LU102463B1

LU102463B1 - Molecular markers closely linked with major qtl for kernel width in maize and the application thereof

Info

Publication number: LU102463B1
Application number: LU102463A
Authority: LU
Inventors: Yanming Zhao; Chengfu Su
Original assignee: Univ Qingdao Technology
Priority date: 2021-02-02
Filing date: 2021-02-02
Publication date: 2021-08-04

Abstract

The present invention relates to a molecular marker closely linked with a major QTL(quantitative trait locus) for kernel width in maize, the a major QTL for kernel width in maize comprises a qKW-1, the qKW-1 is located on chromosome 3 of maize and closely linked with a molecular marker mk1043, the physical position of the molecular marker mk1043 is 30553179; the sequence of the molecular marker mk1043 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 KERNEL -U'02463

WIDTH IN MAIZE AND THE APPLICATION THEREOF Technical Field

[0001] The present invention relates to the field of maize breeding. in particular to a molecular marker closely linked with a major QTL for kernel width 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, 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 vicld was positively correlated with car diameter. kernels per car, hundred-kernel weight, leaf arca index and leat 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 vield 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. OTL

. . . . . a LU102463 mapping and biological polymorphism research. At the same time. the development of SNP markers has promoted the genctic 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 genctic markers related to kernel width at the whole genome level, which will help accelerate the breeding process ol 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 QTI. positioning of maize yield has low marker density. large QTL positioning confidence interval. difficulty in directly predicting candidate genes for positioning QTL, ctc.. the present invention adopts GBS abbreviated genome sequencing technology to construct a high-density SNP genetic map of maize, and carries oul whole genome scanning in combination with the investigated phenotypic traits of maize kernel width 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-l,

[0007] The gKW-1 is located on chromosome 3 of maize and closely linked with a molecular marker mk1043. the physical location of molecular marker is 30553179;

[0008] And the sequence of the molecular marker mk1043 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:

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[0012] (iii) Screening SNP markers according to the sequencing result: U102463

[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 Fo and Fai:

[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 (1).

[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 (i). the DNA concentration is detected by an ultraviolet spectrophotometer after the whole genome DNA 1s extracted. 10018] 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 winQTLeart2.5 software is lem. 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 tn maize in maize kernel width trait breeding.

[0021] The present invention provides a molecular marker closely Inked with the a major OTL for kernel width in maize and the application thercof. 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 width 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 |

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[0023] The embodiment of the present invention adopts the maize inbred line SG-5 as a female parent and the maize inbred linc 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 PI, P2, F2. F2:3 are investigated and recorded.

[0024] The leaves of parent SG-5, SG-7 and hybrid F2 are collected. As a preterred embodiment. the leaves are taken from seeding in six-leaf stage. Each leaf sample was stored at -80°C. 10025] 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] fip://1tp .ensemblgenomes .org/pub/plants/relcase29/fasta/zca mays/dna/Zea mays AGPv3 | 29 dna .toplevel la gz 10030] (1) Using BWA comparison software (parameter: mem-t 4-k 32-M-R). the PI: reads of parents and offspring Clean data are compared with the reference genome:

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

[0032] (iii) Using Perl seript to count the comparison rate and coverage:

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

[0034] (i) Filtering the BWA alignment results: selecting the reads aligned to the unique position on the genome for subsequent analysis 5 [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 loct 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] AH 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/gtl. Meanwhile, perl script was used for drawing to construct the genetic map, and the genetic map was combined with the previously investigaled parents and kernel width traits of F2 and F2:3 10042] The QTL. analysis is carried out by using winQTLeart2.5 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 winOTLeart2.5 software is lem, and the adopted LOD critical value is the threshold value of 1000 times of permutation.

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[0043] The method is adopted to obtain the major QTL of kemel width located on chromosome 3 of maize: gKW-1, and SNP molecular marker mk1043 closely linked with qKW-1 is also obtained. The molecular marker sequence is shown as SEQ ID NO.1, and the base of the marker at the position 30553179 of chromosome 8 of maize is G or A.

[0044] The additive effect of the major QTL gKW-1 is 0.37-0.54mm. and at SNP marker mk1043 locus. the kernel width of maize with homozygous GG genotype is significantly higher than that of maize with AA 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-5 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 | 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 Ilaelll are used for double enzyme digestion, the DNA samples (6-lcat- stage seedlings) are sequenced by the GBS method. and SNP typing is carried out.

[0046] The SNP molecular marker mk1043 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 LU102463 <11@> QINGDAO AGRICULTURAL UNIVERSITY <128> MOLECULAR MARKERS CLOSELY LINKED WITH A MAJOR QTL FOR KERNEL WIDTH IN

MAIZE AND THE APPLICATION THEREOF <168> 1 <178> SIPOSequenceListing 1.0 <218> 1 <211> 1181 <212> DNA <213> maize(Zea mays L.) <221> misc_feature <222> (551)..(551) <223> n=g or a <480> 1 agatctactg catatatcat cctaagataa tggtaagata ttatctacat ctgcaactta 60 ttttteccag attatttatg tttagaacct ctggettgtc cattgaactc tectetattt 120 caacatcatt gttatctctc atattacagt agggttcagc cctaccaatt tcataagaaa 180 agaaccaaca aactcaatca acgagtaggt aagaaaagaa gtggaagttc caacaaaaat 240 agagctattg tagaaacaca gtccattttt aaaccaacaa ttacaaaagg aggaagaagg 300 catacggcga tggaaatttt ttaaatattt gcaggttcca tgtatcttct gatagaaatg 360 atgtatgaac ttggtcctgg aagaagagga aaaaaacatt agaaacacct agtggtagct 420 ataagttact cacaggaata gttagagctc atcctggatg gaaagtaggt aactgactga 480 actgagagtg ggaaacccac ccgcatgatc attccaccca gcaaaatcag agctgaatca 540 gcctacaaaa ntgcacaaca ttacacgatc asagggtact agataggtga cacataatgt 666 aatgagttaa gattatcttt gtaagatggt atataaacag ctttcaaatc atctaatttg 660 gcaaatgtca tgtcaaataa gcacttccaa aaaattgatt aagatactaa taatatttgc 720 agtcctgact tgteectttec aagaatatat attacgggag atggataaat cgtgttgaga 780 tegeapetec agatcaagtt tacagtgata aaagtactgc caaactttca gaatgaagtt 840 ctgaattcca atctatagaa catagattca gtgaaaactt taattgtatt ctagcaatta 900 tggtgacatt tcactagttc ataagatacc ataagcataa tgcaagcaat tagtaaagaa 960 taactgggaa atacatcatt caatttgaga gactaaaatc ccgaaaaatg gagatactgg 1820 aagatgtaaa tagagcagat aactcttttc tttaatggcc aatttaattt ataagtgttt 1080 cattaatatt caaaaaacac t 1101

Claims

. LU1024 Claims U102463

1. A molecular marker closely linked with a major QTIL tor kernel width in maize. characterized in that. the a major QTL for kernel width in maize comprises: A gKW-1. the qKW-1 is located on chromosome 3 of maize and closely linked with a molecular marker mk1043, the physical position of the molecular marker mk1043 is 30553179; the sequence of the molecular marker mk 1043 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: it). Using GBS method to sequence the whole genome DNA obtained in step i); iil). 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 with a major QTL for kernel width in maize according 10 claim 2, characterized in that. 199 hybrid F2 are extracted instep).

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 i). 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.

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6. 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 iv), 15 windows are set in bin-map mode. and R/qtl 1s 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 QTI. for kernel width in maize according to claim 2, characterized in that. in step v), the scarch step length of composite interval mapping method for using winQTLcart2.5 software is tem. 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 OTL for kernel width in maize according to claim 1 in maize kernel width trait breeding is provided.