感病虫害，无法生产和加工的技术问题。本发明筛选得到一种玉米高赖氨酸基因ZmcytMdh4，分析得两种玉米高赖氨酸基因ZmcytMdh4的分子标记，并提供了相应的检测分子标记的引物对。进一步将玉米高赖氨酸基因ZmcytMdh4、玉米高赖氨酸基因ZmcytMdh4的分子标记、引物对在高赖氨酸含量玉米分子标记辅助选择和聚合育种中应用。本发明可大大提高分子标记辅助选择的效率，为选育赖氨酸含量高的优良品系奠定了基础，为高赖氨酸玉米育种提供了技术支持。

Figure 201910413352

The invention discloses a molecular marker of a maize high lysine gene ZmcytMdh4 and its application, aiming to solve the problem that the existing high lysine mutants have weak seedling growth, powdery endosperm, low yield, susceptibility to diseases and insect pests, and cannot be Technical issues of production and processing. The present invention obtains a corn high lysine gene ZmcytMdh4 by screening, analyzes the molecular markers of two corn high lysine genes ZmcytMdh4 , and provides corresponding primer pairs for detecting the molecular markers. The molecular markers and primer pairs of maize high lysine gene ZmcytMdh4 and maize high lysine gene ZmcytMdh4 were further applied in molecular marker-assisted selection and polymerization breeding of maize with high lysine content. The invention can greatly improve the efficiency of molecular marker-assisted selection, lays a foundation for breeding excellent lines with high lysine content, and provides technical support for high lysine corn breeding.

Figure 201910413352

Description

Molecular marker of corn high-lysine gene ZmcytMdh4 and application thereof
Technical Field
The invention relates to the technical field of genetic engineering, in particular to a corn high-lysine geneZmcytMdh4And applications thereof.
Background
Corn is the first large grain crop in China, and is also an important feed, industrial raw material and energy crop. However, most of the proteins in corn nutrients are mucilage and lack the essential lysine required by humans and monogastric animals, which affects the human health and livestock production performance depending on corn products.
Breeding of maize (QPM), a high-quality protein aiming at increasing lysine content, is an important approach to solve this problem.opaqueThe discovery of the mutant-like body opens a new chapter of high-quality protein corn cultivation. Typical high lysine content maize mutants available includeo2、o7、fl2AndZmocd1the positioning and function analysis of the mutant gene make it possible to cultivate high lysine corn variety through gene engineering. However, the existing corn mutant has weak seedling growth, is floury endosperm, has low yield, is susceptible to diseases and insect pests, brings inconvenience to production and processing, and has a great upper limitMaking its use.
Therefore, research and development of a new high-lysine mutant and research on related gene functions of the mutant are urgently needed, which is helpful for deeply understanding a gene network for regulating and controlling the lysine content, so that high-quality protein corn with high grain hardness and high yield is cultivated by gene polymerization by utilizing functional molecular markers of genes.
Disclosure of Invention
The technical problem to be solved by the invention is to provide a corn high-lysine geneZmcytMdh4The molecular marker and the application thereof aim at solving the technical problems that the existing high-lysine mutant is weak in seedling growth, is floury endosperm, is low in yield, is susceptible to diseases and insect pests, and cannot be produced and processed.
In order to solve the technical problems, the invention adopts the following technical scheme:
screening to obtain a corn high-lysine geneZmcytMdh4The nucleotide sequence is shown as SEQ ID NO. 2; the gene codes cytoplasmic malate dehydrogenase 4, can influence the balance of endosperm starch and storage protein, and obviously improves the lysine content.
Analyzing to obtain a corn high lysine geneZmcytMdh4The molecular marker of (1), namely, the mutation of A at-293 bp of 5' UTR to C, and the nucleotide sequence of the site and the flank thereof is shown as SEQ ID NO. 4.
Provides a method for detecting the high lysine gene of the cornZmcytMdh4The nucleotide sequence of the primer pair of the molecular marker is as follows:
SNP(-293)-BspHI-L：AAGCGTCACGCCGTCTCATG；
SNP(-293)-1R：CTTGGGAATCCACTGTCACACGC。
analyzing to obtain a corn high lysine geneZmcytMdh4The molecular marker of (1), namely the deletion of the 59 th to 61 th base GAG in the seventh exon, and the nucleotide sequence of the site and the flank thereof is shown in SEQ ID NO. 6.
Provides a method for detecting the high lysine gene of the cornZmcytMdh4The nucleotide sequence of the primer pair of the molecular marker is as follows:
Exon7-L：CAAGGAAGAAGATGGATGCC；
Exon7-R：TGCTCTCTGGGTGTGGTAGAAT。
the corn high lysine gene is usedZmcytMdh4Or maize high lysine geneZmcytMdh4The molecular marker is applied to the auxiliary selection and the polymerization breeding of the corn molecular marker with high lysine content.
The detection of the high lysine gene of the corn is carried outZmcytMdh4The application of the molecular marker primer pair in high-lysine corn breeding.
Compared with the prior art, the invention has the beneficial technical effects that:
1. the molecular marker is developed based on the functional marker which causes phenotypic variation in the functional gene, the functional marker does not depend on molecular genetic mapping, and the efficiency of molecular marker-assisted selection can be greatly improved by using the functional marker.
2. The invention can be applied to the backcross transformation methodZmcytMdh4The genes are introduced into the parental backgrounds of a plurality of hybridization combinations, and the functional molecular marker is combined for auxiliary selection to obtain the corn plant intermediate material with the lysine content improved to different degrees, thereby laying a solid material and technical foundation for breeding the excellent strains with high lysine content.
3. The invention is thatZmcytMdh4The application of the method in the breeding of the high-lysine corn provides technical support.
Drawings
FIG. 1 is a drawing ofZmcytMdh4Gene structure and nucleotide polymorphism information maps;
in the figure, a isZmcytMdh4Positioning of genes, wherein b is a gene structure and nucleotide polymorphism;
FIG. 2 is a dCAPS marker map of SNP (-293);
in the figure, 1 and 3 indicate that SNP (-293) is A; 2 and 4 represent SNP (-293) as C;
FIG. 3 is a PCR marker map of Indel (3-bp);
in the figure, lanes 2, 4, 6, 8 and 10 are advantageous variations;
FIG. 4 is a nucleotide state diagram of Indel (3-bp) in teosinte and 55 natural populations.
Detailed Description
The following examples are intended to illustrate the present invention in detail and should not be construed as limiting the scope of the present invention in any way.
The instruments and devices referred to in the following examples are conventional instruments and devices unless otherwise specified; the related reagents are all conventional reagents in the market, if not specifically indicated; the test methods involved are conventional methods unless otherwise specified.
Example 1: obtaining of high lysine functional gene
(1) A segregation group containing 30,000 single plants is constructed by utilizing the corn mutant with high lysine content and the Zheng 58 excellent inbred line, and a mutant with the lysine content being obviously improved is obtained.
The inventor screens out a mutant material with defective grain phenotype in a large amount of early-stage field breeding processes. Then, the mutant and a good inbred line Zheng 58 are combined to construct a set of F2Separating the population, and determining the mature seeds of the wild type and the mutant by a near infrared spectroscopy (NIR) method, wherein the results show that the lysine content in the mutant is obviously higher than that in the wild type.
(2) The gene of interest was mapped by map-based cloning to a physical region of approximately 224kb on chromosome 1 of maize, with 2 protein-encoding genes in the candidate segment.
By Chi-Square test, for F2The statistical analysis of the grain phenotype in the segregating population shows that 100 randomly selected F2Phenotype expression of seeds in isolated ears 3: 1 (wild type: mutant = 5494: 1833, χ)2 = 0.002 < χ2 0.05= 3.84), indicating that the mutant is a recessive mutation under the control of a single gene, and that the wild type appears dominant to the mutant.
Firstly, a separate population grouping analysis (BSA) method is adopted to select 10 seeds of a wild type and a mutant in a separate cluster to construct a dominant pool and a recessive pool. The dominant and recessive pools were then screened for markers covering 10 chromosomes of maize, initially defining the target segment between umc1245 and umc2181 on the first chromosome. Further by fine localization, 34080 individuals were used to define the target segment between C368 and Indel-98 with an interval length of approximately 224 Kb.
The comparative analysis with the published genome sequence of B73V4 version shows that the segment contains 2 protein-encoded genes, Zm00001d032695 and Zm00001d 032699.
Example 2: screening of high lysine functional Gene
Sequence analysis and genetic transformation of maize demonstrated that the gene encodes cytoplasmic malate dehydrogenase 4ZmcytMdh4The gene is a gene of interest.
From a query in the MaizeGDB (https:// www.maizegdb.org /) database, Zm00001d032695 was found to encode a cytoplasmic malate dehydrogenase. Constructing a knockout carrier by using the CRISPR/Cas9 technology, sending the knockout carrier to China seedgroup limited company for genetic transformation, and carrying out allelic test on the separated homozygous positive plants and the original mutant and the heterozygous single plant respectively, wherein the result shows thatZmcytMdh4Is a target gene.
ZmcytMdh4The gene structure and nucleotide polymorphism information are shown in FIG. 1:
FIG. 1 (a) shows the process of mapping the ZmcytMdh4 gene, the upper part of the vertical line representing the molecular marker used, the number on the left side of the corresponding lower oblique line representing the number of individuals undergoing recombination, and the right side of the oblique line representing the number of populations; (b) the figure shows the gene structure of the mutant and the wild type and the functional sites of the difference between the two.
Example 3: high lysine functional geneZmcytMdh4Analysis of (2)
(1) In the mutantZmcytMdh4The seventh exon has 3-bp deletion, and the deletion generates frame shift mutation to reduce enzyme activity, accumulate malic acid and increase lysine content.
Analysis of the DNA sequences and coding sequences of the wild type and mutant revealed that 3-bp deletion was present in the seventh exon of the mutant, which finally resulted in deletion of one amino acid in the mutant. The wild-type and mutant proteins were subsequently purified in vitro by prokaryotic expression, and the catalytic activity of the proteins was determined by addition of different substrates and corresponding coenzymes. The results show that the catalytic activity of the mutant is almost lost.
Through the targeted metabolome, the change of energy metabolism related substances in wild type seeds and mutant seeds 12 days after pollination is determined, and the results show that the contents of malic acid and lysine in the mutant are obviously increased. Meanwhile, the malic acid content and the lysine content in the 12 DAP, 20 DAP and mature grains are measured, and the results show that the malic acid content and the lysine content in the mutant are both obviously improved compared with the wild type.
(2) According toZmcytMdh4The nucleotide polymorphisms in wild type and mutant, through candidate gene association analysis, obtain SNP (-293) -A/C which is obviously associated with lysine content on 5' UTR.
First toZmcytMdh4Primers are designed for the full length of the gene and sequenced to obtain wild type and mutantZmcytMdh4, or a nucleotide polymorphism site. And then, using the data of the association group of the strict soldier topic group (http:// www.maizego.org/resources. html) of the university of agriculture in Huazhong, selecting a Q + K model for association analysis, and finally displaying that the SNP (-293) on the 5' UTR is obviously associated with the lysine content in the grains according to the analysis result.
TABLE 1 markers and sequences used in the localization procedure
Figure DEST_PATH_IMAGE001
。
Table 1 is mainly the differentially linked molecular markers used in the preliminary and fine localization processes, wherein C4, 45, 46, C351, C368, C143, C201 and Indel-98 are newly developed SSR (simple sequence repeat) or Indel type molecular markers, and umc2237, bnlg1556, umc1245, umc2181, mmc0041 and phi423298 are SSR molecular markers published in the MaizeGDB database.
TABLE 2 analysis of association of candidate genes
Figure 835201DEST_PATH_IMAGE002
。
aOnly significant association sites are listed;
ballelic variation of SNP (-293), bold indicates favorable allelic variation;
cSGW represents starch grain width; GI represents the length of starch grain, Arg represents arginine content; lys represents the lysine content
dThe p-value is obtained by integrating the group structure and the affinity relationship through a mixed linear model of tassel software; BLUP is the result of two year data integration;
ep-value is the result of ANOVA analysis using BLUP data.
Table 2 shows 4 traits significantly associated with SNP (-293), including SGW (starch grain width), GI (starch grain length), Arg (arginine), and Lys (lysine). N represents the number of individuals used for the trait.
Example 4: maize high lysine geneZmcytMdh4Design of molecular markers of (3)
A PCR functional molecular marker easy to detect is developed aiming at the specificity of 3-bp, and a dCAPS functional molecular marker easy to detect is designed according to SNP (-293).
cytMdh4The PCR primer sequence of the gene functional molecular marker is as follows:
(1) functional SNP site: B73V 4: 234908227, the actual position of the functional SNP site on the chromosome is the reference sequenced B73 genome sequence.
SNP(-293)-BspHI-L：AAGCGTCACGCCGTCTCATG；
SNP(-293)-1R：CTTGGGAATCCACTGTCACACGC；
(2) 3-bp differential functional site: B73V 4: 234902919-.
Exon7-L：CAAGGAAGAAGATGGATGCC；
Exon7-R：TGCTCTCTGGGTGTGGTAGAAT。
Example 5: maize high lysine geneZmcytMdh4Detection of the molecular marker of (1)
Detection of a molecular marker for the A to C mutation within the 5' UTR (-293 bp) comprising the steps of:
(1) preparing a 10 mu L reaction system:
DNA template (30-50 ng/ul) 2ul
Left primer (10uM) 0.5ul
Right primer (10uM) 0.5ul
ddH2O 2ul
2×Hieff®PCR Master Mix 5ul
(2) The PCR amplification procedure was:
1 cycle 95℃ 5min
36 cycles of denaturation at 95 ℃ for 30s, annealing at 58 ℃ for 30s, and extension at 72 ℃ for 30s
Delay 72℃ 10min
Soak 25℃
(3) The enzyme cutting system is as follows:
PCR product 3ul
CutSmart 10×buffer 1ul
BspHI (NEB) 0.5ul
ddH2O 5.5ul
(4) 4% agarose gel is prepared for electrophoresis under conventional conditions, and the difference can be displayed after about 30 min.
The dCAPS marker for SNP (-293) is shown in FIG. 2:
differential design of SNP (-293) site in wild typeBspH I, the wild-type amplification product is normally cleaved by the enzyme containing the cleavage site, and the mutant does not contain the cleavage site and remains intact, thus showing differences in the gel maps. In the figure, 1 and 3 represent wild type, and 2 and 4 represent mutant.
Example 6: maize high lysine geneZmcytMdh4Detection of the molecular marker of (1)
The detection of the molecular marker aiming at the seventh exon 3-bp Indel comprises the following steps:
(1) the reaction system is as follows:
DNA template (30-50 ng/ul) 2ul
Left primer (10uM) 1ul
Right primer (10uM) 1ul
ddH2O 1ul
2×mix for PAGE 5ul
(2) The PCR amplification procedure was:
1 cycle 95℃ 5min
36 cycles of denaturation at 95 ℃ for 30s, annealing at 58 ℃ for 30s, and extension at 72 ℃ for 30s
Delay 72℃ 10min
Soak 25℃
The PCR marker map for Indel (3-bp) is shown in FIG. 3:
designing a primer containing the site aiming at the difference of 3-bp in the wild type and the mutant, wherein the amplification products of the wild type and the mutant are respectively 96bp and 93bp, wherein 1, 3, 5, 7 and 9 represent individuals containing 3-bp, and 2, 4, 6, 8 and 10 represent individuals lacking 3-bp.
Example 7: distribution of SNPs (-293) in the Natural population
And (3) carrying out alignment search on the sequencing data of the associated population by using a strict soldier topic group (http:// www.maizego.org/resources. html) of China university of agriculture to obtain the distribution of the SNP (-293) in the natural population.
The distribution of SNPs (-293) in the natural population is shown in Table 3.
TABLE 3 distribution of SNPs (-293) in the Natural population
Inbred lines Germplasm SNP(-293) Inbred lines Germplasm SNP(-293)
04K5672 temperate - CIMBL58 subtropical C
04K5686 temperate A CIMBL59 subtropical A
04K5702 temperate C CIMBL6 tropical C
05W002 temperate C CIMBL60 subtropical -
05WN230 temperate - CIMBL61 subtropical -
07KS4 temperate C CIMBL62 subtropical A
1323 temperate C CIMBL63 tropical A
1462 temperate - CIMBL65 tropical A
150 temperate C CIMBL66 subtropical A
177 temperate C CIMBL67 tropical A
18-599 temperate C CIMBL68 tropical C
238 temperate A CIMBL69 subtropical A
268 temperate - CIMBL7 tropical A
303WX temperate C CIMBL70 tropical -
3411 temperate - CIMBL71 tropical C
384-2 temperate - CIMBL72 subtropical A
3H-2 temperate C CIMBL73 subtropical A
4019 temperate C CIMBL74 tropical A
4F1 temperate A CIMBL75 subtropical A
501 temperate - CIMBL76 subtropical A
5213 temperate A CIMBL77 tropical C
5237 temperate A CIMBL78 tropical -
526018 temperate - CIMBL79 tropical -
5311 temperate C CIMBL8 subtropical A
647 subtropical A CIMBL80 tropical A
7327 temperate A CIMBL81 tropical A
7381 temperate C CIMBL82 tropical C
7884-4HT temperate A CIMBL83 subtropical C
81162 temperate C CIMBL84 subtropical -
812 temperate A CIMBL85 subtropical A
832 temperate - CIMBL86 tropical A
835A . C CIMBL87 subtropical C
835B . - CIMBL88 subtropical A
8902 temperate A CIMBL89 subtropical A
9642 temperate C CIMBL9 tropical C
975-12 temperate A CIMBL90 tropical A
9782 temperate - CIMBL91 tropical -
A619 temperate - CIMBL92 tropical C
B11 tropical A CIMBL93 subtropical A
B110 temperate A CIMBL94 subtropical A
B111 temperate A CIMBL95 subtropical C
B113 temperate A CIMBL96 tropical A
B114 temperate C CIMBL97 subtropical A
B151 temperate - CIMBL98 subtropical A
B73 temperate C CIMBL99 tropical A
B77 . C CML113 subtropical A
BEM temperate C CML114 subtropical A
BGY temperate A CML115 subtropical C
BK . - CML116 subtropical C
BS16 temperate A CML118 tropical A
BT1 subtropical C CML121 tropical A
BY4839 temperate A CML122 subtropical A
BY4944 temperate A CML130 subtropical -
BY4960 temperate A CML134 subtropical -
BY804 temperate A CML139 subtropical A
BY807 temperate A CML162 tropical A
BY809 temperate A CML163 tropical C
BY813 temperate A CML165 tropical A
BY815 temperate A CML166 tropical -
BY843 temperate C CML168 tropical A
BY855 temperate A CML169 tropical -
BZN temperate - CML170 tropical C
C8605 temperate A CML171 tropical A
CA47 temperate C CML172 tropical A
CF3 temperate C CML189 subtropical C
CHANG3 temperate C CML191 subtropical C
CHANG7-2 temperate A CML192 tropical A
CHENG698 temperate A CML20 tropical A
CHUAN48-2 temperate C CML223 tropical A
CI7 temperate C CML225 tropical A
CIMBL1 subtropical A CML226 tropical A
CIMBL10 tropical A CML228 tropical A
CIMBL100 tropical - CML229 tropical -
CIMBL101 tropical A CML26 tropical -
CIMBL102 tropical C CML27 tropical -
CIMBL103 subtropical A CML28 tropical A
CIMBL104 subtropical A CML282 tropical A
CIMBL105 tropical A CML285 tropical -
CIMBL106 tropical C CML286 tropical -
CIMBL107 tropical C CML287 tropical -
CIMBL108 tropical C CML289 tropical A
CIMBL109 tropical C CML29 tropical -
CIMBL11 tropical A CML290 tropical A
CIMBL110 tropical C CML297 tropical C
CIMBL111 subtropical A CML298 tropical C
CIMBL112 subtropical A CML300 tropical A
CIMBL113 subtropical A CML304 tropical -
CIMBL114 tropical C CML305 tropical A
CIMBL115 tropical C CML307 tropical -
CIMBL116 subtropical A CML31 tropical C
CIMBL117 subtropical C CML32 tropical A
CIMBL118 tropical A CML323 subtropical A
CIMBL119 tropical C CML324 subtropical C
CIMBL12 tropical A CML325 subtropical -
CIMBL120 subtropical A CML326 subtropical C
CIMBL121 subtropical A CML327 tropical C
CIMBL122 subtropical C CML338 subtropical -
CIMBL123 subtropical C CML360 tropical A
CIMBL124 subtropical A CML361 tropical C
CIMBL125 tropical A CML364 tropical A
CIMBL126 subtropical C CML40 tropical -
CIMBL127 subtropical - CML408 tropical A
CIMBL128 tropical A CML411 tropical A
CIMBL129 subtropical - CML412 tropical A
CIMBL13 tropical C CML415 tropical C
CIMBL130 subtropical - CML422 subtropical A
CIMBL131 tropical - CML423 subtropical A
CIMBL132 tropical - CML426 subtropical -
CIMBL133 tropical A CML428 tropical A
CIMBL134 tropical A CML430 tropical A
CIMBL136 tropical C CML431 tropical A
CIMBL137 tropical - CML432 subtropical C
CIMBL138 subtropical C CML433 tropical A
CIMBL139 subtropical A CML451 tropical A
CIMBL14 tropical A CML454 tropical A
CIMBL140 tropical A CML465 tropical A
CIMBL141 subtropical A CML470 subtropical C
CIMBL142 subtropical A CML471 subtropical -
CIMBL143 subtropical C CML473 subtropical C
CIMBL144 subtropical A CML474 subtropical A
CIMBL145 subtropical A CML479 tropical A
CIMBL146 subtropical - CML480 tropical A
CIMBL147 subtropical A CML486 subtropical -
CIMBL148 tropical A CML493 tropical C
CIMBL149 tropical A CML496 tropical -
CIMBL15 tropical A CML497 tropical C
CIMBL150 subtropical A CML50 tropical C
CIMBL151 subtropical C CML51 tropical C
CIMBL152 subtropical - CML69 tropical C
CIMBL153 subtropical A D047 temperate A
CIMBL154 subtropical - D863F temperate C
CIMBL155 tropical A DAN3130 temperate -
CIMBL156 tropical A DAN340 temperate A
CIMBL157 tropical A DAN360 temperate C
CIMBL16 tropical A DAN4245 temperate C
CIMBL17 subtropical C DAN598 temperate -
CIMBL18 tropical A DAN599 subtropical -
CIMBL19 tropical A DE.EX temperate C
CIMBL2 tropical - DH29 temperate A
CIMBL20 tropical A DH3732 temperate A
CIMBL21 tropical A DI-16 . C
CIMBL22 tropical C DONG237 temperate C
CIMBL23 tropical A DONG46 temperate A
CIMBL24 tropical A DSB temperate A
CIMBL25 tropical A E28 . -
CIMBL26 tropical A EN25 temperate -
CIMBL27 tropical A ES40 temperate -
CIMBL28 tropical A FCD0602 temperate -
CIMBL29 tropical - GEMS1 . -
CIMBL3 tropical A GEMS10 . C
CIMBL30 subtropical - GEMS11 . A
CIMBL31 tropical - GEMS12 . -
CIMBL32 tropical A GEMS13 . A
CIMBL33 tropical C GEMS14 . C
CIMBL34 tropical C GEMS15 . -
CIMBL35 tropical A GEMS16 . C
CIMBL36 subtropical A GEMS17 . C
CIMBL37 tropical A GEMS18 . A
CIMBL38 tropical C GEMS19 . -
CIMBL39 tropical A GEMS2 . -
CIMBL4 subtropical A GEMS20 . C
CIMBL40 tropical A GEMS21 . C
CIMBL41 tropical - GEMS23 . C
CIMBL42 subtropical A GEMS24 . -
CIMBL43 tropical C GEMS25 . A
CIMBL44 subtropical C GEMS27 . C
CIMBL45 subtropical C GEMS28 . C
CIMBL46 subtropical A GEMS29 . C
CIMBL47 subtropical A GEMS3 . -
CIMBL48 tropical - GEMS30 . C
CIMBL49 subtropical C GEMS31 . A
CIMBL5 tropical A GEMS32 . C
CIMBL50 subtropical C GEMS33 . -
CIMBL51 subtropical A GEMS35 . C
CIMBL52 tropical A GEMS36 . A
CIMBL53 tropical A GEMS37 . A
CIMBL54 subtropical C GEMS39 . -
CIMBL55 tropical A GEMS4 . C
CIMBL56 subtropical A GEMS40 . -
CIMBL57 subtropical A GEMS41 . -
P6WC . C GEMS42 . -
PH4VC . - GEMS43 . -
Q1261 temperate C GEMS44 . C
QI205 temperate C GEMS45 . C
QI319 subtropical C GEMS46 . C
QTHHSBTS temperate A GEMS47 . A
R08 temperate C GEMS48 . C
R15 temperate - GEMS49 . -
R15X1141 temperate C GEMS5 . -
RY684 temperate A GEMS50 . -
RY697 temperate - GEMS51 . C
RY713 temperate C GEMS52 . C
RY729 temperate A GEMS53 . C
RY732 temperate C GEMS54 . C
RY737 temperate - GEMS55 . C
S22 temperate C GEMS56 . -
S37 tropical A GEMS57 . C
SC55 tropical A GEMS58 . C
SHEN135 temperate A GEMS59 . C
SHEN137 subtropical - GEMS6 . C
SHEN5003 temperate - GEMS60 . -
SI273 temperate C GEMS61 . -
SI434 temperate C GEMS62 . C
SI444 temperate - GEMS63 . -
SI446 temperate C GEMS64 . -
SK . C GEMS65 . C
SW92E114 temperate A GEMS66 . C
SY1032 temperate C GEMS9 . C
SY1035 temperate A GY1007 temperate A
SY1039 temperate - GY1032 temperate A
SY1052 temperate - GY220 temperate A
SY1077 temperate C GY237 temperate -
SY1128 temperate A GY246 temperate C
SY3073 temperate C GY386 temperate A
SY998 temperate C GY386B . A
SY999 temperate A GY462 temperate C
TIAN77 temperate - GY798 temperate -
TIE7922 temperate A GY923 temperate A
TT16 temperate A H127 . A
TX5 temperate - H21 temperate A
TY1 temperate A HAI1134 temperate -
TY10 temperate A HAI268 temperate A
TY11 temperate A HB temperate A
TY2 temperate A HSBN temperate A
TY3 temperate A HTH-17 temperate A
TY4 temperate A HU803 temperate -
TY5 temperate A HUA83-2 temperate A
TY6 temperate A HUANGC temperate C
TY7 temperate A HYS temperate A
TY8 temperate A HZS temperate A
TY9 temperate A IRF291 temperate -
U8112 temperate A IRF314 temperate -
W138 temperate - J4112 temperate -
WH413 temperate - JH59 temperate -
WMR temperate - JH96C temperate -
WU109 temperate A JI53 temperate -
X1141P temperate A JI63 temperate A
XI502 temperate A JI842 temperate A
XUN971 temperate - JI846 temperate A
XZ698 temperate C JI853 temperate A
YAN414 temperate - JIAO51 temperate A
YE107 temperate C JING24 . -
YE478 temperate A JY01 temperate A
YE488 temperate C K10 temperate C
YE515 temperate C K12 temperate C
YE52106 temperate C K14 temperate C
YE8001 temperate A K22 temperate C
YU374 temperate - L3180 . C
YU87-1 temperate C LG001 temperate C
YUN46 . A LIAO138 temperate C
YZ15 temperate - LIAO159 temperate C
Z2018F temperate A LIAO5114 temperate -
ZAC546 temperate C LIAO5262 temperate C
ZB648 temperate A LIAO5263 temperate -
ZH68 temperate A LK11 temperate C
ZHENG22 temperate C LV28 temperate A
ZHENG28 temperate - LX9801 temperate A
ZHENG29 temperate C LXN temperate A
ZHENG30 temperate C LY temperate A
ZHENG32 temperate - LY042 temperate A
ZHENG35 temperate - M153 temperate C
ZHENG58 temperate A M165 temperate C
ZHENG653 temperate - M97 temperate A
ZHI41 temperate C MN temperate A
ZHONG69 temperate C MO113 temperate C
ZI330 temperate - MO17 temperate C
ZONG3 temperate C NAN21-3 subtropical C
ZONG31 temperate A NMJT temperate A
ZZ01 temperate - P138 . C
ZZ03 temperate C P178 subtropical C
Note: "-", deletion; ", No data" - "represents the deletion of the base at the site, and" - "represents that the material has No corresponding data.
Example 7: nucleotide states of Indel (3-bp) in teosintes and 55 natural populations
Sequencing primers containing 3-bp deletion sites were designed, and the genomic DNAs of teosintes and 55 natural populations were sequenced, and then the sequences were aligned and analyzed by BioEdit software.
The sequence of the sequencing primer is as follows:
MDH4-4L：GGTTTCGTTAGGGACACAATGA
MDH4-4R：ACCCACCGAATCCCAAGTT
the nucleotide states of Indel (3-bp) in teosintes and 55 natural populations are shown in FIG. 4.
The left-most letters in fig. 4 represent different material names, wherein the first row is the wild-type sequence and the second row is the mutant sequence; the black box part represents the situation of an indel of 3-bp for wild type, mutant, teosinte and 55 natural population materials, and the results show that only the deletion of 3-bp was detected in the mutant, demonstrating that the deletion is characteristic of the mutant.
While the present invention has been described in detail with reference to the drawings and the embodiments, those skilled in the art will understand that various specific parameters in the above embodiments can be changed without departing from the spirit of the present invention, and a plurality of specific embodiments are formed, which are common variation ranges of the present invention, and will not be described in detail herein.
SEQUENCE LISTING
<110> Henan university of agriculture
Molecular marker of <120> corn high-lysine gene ZmcytMdh4 and application thereof
<130> 2019
<160> 12
<170> PatentIn version 3.2
<210> 1
<211> 6408
<212> DNA
<213> Zea mays
<400> 1
cttagagttt attattgtaa ttgctcttac tcccaaccaa acataggcct tgtttggcac 60
agctgctgct tgttgaaaaa acagcttatc ttagaagctg gtgaaaacag cttctgcttg 120
ttgactgctt ttagtgtatt ctgagaagca gttgaactga taagctgctg cagaagctgg 180
ctgtttggca gaactttaac ttaaatttgt gaagaagcta aaaataagtt gtgtcaaaca 240
tggccacagt agtaagaaaa agtatcgtca catcgcgttt ccctatcggt cgaagtagct 300
ttcaaatctt tagatgtgtg gataaaaaaa cggctggtga tcaaagccat cggtacctat 360
caaacctatc accaacccta tcgtgaatga gtgccatctt cctcacgcca cgcttctcga 420
atctcgagag gcatctcctc cacaacgata atgtcgccgc ttcggtcctg taaaacagca 480
cgcaagaaaa atgcagaaaa tcaatcaagc tccatccagc ttgtcaagct gccatgctgt 540
ctaactgaca gaaggacccc accatcttgc tccactccgt acaactgtac ctgtacgtag 600
tattcatcgc cgaagccgtg ggcggtgggc cagcgcccac agcgtcaagc gtcacgccgt 660
cacttgagag tctcctgcct aaagacacac caaccaatca gagccaacat agcacactga 720
cgtcaggggc ccgctgctgt cgggccaatt gtcagttact gataagcacg ctgtcattat 780
cggggcgtgt gacagtggat tcccaaggct tccccgtcac agcgacgttg ttaaatagtg 840
gccacccatt gccgcgttct cttcgctatc ccttggttgc cgcagcctcc aaagccactc 900
ccaaaccccc gcttccagaa ccttctcgaa gctccccgcc gcctcctcca ctcgcaccga 960
tggcgaagga accaatgcgc gtgctcgtca ctggcgccgc aggtacggtg ctgagccttc 1020
cctccttccc gatccgcgta gtatccggtg tcgtctggtc ccggctcgga tctggcctag 1080
tccgcccgag cctgagcccg agctcgatcc gatcctgtgt ggtttagagt gtgcttaact 1140
cgctagatct cagtttttat cgctcgatct gatggtgttt agggcgtttg ggttgacgta 1200
gttttggcca gtagaactgt agaagggctc actgtcactc tccttgttca gtcggtgaag 1260
ttgaaccgat ccgcggtgct agctgatctg atcagtggtt gggcttgatg ggtagatcct 1320
cgtggtagtc acttgatcca gttgactcgc tagctgcact taattaactg cttactcttt 1380
agatagttga ttttactgct gcgatctcta gttgaacctt aagggctccg ggatagcagc 1440
aggttcggat ccggtggttt tggagtcaac ggcgttttgg agcgagtggt tttgatatat 1500
ttcatcccat cagactatct ggtgggactt aagttacaga tctaggtact ccacccatgg 1560
agtttacgtg ctaaacttgc atgaattctg tctacacgat cactgcgtga gtgatgtaac 1620
cttttagctg ttcagatagg tgccttaata atcatgtaat gtaatggaac ttcctcaaac 1680
attaaggatg gtggtgtagg catccagggg tccaggctgc agttgtttgg ggcccatgct 1740
tgacgcgttc actcttttca tcttttgtag catgtcatct gagttcttgc atttctatta 1800
tcataaccac agggtttggt atggattagc caaaagttag ttaatatcct gcttatttat 1860
ggtagcatat gtctggcatt cagttataaa attgcacagc gtgcttgaca cttttcttgt 1920
atattattga gctattctta gggggtgttt ggtttgcccc tgctaaaatt tagcccctat 1980
cacatcgaat gtttgaacct ccgttcgggg tattaaatgt agtcggatta taaaactaat 2040
tttcagccga agattaaaaa gtgagacgaa ttagtacagt tggttgggtc aatattttat 2100
actcctattt gaaagtcaaa cgcttgatgt gacccgggct aaactttagt ccacagaacc 2160
aaacaccccc ttataagcat ctactgtgat acaacaattg tctactgctt gtgtcatatt 2220
tgatggtatt agagttggtg tacaatgcgt atttactagt tccattttta tatgcttgta 2280
ctgagttaaa atatgatgct tgttcttatg gtgatagtga ccttccctca cctgttccgg 2340
tacctgggat gatatttgct aattcaataa ttgtcatatt gttttaaaga tctactccat 2400
atcagcagta aaggtgtctc catgtagcat aatagaatga ggagcaagag tatgttctgg 2460
ctttgaggtt gccacatgct atgataggat agggtgttgc attcttcttt gcatggacac 2520
gtacgtcatt gtgcaattca caaaaggtgc catggatacc atgtgctttc atagcaaaca 2580
attttgtcat gaaaatctta gtcgacgagt gattgtcatt gtgacagaga gggtgaattt 2640
ggatccaaaa tcttactact gatttgcgcc caacatatgg gtgaggtttt tttttgggtt 2700
ttattttctt attttaagtt gtaaacccaa tgtcatgttg taagcctgag gatggagatt 2760
agtcattccc aaatttgaag cagcatgaca aaaattggtg gaggatgttg acgctaccaa 2820
ccaatgaccc atcaaaaatt ttctggctag ttttacatcc gaagaatgat ctcactttct 2880
ggtgaccact atgggtattg atatatttgt ttagttggtg gctccttgtt caggaaagta 2940
ctgtaggatg tgccattagg tctttagatg aacctagtag atgcagtact accttctaga 3000
acttctgggg ttaaaaacac cgattctggt cattttaagt agcttttcaa ttgaatttct 3060
cattcccaca atgttttaca atactatctg ataaaattgc ttgccataat gggaattgag 3120
aggaactagt ctgtggttac gtatatttta gttctcagaa gttagcactc aaccatactg 3180
atttgcatct ttggtgctga cttcctatta caatttcatt ctctcactgt taccacagta 3240
aaagggattt gtacaaaggg acattttgaa cggatctttc tctaatttgt gctcatcttt 3300
aggtgtgttg agctaatttg tttgttgttt gataggacaa attggatatg ccctggttcc 3360
catgattgct aggggtgtta tgcttggtgc agaccagcct gttattctgc acatgcttga 3420
cattccgcca gcagctgaag cccttaatgg tgttaagatg gagttggttg atgctgcgtt 3480
tcctcttctg aagggtaagc tgatgtgcta atttagagag tatggcctct tctgtctttt 3540
atatttgaat tgttgttctt gctggtgtat aaatgtttgg taaactatag gagttgttgc 3600
tacaactgat gttgtggaag cctgcactgg ggtcaatgtg gccgtcatgg ttggtggatt 3660
ccccaggaag gagggaatgg aaaggaaaga tgttatgtcg aaaaatgttt caatctacaa 3720
atcccaagca tctgcccttg aagcccatgc agctcccaac tgcaaggtat tggctacctt 3780
caactttttt agtagcagac tagttatcat gcactgaatt tggtcatttt ggtgctgata 3840
gttttgtcaa tcgctcaatt ccgtcatgta agtagtggtg gtgctgtcat catgcactcg 3900
atttagctca tctgggtggt gaacggtgac atacataaat gaccattatg aaatggactt 3960
gccctttttt atgagttttt tttcctgttt tgcatgctcc acactatgac actatgcccc 4020
tgtttgtttc ggctttttgt agcttctggc caccaaaagc tgctgcggac tgccaaacac 4080
tcagcttttc agccagcttc tataaaattc atttgggtaa aagccattca aaatcaacat 4140
aaacatataa tcggttgagt cgtcgcaata atagtaatcc gtcactttat agatcctgag 4200
ccttatgaac aactttatct tcctccgcac gtaatcctaa tgatactcag attctctaca 4260
cagtcaaatt ctccccatag ctagattttc ggaaaagctg gtaaaaaaaa gctgaaccaa 4320
acagacccta tgtcacagcc aatctgccgc tgtgttctgc cttaacaagt aaaaaagggc 4380
agaccttccc ccgcaaatgc attaacaagt agcctcaaca aatattgaat caaagtatct 4440
gaatttcagc agttatgtac ccttagtttc ctgagaagcc atgttgggtt cattatgttt 4500
gtttccttgc ttttttcatc tgtagatttt caatacctgt tctgtgctgt gcaggttctg 4560
gtggttgcca acccagcaaa caccaatgct cttatcttga aagaatttgc tccatctatt 4620
ccagagaaga acgttacttg cctgacccgt ctggaccaca acagggctct tggccagatt 4680
tctgagagac ttaatgtcca agttagtgat gtgaagaatg ttatcatctg gggtaaccac 4740
tcgtcctctc agtaccctga tgttaaccat gccacagtga agacttccac tggggagaag 4800
cctgttcgtg agcttgtctc tgatgacgag tggtaaatca gaaattttca aaatacttgg 4860
atatcactgt ttgtcgctat tttcctggta ggtcagaagt tggaattaat ggtttcactc 4920
ttttttttat aacaggctta atggtgaatt catcacaact gtccaacagc gtggtgctgc 4980
aatcattaaa gcgagaaagt tctccagtgc tctctcggct gctagctctg cttgtgacca 5040
cattcgtgat tgggttcttg gaacacctga ggtcagcttc agttctttgc tccttttgat 5100
tcttttagtt ttaactttta aatgtgaact tgtatcaaca tcttcagttc tttttattgt 5160
agggtacatt tgtttccatg ggtgtgtact ctgatggttc atacggtgtt ccttctggac 5220
tcatttattc attcccagta acatgcagtg gtggtgaatg gaaaatcgtt caaggtatga 5280
ttaatgtagt tttgagaggc attaacacag ctatttagtg cctttccttg ttattgtttt 5340
tttgattggt aattaatgtt gcagtttctt ccataactct tgcttctcgg tgtttggaag 5400
tctgcacatg tagttatgtt tggcctaaag ttttcaatta gaaaaaaaaa cctgccatcc 5460
tactcaatag gaagtgatta tggccctgta tttttttaat caaacagaga taatagaaac 5520
tcctaaaaat ctctagctga tccaaaccca ccgaatccca agttgtttgg ttttcaggca 5580
tatataactt ttcttaggta cttaggtatg cactatgtct agatatagca atctagaaaa 5640
aatcaaaata acttacaatt tgaaaagcaa aaccgagctt taactatcca tccacacagg 5700
atccagctaa taaaaagata tagttggcac ccaaagttat tgcagaagtt tatactgcac 5760
cacgctgact tgttctacat aagctaacaa gctattatct gcccaacacc aaaagttcaa 5820
acaaaagtgt ttttttatca taagttacaa tgcacctggc ttcctaagtt taaaccttcg 5880
aagttgcctg atgttgtgtc atgtcatggt aggcctcccg attgatgagt tctcaaggaa 5940
gaagatggat gccactgccc aagagctgac ggaggagaag acgcttgctt actcatgcct 6000
cgagtaaatt ctaccacacc cagagagcaa ttgccacccc aatgttttga ataaaaaaag 6060
aaactccaac tccattcata gattctcact cagaactgct gcacgtcgca gtgcaggcgt 6120
gttgctggct tcgcccagct tctttttttg cctgtgattt gcacgacatt tatatttatt 6180
gaaatcttgc agaatcgctt gacgatgaaa gtggtagcat acttttgttg ctttcctggt 6240
gcctaactat cttttaaagt ttcatttaga tcctttctgg attccgtaga tcccccctcg 6300
attctacagt ttattagcct gttggaaaat tcattgtgtc cctaacgaaa cctttattgt 6360
tgggtgcgtc cccccatcct ctcccggtct cccccctctt tttctgtc 6408
<210> 2
<211> 6342
<212> DNA
<213> Zea mays
<400> 2
gacgacgtat agtcgcggtg aagattgaga tgaagaaagc tatgctactg gctattcata 60
ggacaaaaaa gtaaagtaca gattgattgc tttaattaat ggtgtggaca atcgaccata 120
ccacataaat aaaaatgtca tgtaagcaga cttagagttt attattgtaa ttgctcttac 180
tcccaagtcc caaccaaaca tagtagtaag aaaaagtatc gtcacatcgc gtttccctat 240
cggtcgaagt agctttcaaa tctttagatg tgtggataaa aaaacggctg gtgatcaaag 300
ccatcggtac ctatcaaacc tatcaccaac cctatcgtga atgagtgcca tcttcctcac 360
gccacgcttc tcgaatctcg agagacatct cctccacaac gataatgtcg ccgcttcggt 420
cctgtaaaac agctcgcaag aaaaatgcag aaatcaatcg agcatcatcc agcttgccaa 480
gctgccatgc tgtctaactg acagaaggac cccaccatct tgctccactc cgtactactg 540
tacgtagtat tcatcgccga agccgtgggc ggtgggccag cgcccacagc gtcaagcgtc 600
acgccgtcac ttgcgagtct cctgcctaaa gacacaccaa ccaatcagag ccaacatagc 660
acactgacgt caggggcccg ctggtgtcgg gccaattgtc agttactgat aagcacgctg 720
tcattatcgg ggcgtgtgac agtggattcc caaggcttcc ccgtcacagc gacgttgtta 780
aatagtggcc acccattgcc gcgttctctt cgctatccct tggttgccgc agcctccaaa 840
gccactccca aacccccgct tccagaacct tctcgaagct ccccgccgcc tcctccactc 900
gcaccgatgg cgaaggaacc aatgcgcgtg ctcgtcactg gcgccgcagg tacggtgctg 960
aaccttccct ccttcccgat ccgcgtagta tccggtgtcg tctggtcccg gctcggatct 1020
ggcctagtcc gcccgagcct gagcccgagc tcgatccgat cctgtgtggt ttagagtgtg 1080
cttaactcgc tagatctcag tttttatcgc tcgatctgat ggtgtttagg gcgtttgggt 1140
tgacgtagtt ttggccagta gaactgtaga agggctcact gtcactctcc ttgttcagtc 1200
ggtgaagttg aaccgatccg cggtgctagc tgatctgatc agtggttggg cttgatgggt 1260
agatcctcgt ggtagtcact tgatccagtt gactcgctag ctgcacttaa ttaactgctt 1320
actctttaga tagttgattt tactgctgcg atctctagtt gaaccttaag ggctccggga 1380
tagcagcagg ttcggatccg gtggttttgg agtcaacggc gttttggagc gagtggtttt 1440
gatatatttc atcccatcag actatctggt gggacttaag ttacagatct aggtactcca 1500
cccatggagt ttacgtgcta aacttgcatg aattctgtct acacgatcac tgcgtgagtg 1560
atgtaacctt ttagctgttc agataggtgc cttaataatc atgtaatgta atggaacttc 1620
ctcaaacatt aaggatggtg gtgtaggcat ccaggggtcc aggctgcagt tgtttggggc 1680
ccatgcttga cgcgttcact cttttcatct tttgtagcat gtcatctgag ttcttgcatt 1740
tctattatca taaccacagg gtttggtatg gattagccaa aagttagtta atatcctgct 1800
tatttatggt agcatatgtc tggcattcag ttataaaatt gcacagcgtg cttgacactt 1860
ttcttgtata ttattgagct attcttaggg ggtgtttggt ttgcccctgc taaaatttag 1920
cccctatcac atcgaatgtt tgaacctccg ttcggggtat taaatgtagt cggattataa 1980
aactaatttt cagccgaaga ttaaaaagtg agacgaatta gtacagttgg ttgggtcaat 2040
attttatact cctatttgaa agtcaaacgc ttgatgtgac ccgggctaaa ctttagtcca 2100
cagaaccaaa caccccctta taagcatcta ctgtgataca acaattgtct actgcttgtg 2160
tcatatttga tggtattaga gttggtgtac aatgcgtatt tactagttcc atttttatat 2220
gcttgtactg agttaaaata tgatgcttgt tcttatggtg atagtgacct tccctcacct 2280
gttccggtac ctgggatgat atttgctaat tcaataattg tcatattgtt ttaaagatct 2340
actccatatc agcagtaaag gtgtctccat gtagcataat agaatgagga gcaagagtat 2400
gttctggctt tgaggttgcc acatgctatg ataggatagg gtgttgcatt cttctttgca 2460
tggacacgta cgtcattgtg caattcacaa aaggtgccat ggataccatg tgctttcata 2520
gcaaacaatt ttgtcatgaa aatcttagtc gacgagtgat tgtcattgtg acagagaggg 2580
tgaatttgga tccaaaatct tactactgat ttgcgcccaa catatgggtg aggttttttt 2640
ttgggtttta ttttcttatt ttaagttgta aacccaatgt catgttgtaa gcctgaggat 2700
ggagattagt cattcccaaa tttgaagcag catgacaaaa attggtggag gatgttgacg 2760
ctaccaacca atgacccatc aaaaattttc tggctagttt tacatccgaa gaatgatctc 2820
actttctggt gaccactatg ggtattgcta tatttgttta gttggtggct ccttgttcag 2880
gaaagtactg taggatgtgc cattaggtct ttagatgaac ctagtagatg cagtactacc 2940
ttctagaact tctggggtta aaaacaccga ttctggtcat tttaagtagc ttttcaattg 3000
aatttctcat tcccacaatg ttttacaata ctatctgata aaattgcttg ccatactggg 3060
aattgagagg aactagtctg tggttacgta tattttagtt ctcagaagtt agcactcaac 3120
catactgatt tgcatctttg gtgctgactt cctattacaa tttcattctc tcactgttac 3180
cacagtaaaa gggatttgta caaagggaca ttttgaacgg atctttctct aatttgtgct 3240
catctttagg tgcgttgagc taatttgttt gttgtttgat aggacaaatt ggatatgccc 3300
tggttcccat gattgctagg ggtgttatgc ttggtgcaga ccagcctgtt attctgcaca 3360
tgcttgacat tccgccagca gctgaagccc ttaatggtgt taagatggag ttggttgatg 3420
ctgcgtttcc tcttctgaag ggtaagctga tgtgctaatt tagagagtat ggcctcttct 3480
gtcttttata tttgaattgt tgttcttgct ggtgtataaa tgtttggtaa actataggag 3540
ttgttgctac aactgatgtt gtggaagcct gcactggggt caatgtggcc gtcatggttg 3600
gtggattccc caggaaggag ggaatggaaa ggaaagatgt tatgtcgaaa aatgtttcaa 3660
tctacaaatc ccaagcatct gcccttgaag cccatgcagc tcccaactgc aaggtattgg 3720
ctaccttcaa cttttttagt agcagactag ttatcatgca ctgaatttgg tcattttggt 3780
gctgatagtt ttgtcaatcg ctcaattccg tcatgtaagt agtggtggtg ctgtcatcat 3840
gcactcgatt tagctcatct gggtggtgaa cggtgacata cataaatgac cattatgaaa 3900
tggacttgcc cttttttatg agtttttttt cctgttttgc atgctccaca ctatgacact 3960
atgcccctgt ttgtttcggc ttttcgtagc ttctggccac caaaagctgc tgcggactgc 4020
caaacactca gcttttcagc cagcttctat aaaattcatt tgggtaaaag ccattcaaaa 4080
tcaacataaa catataatcg gttgagtcgt tgcaataata gtaatccgtc actttataga 4140
tcttgagcct tatgaacaac tttatcttcc tccgcacgta atcctaatga tactcagatt 4200
ctctacacag tcaaattctc cccatagcta gattttcgga aaagctggta aaaaaaagct 4260
gaaccaaaca gaccctatgt cacagccaat ctgccgctgt gttctgcctt aacaagtaaa 4320
aaagggcaga ccttcccccg caaatgcatt aacaagtagc ctcaacaaat attaaatcaa 4380
agtatctgaa tttcagcagt tatgtaccct tagtttcctg agaagccatg ttgggttcat 4440
tatgtttgtt tccttgcttt tttcatctgt agattttcaa tacctgttct gtgctgtgca 4500
ggttctggtg gttgccaacc cagcaaacac caatgctctt atcttgaaag aatttgctcc 4560
atctattcca gagaagaacg ttacttgcct gacccgtctg gaccacaaca gggctcttgg 4620
ccagatttct gagagactta atgtccaagt tagtgatgtg aagaatgtta tcatctgggg 4680
taaccactcg tcctctcagt accctgatgt taaccatgcc acagtgaaga cttccactgg 4740
ggagaagcct gttcgtgagc ttgtctctga tgacgagtgg taaatcagaa attttcaaaa 4800
tacttggata tcactgtttg tcgctatttt cctggtaggt cagaagttgg aattaatggt 4860
ttcactcttt tttttataac aggcttaatg gtgaattcat cacaactgtc caacagcgtg 4920
gtgctgcaat cattaaagcg agaaagttct ccagtgctct ctcggctgct agctctgctt 4980
gtgaccacat tcgtgattgg gttcttggaa cacctgaggt cagcttcagt tctttgctcc 5040
ttttgattct tttagtttta acttttaaat gtgaacttgt atcaacatct tcagttcttt 5100
ttattgtagg gtacatttgt ttccatgggt gtgtactctg atggttcata cggtgttcct 5160
tctggactca tttattcatt cccagtaaca tgcagtggtg gtgaatggaa aatcgttcaa 5220
ggtatgatta atgtagtttt gagaggcatt aacacagcta tttagtgcct ttccttgtta 5280
ttgttttttt gattggtaat taatgttgca gtttcttcca taactcttgc ttctcggtgt 5340
ttggaagtct gcacatgtag ttatgtttgg cctaaaattt tcaattagaa aaaaaaacct 5400
gccatcctac tcaataggaa gtgattatgg ccctgtattt ttttaatcaa acagagataa 5460
tagaaactcc taaaaatctc tagctgatcc aaacccaccg aatcccaagt tgtttggttt 5520
tcaggcatat ataacttttg ttaggtactt aggtatgcac tatgtctaga tatagcaatc 5580
tagaaaaaat caaaataact tacaatttga aaagcaaaac cgagctttaa ctatccatcc 5640
acacaggatc cagctaataa aaagatatag ttggcaccca aagttattgc agaagtttat 5700
actgcaccac gctgacttgt tctacataag ctaacaagct attatctgcc caacaccaaa 5760
agttcaaaca aaagtgtttt tttatcataa gttacaatgc acctggcttc ctaagtttaa 5820
accttcgaag ttgcctgatg ttgtgtcatg tcatggtagg cctcccgatt gatgagttct 5880
caaggaagaa gatggatgcc actgcccaag agctgacgga gaagacgctt gcttactcat 5940
gcctcgagta aattctacca catccagaga gcaattgcca ccccaatgtt ttgaataaaa 6000
aaagaaactc caactccatt catagattct cactcagaac tgctgcacgt cgcagtgcag 6060
gcgtgttgct ggcttcgccc agcttctttt tttgcctgtg atttgcacga catttatatt 6120
tattgaaatc ttgcagaatc gcttgacgat gaaagtggta gcatactttt gttgctttcc 6180
tggtgcctaa ctatctttta aagtttcatt tagatccttt ctggattccg tagatccccc 6240
ctcgattcta cagtttatta gcctgttgga aaattcattg tgtccctaac gaaaccttta 6300
ttgttgggtg cgtcccccca tcctctcccg gtctcccccc tc 6342
<210> 3
<211> 161
<212> DNA
<213> Zea mays
<400> 3
aagcgtcacg ccgtcacttg agagtctcct gcctaaagac acaccaacca atcagagcca 60
acatagcaca ctgacgtcag gggcccgctg gtgtcgggcc aattgtcagt tactgataag 120
cacgctgtca ttatcggggc gtgtgacagt ggattcccaa g 161
<210> 4
<211> 159
<212> DNA
<213> Zea mays
<400> 4
aagcgtcacg ccgtcacttg cgagtctcct gcctaaagac acaccaacca atcagagcca 60
acatagcaca ctgacgtcag gggcccgctg gtgtcgggcc aattgtcagt tactgataag 120
cacgctgtca ttatcggggc gtgtgacagt ggattccca 159
<210> 5
<211> 96
<212> DNA
<213> Zea mays
<400> 5
caaggaagaa gatggatgcc actgcccaag agctgacgga ggagaagacg cttgcttact 60
catgcctcga gtaaattcta ccacacccag agagca 96
<210> 6
<211> 93
<212> DNA
<213> Zea mays
<400> 6
caaggaagaa gatggatgcc actgcccaag agctgacgga gaagacgctt gcttactcat 60
gcctcgagta aattctacca cacccagaga gca 93
<210> 7
<211> 20
<212> DNA
<213> Artificial Synthesis
<400> 7
aagcgtcacg ccgtctcatg 20
<210> 8
<211> 23
<212> DNA
<213> Artificial Synthesis
<400> 8
cttgggaatc cactgtcaca cgc 23
<210> 9
<211> 20
<212> DNA
<213> Artificial Synthesis
<400> 9
caaggaagaa gatggatgcc 20
<210> 10
<211> 22
<212> DNA
<213> Artificial Synthesis
<400> 10
tgctctctgg gtgtggtaga at 22
<210> 11
<211> 22
<212> DNA
<213> Artificial Synthesis
<400> 11
ggtttcgtta gggacacaat ga 22
<210> 12
<211> 19
<212> DNA
<213> Artificial Synthesis
<400> 12
acccaccgaa tcccaagtt 19

Claims (5)

1.一种玉米高赖氨酸基因ZmcytMdh4，其核苷酸序列如SEQ ID NO.2所示。1. A maize high lysine gene ZmcytMdh4 , the nucleotide sequence of which is shown in SEQ ID NO.2. 2.一种引物组合，其特征在于，所述引物组合用于扩增一种分子标记，所述分子标记为玉米高赖氨酸基因ZmcytMdh4的5’UTR的-293bp处的SNP位点，多态性为A或C，所述玉米高赖氨酸基因ZmcytMdh4为SEQ ID NO.2核苷酸序列，所述引物组合含有两条引物核苷酸序列为：2. a primer combination, it is characterised in that the primer combination is used to amplify a molecular marker, the molecular marker is the SNP site at the -293bp at the 5' UTR of the corn high lysine gene ZmcytMdh4 , and more The state is A or C, the maize high lysine gene ZmcytMdh4 is the nucleotide sequence of SEQ ID NO.2, and the primer combination contains two primer nucleotide sequences: SNP(-293)-BspHI-L：AAGCGTCACGCCGTCTCATG；SNP(-293)-BspHI-L:AAGCGTCACGCCGTCTCATG; SNP(-293)-1R：CTTGGGAATCCACTGTCACACGC。SNP(-293)-1R: CTTGGGAATCCACTGTCACACGC. 3.一种引物组合，其特征在于，所述引物组合用于扩增一种分子标记，所述分子标记为玉米高赖氨酸基因ZmcytMdh4的第七个外显子中第59到61位碱基GAG的缺失，所述玉米高赖氨酸基因ZmcytMdh4为SEQ ID NO.2核苷酸序列，所述引物组合含有两条引物核苷酸序列为：3. A primer combination, characterized in that, the primer combination is used to amplify a molecular marker, and the molecular marker is the 59th to 61st bases in the seventh exon of the maize high lysine gene ZmcytMdh4 Based on the deletion of GAG, the maize high lysine gene ZmcytMdh4 is the nucleotide sequence of SEQ ID NO.2, and the primer combination contains two primer nucleotide sequences: Exon7-L：CAAGGAAGAAGATGGATGCC；Exon7-L: CAAGGAAGAAGATGGATGCC; Exon7-R：TGCTCTCTGGGTGTGGTAGAAT。Exon7-R:TGCTCTCTGGGTGTGGTAGAAT. 4.权利要求1所述玉米高赖氨酸基因ZmcytMdh4、权利要求2或权利要求3所述引物组合在高赖氨酸含量玉米分子标记辅助选择和聚合育种中的应用。4. The application of the high lysine gene ZmcytMdh4 of claim 1 and the primer combination of claim 2 or claim 3 in molecular marker-assisted selection and polymerization breeding of high lysine content corn. 5.权利要求2或权利要求3所述引物组合在高赖氨酸玉米育种中的应用。5. Application of the primer combination of claim 2 or claim 3 in high lysine corn breeding.
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