LU500540B1 - MOLECULAR MARKERS CLOSELY LINKED WITH A MAJOR QTL FOR KERNEL LENGTH IN MAIZE AND THE APPLICATION THEREOF - Google Patents

Abstract

The present invention relates to a molecular marker closely linked with a major QTL(quantitative trait locus) for kernel length in maize, wherein the major QTL for kernel length in maize comprised a qKL-2, the qKL-2 is located on chromosome 9 of maize and closely linked with a molecular marker mk3110, the physical position of the molecular marker mk3110 is 134758134; the sequence of the molecular marker mk3110 is shown in SEQ ID NO.1. The present invention also provides a method for obtaining the molecular marker closely linked with a major QTL for kernel length in maize.

Description

MOLECULAR MARKERS CLOSELY LINKED WITH A MAJOR QTL FOR KERNEF/500540

LENGTH IN MAIZE AND THE APPLICATION THEREOF Technical Field

The present invention relates to the field of maize planting, in particular to a molecular marker closely linked with a major QTL for kernel length in maize and the application thereof. Background

[0002] Maize is an important food and feed crop and one of the three major crops in the world. Maize can be used as human food, animal feed, pharmaceutical and industrial products. With the increasing population in the world, how to produce more food on the limited cultivated land area has become the main problem at present. Maize is the largest crop in China and plays an important role in ensuring national food security (Li Shaokun et al., 2017). It is planted in 31 provinces, cities and autonomous regions of China, as a crop for both kernel and feed, it has a great influence on the development of the whole national economy.

[0003] Through the analysis of maize hybrids and their parents in different years of China, it was found that the yield was positively correlated with ear diameter, kernels per ear, hundred-kernel weight, leaf area index and leaf orientation value, among which hundred-kernel weight, leaf area index and leaf orientation value contribute greatly to the yield (Li Congfeng, 2009). Because the reduction of kernel weight can not be compensated by other yield factors, kernel weight has become the major bottleneck affecting yield, and it is also one of the key traits of high-yield breeding at present. As an important factor for maize yield, kernel weight has been proven to be significantly positively correlated with the yield per mu. Increasing kernel weight can effectively increase maize yield (Gupta et al, 2006). The development of molecular markers has gone through the first generation (represented by RFLP) and the second generation (represented by SSR). The new generation high-throughput sequencing technology and abundant genotyping technology have given birth to the rapid development of the third generation SNP. Compared with AFLP, RFLP, RAPD and SSR markers, SNP (Single Nucleotide Polymorphism) has the advantages of high density, strong representativeness, good genetic stability and easy automatic analysis and detection. It has been widely used in the construction of plant genetic linkage map, QTL mapping and biological polymorphism research. At the same time, the development of SNP markers has promoted the genetic study of complex quantitative traits of plants, such as genetic map, gene mapping and association analysis. Studies have shown that most SNP variations are closely related to gene function, and these SNP loci can be discovered and applied to crop genetics and breeding through gene mapping and association analysis. There is a significant/500540 correlation between kernel length and kernel weight, so it is necessary to dig genetic markers related to kernel length at the whole genome level, which will help accelerate the breeding process of high-yield maize.

[0004] In view of this, the present invention is specially proposed.

Summary of the Invention

[0005] In view of the problems that the traditional genetic map used for QTL positioning of maize yield has low marker density, large QTL positioning confidence interval, difficulty in directly predicting candidate genes for positioning QTL, etc., the present invention adopts GBS abbreviated genome sequencing technology to construct a high-density SNP genetic map of maize, and carries out whole genome scanning in combination with the investigated phenotypic traits of maize kernel length to obtain SNP markers closely linked with QTL of target traits.

The present invention provides a molecular marker closely linked with a major QTL for kernel length in maize, wherein the a major QTL for kernel length in maize including qKL-2,

The qKL-2 is located on chromosome 9 of maize and closely linked with a molecular marker mk3110, the physical location of molecular marker is 134758134;

[0008] And the sequence of the molecular marker mk3100 is shown as SEQ ID NO. 1.

[0009] On the other hand, the present invention also provides a method for obtaining a molecular marker closely linked with the a major QTL for kernel length in maize, the method comprises the following steps:

[0010] (1) Using CTAB method to extract whole genome DNA from parent SG-5 and SG7, and hybrid F, single plant leaves;

(ii) Using GBS method to sequence to the whole genome DNA obtained in step i;

[0012] (iii) Screening SNP markers according to the sequencing result;

(iv) Using bin-map method to bin divide the screened SNP markers to construct a genetic map; combining the constructed genetic map with the kernel length trait phenotype of F2 and F2:3;

(v) Using winQTLcart2.5 software composite interval mapping for QTL analysis to obtain a SNP molecular marker closely linked with QTL.

[0015] However, 199 hybrid F2 are extracted in step (i). LUS00540

[0016] However, in step (i), 1% agarose gel is used to detect the DNA degradation and pollution after the whole genome DNA is extracted.

[0017] However, in step (i), the DNA concentration is detected by an ultraviolet spectrophotometer after the whole genome DNA is extracted.

[0018] Furthermore, in step (iv), 15 windows are set in bin-map mode, and R/qtl is used for inheritance distance calculation and perl script is used for drawing.

[0019] However, in step (v), the search step length of composite interval mapping method for using winQTLcart2.5 software is 1cm, and the adopted LOD critical value is the threshold value of 1000 times of permutation

The present invention also provides an application of the molecular marker closely linked with a major QTL for kernel length in maize in maize kernel length trait breeding.

The present invention provides a molecular marker closely linked with a major QTL for kernel length in maize and the application thereof. Genome-wide scanning was carried out to analyze the chromosome region and genetic effect of major QTL, and SNP markers closely linked with target QTL are obtained, which laid a foundation for prediction, cloning and molecular marker-assisted breeding of QTL candidate genes for maize kernel length traits.

Detailed Description of the Invention

The following embodiments are used to further illustrate the present invention, but the present invention is not limited to the described embodiments surrounded by.

Embodiment 1

The embodiment of the present invention adopts the maize inbred line SG-5 as a female parent and the maize inbred line SG-7 as a male parent to configure a hybrid group, the F2 and F2:3 segregation populations containing 199 individual plants are constructed through reproduction in South to increase generations, and the kernel length traits of P1, P2, F2, F2:3 are investigated and recorded.

[0024] The leaves of parent SG-5, SG-7 and hybrid F2 are collected. As a preferred embodiment, the leaves are taken from seeding in six-leaf stage. Each leaf sample was stored at -80°C.

[0025] Using leaves as samples, the whole genome DNA of parents and F2 population 4500540 extracted through CTAB method. CTAB method is a conventional technical means in this field, so it will not be described in detail here. To ensure that the extracted whole genome DNA can be used in the subsequent steps, 1% agarose gel electrophoresis was used to detect the degradation and pollution of DNA, and ultraviolet spectrophotometer was used to detect the concentration of DNA, and the unqualified samples are reprocessed to obtain the whole genome DNA which can be used in the subsequent steps.

[0026] The whole genome DNA is sequenced by GBS method, and the suitable genetic markers are screened according to the sequencing results.

[0027] Wherein the screening of genetic markers is carried out according to the following steps.

[0028] Compare the sequencing data with the reference genome. The download address of the reference genome is as follows:

[0029] ftp://ftp.ensemblgenomes.org/pub/plants/release29/fasta/zea_mays/dna/Zea mays. AGPv3.29.dna .toplevel fa .gz

[0030] (i) Using BWA comparison software (parameter: mem-t 4-k 32-M-R), the PE reads of parents and offspring Clean data are compared with the reference genome;

[0031] (11) Using SAMtools to format the comparison results into SAM/BAM files;

[0032] (iit) Using Perl script to count the comparison rate and coverage;

[0033] (iv) Using SAMtools comparison to sort the results (parameter: sort) for variation detection. Group SNP detection:

[0034] (1) Filtering the BWA alignment results: selecting the reads aligned to the unique position on the genome for subsequent analysis

[0035] (ii) SNP detection: GATK(-type UnifiedGenotyper) is used to detect population SNP in filtered BAM files

[0036] (iii) SNP filtering: to reduce false positive SNPs caused by sequencing errors, parents and offspring require that the SNP base support number is not less than 4.

[0037] (iv) SNP related information statistics: the number of heterozygous SNPs, the number of homozygous SNPs and the ratio of heterozygous SNPs

[0038] Development of markers between parents: LU500540

[0039] Based on the genotype detection results of maize parents, the development of polymorphism markers between parents is carried out. Filtering out the loci with missing parental information; screening loci with homozygous parents and polymorphism between 5 parents (for example, at a certain SNP site, the genotype of parent 1 is 'GG', the genotype of parent 2 is 'AA', both parents are homozygous, and the genotypes between parents are different).

In this embodiment, a total of 133,936 polymorphic loci are obtained through the above screening steps, among which the available marker type of F2 population is ‘aaxbb’ and polymorphic markers is 68,882.

[0041] All the selected genetic markers were divided into bins through bin-map method, in which 15 windows are set, and the genetic distance is calculated by using R/qtl. Meanwhile, perl script was used for drawing to construct the genetic map, and the genetic map was combined with the previously investigated parents and kernel length traits of F2 and F2:3.

[0042] The QTL analysis is carried out by using winQTLcart2.5 software composite interval mapping method to obtain SNP molecular markers which are closely linked with the QTL, wherein, the search step length of composite interval mapping method for using winQTLcart2.5 software is lcm, and the adopted LOD critical value is the threshold value of 1000 times of permutation.

[0043] The method is adopted to obtain the major QTL of kernel length located on chromosome 9 of maize: qKL-2, and SNP molecular marker mk3110 closely linked with qKL-2 is also obtained. The molecular marker sequence is shown as SEQ ID NO.1, and the base of the marker at the position 134758134 of chromosome 8 of maize is T or G.

[0044] The additive effect of the major QTL qKL-2 is 0.23-0.26mm, and at SNP marker mk3110 locus, the kernel length of maize with homozygous TT genotype is significantly higher than that of maize with GG genotype.

Embodiment 2

The F1 generation seeds are obtained through cross combination of maize inbred line SG-5 and maize inbred line SG-7, SG-5 is used as receptor parent and SG-7 as donor parent, and molecular marker-assisted selection is carried out according to SNP marker mk3110 obtained in embodiment 1 to obtain BC3F1 generation, and 6 families with known kernel length in BC3F1 generation are selected, among which 3 families are long-kernel maize and the other 3 familib4/500540 are short-kernel maize .

[0045] The DNA genomes of the above 6 families are respectively extracted, and restriction enzymes Msel and Haell are used for double enzyme digestion, the DNA samples (6-leaf-stage seedlings) are sequenced by the GBS method, and SNP typing is carried out.

The SNP molecular marker mk3110 developed in embodiment 1 is detected, and used to distinguish the long and short kernel traits of maize, and the identification results of kernel length are consistent with the results of molecular markers.

The above are only the preferred embodiments of the present invention, and is not intended to limit the present invention. Any modification, equivalent substitution and simple improvement made in the essence of the present invention should be included in the protection scope of the present invention.

SEQUENCE LISTING LU500540 <110> QINGDAO AGRICULTURAL UNIVERSITY <120> MOLECULAR MARKERS CLOSELY LINKED WITH A MAJOR QTL FOR KERNEL LENGTH IN

MAIZE AND THE APPLICATION THEREOF <160> 1 <170> SIPOSequenceListing 1.0 <218> 1 <211> 1101 <212> DNA <213> maize(Zea mays L.) <221> misc feature <222> (551)..( 551) <223> n = t or g <400> 1 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 1181 g

Claims (8)

Claims LU500540 1. À molecular marker closely linked with a major QTL for kernel length in maize, characterized in that, wherein the major QTL for kernel length in maize comprised: A qKL-2, the qKL-2 is located on chromosome 9 of maize and closely linked with a molecular marker mk3110, the physical position of the molecular marker mk3110 is 134758134; the sequence of the molecular marker mk3110 is shown in SEQ ID NO 1. 2. A method for obtaining the molecular marker closely linked with the a major QTL for kernel length in maize, characterized in that, the method comprises the following steps: 1). Using CTAB method to extract whole genome DNA from parent SG-5 and SG7, and hybrid F2 single plant leaves; 11). Using GBS method to sequence the whole genome DNA obtained in step 1); 111). Screening SNP markers according to the sequencing result; iv). Using bin-map method to bin divide the screened SNP markers to construct a genetic map; and combining the constructed genetic map with the kernel length trait phenotype of F2 and F2:3; v). Using winQTLcart2.5 software composite interval mapping for QTL analysis to obtain a SNP molecular marker closely linked with QTL. 3. The method for obtaining the molecular marker closely linked with a major QTL for kernel length in maize according to claim 2, characterized in that, 199 hybrid F2 are extracted in step 1). 4. The method for obtaining the molecular marker closely linked with a major QTL for kernel length in maize according to claim 2, characterized in that, in step 1), 1% agarose gel is used to detect the DNA degradation and pollution after the whole genome DNA is extracted. 5. The method for obtaining the molecular marker closely linked with a major QTL for kernel length in maize according to claim 2, characterized in that, in step 1), the DNA concentration is detected by an ultraviolet spectrophotometer after the whole genome DNA is extracted . 6. The method for obtaining the molecular marker closely linked with a major QTL for kernel length in maize according to claim 2, characterized in that, in step iv), 15 windows are set in bin-map mode, and R/qtl is used for inheritance distance calculation and perl script is used for drawing. 7. The method for obtaining the molecular marker closely linked with a major QTL for kerneH500540 length in maize according to claim 2, characterized in that, in step v), the search step length of composite interval mapping method for using winQTLcart2.5 software is 1cm, and the adopted LOD critical value is the threshold value of 1000 times of permutation. 8. An application of the molecular marker closely linked with a major QTL for kernel length in maize according to claim 1 in maize kernel length trait breeding is provided. Claims HU500540 1. A closely related molecular marker has a maize QTL kernel length master effect, characterized in that it is, said maize QTL kernel length master effect comprises qKL-2, qKL-2 is located on the chromosome 9 of maize and is closely related to molecular marker mk3110, said molecular marker mk3110 having a physical location of 134758134; the marker sequence is shown in SEQ ID NO.1. 2. A method for obtaining closely related molecular markers has a master effect of corn kernel length QTL, characterized in that it is, method comprises the following steps: (1) Parents SG-5 and SG-7 and single sheets of the F2 hybridization were taken and the whole genomic DNA was extracted by the CTAB method; (2) Sequencing of all of the genomic DNA obtained in step (1) using the GBS method; (3) Screening for genetic markers based on sequencing results; (4) Using bin-map, all selected genetic markers were bin-delimited and genetic maps were constructed; and the genetic maps were combined with the F2 and F2:3 grain length phenotypic traits; (5) QTL analysis was performed using the composite interval mapping method of winQTLcart2.5 software to obtain molecular marker SNPs closely related to QTLs. 3. A method for obtaining molecular markers closely related to a maize kernel length QTL master effect according to Claim 2, characterized in that it is, the F2 hybridization number extracted in step (1) is of 199. 4. A method for obtaining molecular markers closely related to a maize kernel length QTL master effect according to Claim 2, characterized in that it is, step (1), after extraction of the whole genomic DNA , a 1% agarose gel is used to detect DNA degradation and contamination. 5. A method for obtaining molecular markers closely related to a maize grain length QTL master effect according to Claim 2, characterized in that it is, step (1), after extraction of the genomic DNA whole, the DNA concentration is detected using a UV spectrophotometer. 6. A method for obtaining molecular markers closely related to a maize kernel length QTL master effect according to Claim 2, characterized in that it is, step (4), setting the window in the bin-map approach is 15 and genetic distance is calculated using R/qtl and drawing is done using perl script. 7. A method for obtaining molecular markers closely related to a maize kernel length QTL master effect according to Claim 2, characterized in that it is, step (5), the step size of the search employed using the composite interval mapping method of the winQTLcart2.5 software is 1 cM and the LOD threshold value used is a threshold of 1000 permutations. 8. Application of molecular markers closely related to a maize kernel length QTL master effect in horticultural seeds of the maize kernel length traits in Claim 1.