CN111118206A - Molecular marker closely linked with major QTL (quantitative trait locus) of corn grain length and application - Google Patents
Molecular marker closely linked with major QTL (quantitative trait locus) of corn grain length and application Download PDFInfo
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- CN111118206A CN111118206A CN202010139514.0A CN202010139514A CN111118206A CN 111118206 A CN111118206 A CN 111118206A CN 202010139514 A CN202010139514 A CN 202010139514A CN 111118206 A CN111118206 A CN 111118206A
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Abstract
The invention relates to a molecular marker closely linked with a corn kernel length major QTL, wherein the corn kernel length major QTL comprises qKL-2, qKL-2 which is positioned on chromosome 9 of corn and is closely linked with a molecular marker mk3110, and the physical position of the mk3110 molecular marker is 134758134; the sequence of the marker is shown in SEQ ID NO. 1. The invention also provides a method for obtaining the molecular marker closely linked with the major QTL of the corn grain length, which comprises the following steps: (1) taking parent SG-5 and SG7 and hybrid F2Extracting whole genome DNA from a single leaf by adopting a CTAB method; (2) sequencing the whole genome DNA obtained in the step (1) by adopting a GBS method; (3) screening SNP markers according to the sequencing result; (4) dividing all the screened SNP markers into bins by adopting a bin-map mode, and constructing a genetic map; (5) using constructed genetic mapsIn combination with F2And F2:3The grain length trait phenotype of (a); QTL analysis is carried out by using a winQTLctart 2.5 software composite interval mapping method to obtain SNP molecular markers which are closely linked with the QTL.
Description
Technical Field
The invention relates to the field of corn breeding, in particular to a molecular marker closely linked with a major QTL of corn grain length and application thereof.
Background
Corn is an important food and feed crop and is one of three crops in the world. Corn is used as a food product for humans, as a feed product for animals, as a pharmaceutical product, and as an industrial product. With the increasing world population, how to produce more food on a limited arable area becomes a major current problem. Corn is the first crop in China and plays an important role in guaranteeing national food safety (Lishaohu et al, 2017). The crops are planted in 31 provincial and municipal autonomous regions in China, and have great influence on the development of the whole national economy as the crops for both food and feed.
Through the analysis of corn hybrids and parents thereof in different ages in China, the yield is found to be in a positive correlation with the ear thickness, the ear number, the grain weight, the leaf area index and the leaf direction value, wherein the grain weight, the leaf area index and the leaf direction value have large contribution to the yield (lee-frontier, 2009). Since grain weight reduction cannot be compensated by other yield factors, grain weight becomes a main contradiction affecting yield, and is also one of key traits of high-yield breeding at present. Grain weight is an important constituent factor of corn yield traits, and is proved to be in direct correlation with yield per mu, and the corn yield can be effectively improved by increasing the grain weight (Gupta et al, 2006). The development of molecular markers goes through the courses of the first generation (RFLP as a representative) and the second generation (SSR as a representative), and the rapid development of the third generation of SNP is promoted by a new generation of high-throughput sequencing technology and rich genotyping technology. Compared with AFLP, RFLP, RAPD and SSR markers, SNP (single nucleotide polymorphism), namely single nucleotide polymorphism, has the advantages of high density, strong representativeness, good genetic stability, easiness in realizing automatic analysis and detection and the like, and is widely applied to the aspects of plant genetic linkage map construction, QTL positioning, biological polymorphism research and the like. Meanwhile, the development of SNP markers promotes genetic researches on complex quantitative traits of plants, such as genetic maps, gene positioning, association analysis and the like. The research proves that: most SNP variation is closely related to gene function, and the SNP site information can be discovered and applied to crop genetic breeding through gene positioning and association analysis. Grain length is significantly related to grain weight, and therefore, it is necessary to explore genetic markers related to grain length at the whole gene level, which helps to accelerate the high-yield corn breeding process.
Therefore, the invention is especially provided.
Disclosure of Invention
Aiming at the problems that the traditional corn yield QTL positioning method is low in marker density of a genetic map, large in QTL positioning confidence interval and difficult to directly predict candidate genes of the positioning QTL and the like, the invention adopts a GBS simplified genome sequencing technology to construct a corn high-density SNP genetic map and performs whole genome scanning by combining with the inspected corn grain length phenotypic characters to obtain SNP markers tightly linked with the target character QTL.
The invention provides a molecular marker closely linked with a major QTL of corn grain length, wherein the major QTL of corn grain length comprises qKL-2,
qKL-2 is located on chromosome 9 of maize and is closely linked to the molecular marker mk3110, which is 134758134 in physical position for mk 3110;
the sequence of the marker is shown in SEQ ID NO. 1.
In another aspect, the present invention provides a method for obtaining a molecular marker closely linked to a major QTL of corn grain length, comprising the steps of:
(1) taking parent SG-5 and SG7 and hybrid F2Extracting whole genome DNA from a single leaf by adopting a CTAB method;
(2) sequencing the whole genome DNA obtained in the step (1) by adopting a GBS method;
(3) screening a genetic marker according to a sequencing result;
(4) dividing all the screened genetic markers into bins by adopting a bin-map mode, and constructing a genetic map; and combining the genetic map with F2And F2:3The grain length phenotype trait of (4) is combined;
(5) QTL analysis is carried out by using a winQTLctart 2.5 software composite interval mapping method to obtain SNP molecular markers which are closely linked with the QTL.
Preferably, the hybrid F extracted in step (1)2The number of (2) is 199.
Preferably, in step (1), after the whole genome DNA is extracted, 1% agarose gel is used to detect DNA degradation and contamination.
Preferably, in step (1), after the whole genome DNA is extracted, the DNA concentration is detected using an ultraviolet spectrophotometer.
Preferably, in step (4), the number of windows in the bin-map mode is set to 15, and the genetic distance is calculated using R/qtl and plotted using perl script.
Preferably, in step (5), the search step size of the employed winqtlcart2.5 software composite interval mapping method is 1cM, and the LOD threshold is a threshold of 1000 times permatation.
The invention also provides application of the molecular marker closely linked with the major QTL of the corn grain length in breeding of the corn grain length traits.
The molecular marker tightly linked with the major QTL of the corn grain length and the application thereof provided by the invention can be used for obtaining the SNP marker tightly linked with the target QTL by scanning the whole genome of the grain length character and analyzing the chromosome region and the genetic effect of the major QTL, thereby laying a foundation for candidate gene prediction and cloning of the corn grain length character QTL and molecular marker-assisted breeding.
Detailed Description
The present invention is further illustrated by the following examples, which are not intended to limit the scope of the invention.
Example 1
In the embodiment of the invention, a corn inbred line SG-5 is used as a female parent, a corn inbred line SG-7 is used as a male parent, a hybrid combination is configured, and F containing 199 single plants is constructed by south propagation and generation addition2And F2:3Segregating the population and examining record P1、P2、F2、F2:3The grain length property of (1).
Collecting parent SG-5 and SG-7 and hybrid F2Leaf of the individual plant. As a preferred embodiment, the leaves are taken from 6-leaf stage seedlings. The leaf samples were stored at-80 deg.C
Extracting parent and F by CTAB method with leaves as sample2The whole genomic DNA of the cohort. The CTAB method is a conventional technique in the art and will not be described in detail herein. In order to ensure that the extracted whole genome DNA can be used in the subsequent steps, the degradation and pollution conditions of the DNA are detected by adopting a 1% agarose gel electrophoresis mode, the concentration of the DNA is detected by an ultraviolet spectrophotometer by adopting a spectrophotometry method, and unqualified samples are treated again to obtain the whole genome DNA which can be applied in the subsequent steps.
Sequencing each obtained whole genome DNA by adopting a GBS method, and screening a proper genetic marker according to a sequencing result.
Wherein the selection of the genetic marker is performed according to the following steps.
The sequencing data was aligned to the reference genome. Wherein the reference genome download addresses are as follows:
ftp://ftp.ensemblgenomes.org/pub/plants/release-29/fasta/zea_mays/dna/Zea_mays.AGPv3.29.dna.toplevel.fa.gz
(1) using BWA alignment software (parameter: mem-t 4-k 32-M-R), aligning the PEREADS of parent and offspring Clean data with reference genome;
(2) carrying out format conversion on the comparison result by using SAMtools, and converting the comparison result into SAM/BAM files;
(3) using a Perl script to count the comparison rate and the coverage;
(4) the alignment results were ranked using SAMtools (parameter: sort) for mutation detection.
Group SNP detection:
(1) the BWA alignment results were filtered: selecting reads which are compared to the unique position on the genome, and carrying out subsequent analysis;
(2) SNP detection, adopting GATK (-type UnifiedGenotyper) to detect group SNP of the filtered BAM file;
(3) SNP filtration: in order to reduce false positive SNP caused by sequencing errors, parents and filial generations require that the number of SNP base supports is not less than 4.
(4) Statistics of related information of SNP: number of heterozygous SNPs, number of homozygous SNPs, heterozygous SNP ratio.
And (3) inter-parent label development:
and (3) carrying out inter-parent polymorphism marker development based on the detection result of the corn parent genotype. Filtering out sites with parent information deletion; screening the sites with homozygous parents and polymorphism (for example, at a certain SNP site, the genotype of parent 1 is GG, the genotype of parent 2 is AA, the genotypes of the parents are homozygous, and the genotypes of the parents are different).
In this example, a total of 133,936 polymorphic sites were obtained by the screening procedure described above, where F2The types of available markers in the population are "aa × bb" type, and the number of polymorphic markers is 68,882.
Dividing all the screened genetic markers into bins by adopting a bin-map method, wherein a window is set to be 15, calculating genetic distance by using R/qtl, drawing by using perl script to construct a genetic map, and comparing the genetic map with the parent and F examined previously2And F2:3The grain length trait of (1) is combined.
QTL analysis is carried out by using a winQTLctart 2.5 software composite interval mapping method to obtain SNP molecular markers which are closely linked with the QTL, wherein the search step length of the winQTLctart 2.5 software composite interval mapping method is 1cM, and the adopted LOD critical value is the threshold value of 1000 times of permatation.
The method is adopted to obtain the major QTL of the grain length of the maize chromosome 9, namely qKL-2, and obtain the SNP molecular marker mk3110 closely linked with qKL-2. The sequence of the molecular marker is shown in SEQ ID NO.1, and the base of the marker at the position 134758134 of the maize chromosome 9 is T or G.
The additive effect of the major QTL qKL-2 is 0.23-0.26mm, and the grain length of the corn with the genotype of homozygous TT at the site of the SNP marker mk3110 is obviously longer than that of the corn with the genotype of GG.
Example 2
Hybridizing and combining a corn inbred line SG-5 and a corn inbred line SG-7 to obtain F1Generating seeds, carrying out continuous backcross by taking SG-5 as a receptor parent and SG-7 as a donor parent, and carrying out molecular marker assisted selection according to the SNP marker mk3110 obtained in example 1 to obtain BC3F1Generation and selection of BC3F1The 6 families with known grain length are selected, wherein 3 families are corn long grains and 3 families are corn short grains.
DNA genomes of the 6 families are respectively extracted, restriction enzymes MseI and HaeIII are used for double enzyme digestion, a DNA sample (6-leaf seedling) is sequenced by adopting a GBS method, and SNP typing is carried out.
The SNP molecular marker mk3110 developed in example 1 was detected, and by using this marker, the long-grain and short-grain traits of maize could be distinguished, and the result of grain length identification agreed with the molecular marker result.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and simplifications made in the spirit of the present invention are intended to be included in the scope of the present invention.
Sequence listing
<110> Qingdao agricultural university
<120> molecular marker closely linked with major QTL of corn grain length and application
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<170>SIPOSequenceListing 1.0
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gcaaatggca caagaaacta gtccctaccc agcgcagcaa agtgtgaaat gacataagac 60
ataaccggga agactcatct ttacgccttc acttgaggag gagcatcgcc ttggagggcg 120
ggatctgatt tgacggcatc tagtgtcttg aagaactcct caggaagagc aacgggccgg 180
tgctgaggaa catgcttagg aattggggtg tcgtcttcaa tcacctcaca ttcgtagtca 240
tcagggacac cccacggatc ccattctgcc attcattcaa acaaagaatc acattaaaac 300
gaactgaagc acaaaatttg catcagcaaa caaatagtac atgcaaacat attacgctgg 360
aattgaaaac ctagaacatc agtttgcaga taagcagcat tgcaatatag catcagatgt 420
gttgttcata atcaaaagga atagatgaga agtgaaccag tgtaagtatt acccgtgtta 480
cagtaacaat aatttgcaag cgataagttg aagatatcct caaacccatg cactcaatag 540
gactaatcgt nattggtcca aaccaacttg aaattaatca cgactgattg cgattaattg 600
gacaacttga aaacagtgca ctcaatagaa cttttgaaac taaaaagtcc attcttcaat 660
tatcacactg atataaaatc aaattctgca gctacatgta catggcaaaa catatatgaa 720
aactgttgaa gtataaaaca tatatgaaaa cagtgcactg gtatttgcag tgtgttacga 780
gtttgatttg cattcctgct aacaccaaag actagagttt gatttgcatt cctgctaacg 840
ccaaagacta gggtccgttc gaatgtagat tttggagtct ggaggtgctg gaattgaatt 900
gggttcagta ctaaaccagc attggtattg tggcacaata caaattctat tgtttggatg 960
tacattgaat tggatgttgg aatttgggag gggcaccgga cgcgaggaag aaagggtgtt 1020
gtgtcagtgg caccaagcca ctagccatgg ggaagaaccg aagggcacca ggccaccagc 1080
catggggaac aaggggtgct g 1101
Claims (8)
1. A molecular marker closely linked with a maize grain length major QTL, characterized in that the maize grain length major QTL comprises qKL-2,
qKL-2 is located on chromosome 9 of maize and is closely linked to the molecular marker mk3110, which is 134758134 in physical position for mk 3110;
the sequence of the marker is shown in SEQ ID NO. 1.
2. The method for obtaining the molecular marker closely linked with the major QTL of the corn grain length is characterized by comprising the following steps of:
(1) taking parent SG-5 and SG7 and a hybrid F2 single plant leaf, and extracting whole genome DNA by adopting a CTAB method;
(2) sequencing the whole genome DNA obtained in the step (1) by adopting a GBS method;
(3) screening a genetic marker according to a sequencing result;
(4) dividing all the screened genetic markers into bins by adopting a bin-map mode, and constructing a genetic map; and combining the genetic map with F2And F2:3The grain length phenotype trait of (4) is combined;
(5) QTL analysis is carried out by using a winQTLctart 2.5 software composite interval mapping method to obtain SNP molecular markers which are closely linked with the QTL.
3. The method for obtaining the molecular marker tightly linked with the major QTL of the corn grain length as claimed in claim 2, wherein the number of the hybrids F2 extracted in the step (1) is 199.
4. The method for obtaining the molecular marker tightly linked with the major QTL of corn kernel length as claimed in claim 2, wherein in step (1), after the whole genome DNA is extracted, 1% agarose gel is used for detecting the DNA degradation and contamination.
5. The method for obtaining the molecular marker tightly linked with the major QTL of the corn grain length as claimed in claim 2, wherein in the step (1), after the whole genome DNA is extracted, an ultraviolet spectrophotometer is used for detecting the DNA concentration.
6. The method for obtaining molecular markers closely linked to major QTL of corn kernel length as claimed in claim 2, wherein in step (4), the windows are set to be 15 in a bin-map manner, and R/QTL is used for genetic distance calculation and perl script is used for drawing.
7. The method for obtaining molecular markers closely linked to the major QTL of corn kernel length as claimed in claim 2, wherein in step (5), the search step size of the composite interval mapping method using the winQTLhart 2.5 software is 1cM, and the LOD threshold is 1000 times of persistence threshold.
8. The use of the molecular markers in claim 1 that are tightly linked to major QTLs for maize grain length trait breeding.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106636080A (en) * | 2016-10-14 | 2017-05-10 | 中国科学院微生物研究所 | Expression vector for constructing rice leaf blight bacterial III-type secretion system to transfer target protein into plants and application of expression vector |
| CN110106278A (en) * | 2019-05-15 | 2019-08-09 | 湖北康农种业股份有限公司 | The molecular labeling and application of corn 100-grain weight and grain length character close linkage |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106636080A (en) * | 2016-10-14 | 2017-05-10 | 中国科学院微生物研究所 | Expression vector for constructing rice leaf blight bacterial III-type secretion system to transfer target protein into plants and application of expression vector |
| CN110106278A (en) * | 2019-05-15 | 2019-08-09 | 湖北康农种业股份有限公司 | The molecular labeling and application of corn 100-grain weight and grain length character close linkage |
Non-Patent Citations (1)
| Title |
|---|
| CHENGFU SU等: "High Density Linkage Map Construction and Mapping of Yield Trait QTLs in Maize ( Zea mays) Using the Genotyping-by-Sequencing (GBS) Technology", 《FRONT PLANT SCI》 * |
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