LU500539B1

LU500539B1 - Molecular markers closely linked with a major qtl for kernel width in maize and the application thereof

Info

Publication number: LU500539B1
Application number: LU500539A
Authority: LU
Inventors: Yanming Zhao; Chengfu Su
Original assignee: Univ Qingdao Agricultural
Priority date: 2021-08-16
Filing date: 2021-08-16
Publication date: 2022-02-16

Abstract

The present invention relates to a molecular marker closely linked with major QTL (quantitative trait locus) for kernel width in maize, wherein the major QTL for kernel width in maize comprises a qKW-2, the qKW-2 is located on chromosome 8 of maize and closely linked with a molecular marker mk2814, the physical position of the molecular marker mk2814 is 151071300; the sequence of the molecular marker mk2814 is shown in SEQ ID NO.1. The present invention also provides a method for obtaining the molecular marker closely linked with the a major QTL for kernel width in maize.

Description

MOLECULAR MARKERS CLOSELY LINKED WITH A MAJOR QTL FOR !U500539

KERNEL WIDTH IN MAIZE AND THE APPLICATION THEREOF Technical Field

[0001] The present invention relates to the field of maize planting, in particular to a molecular marker closely linked with a major QTL for kernel width in maize and the application thereof. Background Art

[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, cites 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 proved 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

; ; ; ; : LU500539 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 correlation between kernel width and kernel weight, so it is necessary to dig genetic markers related to kernel width 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 kernel width in maize to obtain SNP markers closely linked with QTL of target traits.

[0006] The present invention provides a molecular marker closely linked with a major QTL for kernel width in maize, wherein the a major QTL for kernel width in maize comprises qKW-2,

[0007] The qKW-2 is located on chromosome 8 of maize and closely linked with a molecular marker mk2814, the physical location of molecular marker is 151071300;

[0008] And the sequence of the molecular marker mk2814 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 width in maize, the method comprises the following steps:

[0010] (i) Using CTAB method to extract whole genome DNA from parent SG-5 and SG7, and hybrid F2 single plant leaves;

[0011] (ii) Using GBS method to sequence to the whole genome DNA obtained in step 1;

[0012] (iii) Screening SNP markers according to the sequencing result; LU500539

[0013] (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 width trait phenotype of F2 and F2:3;

[0014] (v) Using winQTLcart2.5 software composite interval mapping for QTL analysis to obtain a SNP molecular marker closely linked with QTL

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

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

[0017] Preferably, in step (1), the DNA concentration is detected by an ultraviolet spectrophotometer after the whole genome DNA is extracted.

[0018] Preferably, 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] Preferably, in step (v),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

[0020] The present invention also provides an application of the molecular marker closely linked with the a major QTL for kernel width in maize in kernel width in maize trait breeding.

[0021] The present invention provides a molecular marker closely linked with the a major QTL for kernel width 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 kernel width in maize traits.

Detailed Description of the Invention

[0022] 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 LU500539

[0023] 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 width 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 is 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] (1) 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] Gi) Using SAMtools to format the comparison results into SAM/BAM files;

[0032] (iii) Using Perl script to count the comparison rate and coverage; LU500539

[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 5 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:

[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 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).

[0040] 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 width 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 1cm, and the adopted LOD critical value is the threshold value of LU500539 1000 times of permutation.

[0043] The method is adopted to obtain the major QTL of kernel width located on chromosome 8 of maize: qKW-2, and SNP molecular marker mk2814 closely linked with qKW-2 is also obtained. The molecular marker sequence is shown as SEQ ID NO.1, and the base of the marker at the position 151071300 of chromosome 8 of maize is C or T.

[0044] The additive effect of the major QTL qKW-2 is 0.34-0.43mm, and at SNP marker mk2814 locus, the kernel width of maize with homozygous TT genotype is significantly higher than that of maize with CC genotype.

Embodiment 2

[0044] The F1 generation seeds are obtained through cross combination of maize inbred line SG-5 and maize inbred line SG-7, SG-S is used as receptor parent and SG-7 as donor parent, and molecular marker-assisted selection is carried out according to SNP marker mk2814 obtained in embodiment 1 to obtain BC3F1 generation, and 6 families with known kernel width in BC3F1 generation are selected, among which 3 families are wide-kernel maize and the other 3 families are narrow-kernel maize.

[0045] The DNA genomes of the above 6 families are respectively extracted, and restriction enzymes Msel and Haelll 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.

[0046] The SNP molecular marker mk2814 developed in embodiment 1 is detected, and used to distinguish the wide and narrow kernel traits of maize, and the identification results of kernel width are consistent with the results of molecular markers.

[0047] 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 LU500539 <110> QINGDAO AGRICULTURAL UNIVERSITY <120> MOLECULAR MARKERS CLOSELY LINKED WITH A MAJOR QTL FOR KERNEL WIDTH 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 <223> n=c or t <400> 1 gacagtctgg tggcacaccg gacagaccgg tgaattatag tcgagcggcc tctgagaaac 60 ccgaagctga ggagtttgga gtcgatcgac cctgggcacc ggacactgtc aggtggcaca 120 ccggatagtc tggtgcgcca gtccagggtt ctcttcggtt tettttgete ctttcttttg 180 aaccataact tgtaactttt tattggtttg tgttgaacct ttagcacctg tagaatatat 240 aatctaaagc aaactagtta gtccaattat ttgtgttggg catttcaacc accaaaatca 300 ttttaggaaa aggtttgacc ctatttccct ttcaatcttc cagcttgagc ccttggatgec 360 ataaacctgc aaggtaatta agcttgggtt taaggtaccc aactcttgtt gggtcctaca 420 aggttagtta caacaacttt tggaacccaa atacagttct tgtctccctt gcatttagat 480 cccaattttc tagcaactac tttggcattt ttacatgaaa gtacaaaaga agcattacat 540 gcatgataaa nagtattagg accagtgcat gctttcctag gcgcatgaga aacaacatga 600 ttacgcctag acctatttct accataagca tatgtagagc tagatgcaaa catagcatga 660 agattaaatg cagcagcatc atgagaaaga atattatcat aacacctatc attatgagca 720 cgactagtaa atttcttatc ataaagatag gcatagttct tttgagaact actaaccata 780 ggagccttcc ctttctcctt gttgagacca gaagtccttt ggcttgttaa gttcttggtt 840 tccttttgga aaccaagtcc atccttaatt gaggggtgtc taccaacagt gtagacatcc 900 ctagcaaatt ttagtttctc ataactttct ttgcaagtct taagttgagc actaagactt 960 gcaacatcat tgtttaactt agcaatagta gaaacatgtt cattacaagc atcaacatca 1020 aagtctttac atctattgca aataacaaca tgctctacac atgaactagt tacattaatt 1080 ttctctagct tagcattcaa a 1181

Claims

Claims LU500539

1. A molecular marker closely linked with a major QTL for kernel width in maize, characterized in that, the major QTL for kernel width in maize comprises: A qKW-2, the qKW-2 is located on chromosome 8 of maize and closely linked with a molecular marker mk2814, the physical position of the molecular marker mk2814 is 151071300; the sequence of the molecular marker mk2814 is shown in SEQ ID NO. 1.

2. A method for obtaining the molecular marker closely linked with a major QTL for kernel width 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 width 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 a major QTL for kernel width in maize according to claim 2, characterized in that, 199 hybrid F2 are extracted in step i).

4. The method for obtaining the molecular marker closely linked with a major QTL for kernel width 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 width 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 LU500539 kernel width 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 kernel width 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 the a major QTL for kernel width in maize according to claim 1 in kernel width in maize trait breeding is provided.