CN114959104A - SNP loci and corresponding KASP markers for assisting in selection of improved tobacco variety 'K326' leaf width traits - Google Patents

Abstract

The invention discloses a group of SNP loci for assisting in selecting and improving the leaf width of a tobacco variety 'K326', which is characterized in that: the SNP loci are 3 loci of tobacco 430K SNP chip probes numbered AX-117602251, AX-117602267 and AX-117598761, and the 3 SNP loci and DNA single-stranded sequences of 50nt at the upstream and the downstream are respectively shown as SEQ ID 1, SEQ ID2 and SEQ ID 3. The SNP locus corresponding to the tobacco leaf width character and the corresponding KASP detection primer provided by the invention can improve the selection efficiency in the oriented improvement of the K326 leaf width character and accelerate the new variety breeding process.

Description

SNP loci and corresponding KASP markers for assisting in selection of improved tobacco variety 'K326' leaf width traits

Technical Field

The invention belongs to the field of tobacco genetic breeding, and relates to a group of KASP molecular markers of SNP locus genotypes related to the width of tobacco leaves and application thereof.

Background

Common tobacco is one of important economic crops, mainly harvested leaves are taken as main crops, the current domestic tobacco planting area is about 1500 ten thousand mu, and the main popularized and cultivated varieties comprise Yunyan 87, K326, safflower Dajinyuan and the like. The K326 variety is introduced from the United states in 1985 and is the second largest main cultivated variety in the cultivated area in China at present, however, the K326 has the defect of insufficient width of upper leaves in production, so that the leaf opening is insufficient, and the yield and the quality of the upper leaves of the tobacco leaves are directly influenced. This problem is of wide interest to breeders and is expected to be solved. However, the width of the tobacco leaves is taken as a typical complex trait controlled by multiple genes and the environment, genetic analysis shows that the tobacco leaves can be controlled by 2-3 major genes plus multiple genes, the genetic rate of the major genes is about 60.61-90.17%, and the additive effect is mainly used among the genes. By utilizing a QTL positioning method, 12 QTLs related to leaf widths and 4 QTLs related to leaf widths are respectively positioned in genetic groups (Honghuadajinyuan/broadleaf hicks and Y3/K326) derived from two different flue-cured tobacco combinations by children army and the like and are mainly distributed in linkage groups No. 4, No. 14, No. 17 and No. 22 (children army, crop academy 2012; children army, northwest academy of plants 2018). The selection of plum madder (plum madder, southwest university, 2015) using safflower large gold dollars and SWU109 to construct F2 population was mapped to obtain 4 and leaf width-related QTLs, all distributed in linkage groups No. 1, No. 17 and No. 23. Some QTL related to leaf width are obtained in the initial research, but SSR markers are mainly selected in the positioning research, QTL regions obtained in the positioning research are linkage groups and are not directly anchored to physical sequences of tobacco genomes, and genetic improvement of research results on the leaf width characters of K326 varieties is limited due to the difference of genetic backgrounds among parents of the positioning groups.

The chromosome fragment substitution lines (CSSLs) only substitute partial fragments of the chromosome of the receptor parent into the chromosome fragment of the donor parent, so the genetic background is clear, the QTL positioning, the genetic effect analysis and the functional gene mining are accurate, the chromosome fragment substitution lines are often used as ideal materials for realizing the linkage of molecular marker technology and crop breeding, and are widely used in the research of mining important character associated sites of crops such as rice, corn, wheat, soybean, cotton and the like. However, so far, there are no reports on the utilization of tobacco CSSLs to dig mutation sites related to the tobacco leaf width trait, develop molecular markers, and utilize the same.

Disclosure of Invention

The technical problem to be solved by the invention is as follows: the method overcomes the defects of the existing research result aiming at the tobacco leaf width QTL positioning, utilizes the constructed tobacco chromosome fragment substitution line material, combines the drawn tobacco haplotype map and the tobacco 430K SNP chip genotyping means to screen and identify the SNP locus genotype related to the tobacco leaf width, develops the KASP marker which can facilitate the rapid and high-throughput genotyping, and is used for genetic improvement of the tobacco leaf width character.

The technical scheme of the invention is as follows: and a group of SNP sites for assisting in selecting the leaf width of the improved tobacco variety 'K326', wherein the SNP sites are 3 sites of the tobacco 430K SNP chip with the probe numbers of AX-117602251, AX-117602267 and AX-117598761.

Specifically, the 3 loci are all located on chromosome chry 09 (Jiangyitong, China tobacco science and newspaper, 2022) of safflower large gold reference genome (V1.2) sequenced by Chinese tobacco genome plan, the genotypes corresponding to the tobacco variety K326 and the narrow-leaf genome fragment substitution material are respectively "CC", "AA", "GG", and the genotype corresponding to the K326 chromosome substitution wide-leaf genome material is respectively "AA", "GG" and "CC".

The DNA single-stranded sequences of the 3 SNP loci and the upstream and downstream 50nt are respectively shown as SEQ ID 1, SEQ ID2 and SEQ ID 3.

AX-117602251：

TGGCATATAGCTCCAGATACTTCTTATGAAACTTCCGCCAATCAATCCCA[C/A]CATCAGCAATAGGCTCCCCAGATGGGACAAAGTCACGAAACCACTCGCGT

AX-117602267：

ATCAGTTGTCATCTATTAGAAGCTTGGTAACACTAGGGATTCGTGACGGC[A/G]TGCTCACTAATGCTGGGCTCATTATATTCAACCCTCCACCATCTATGACG

AX-117598761：

TGATATCAAGTCAGAGCATGCACCTAGGAGTCCTAGCTACAGCATCTCAT[G/C]CTGTTACAACCCAGACCTTGTTTGTTGTTTACTACAAGCCGAGGTTTGTC

The gene type of the AX-117602251, AX-117602267 and AX-117598761 mutation sites is selected to be AAGGCC.

Selecting the group of SNP loci for assisting in selecting the leaf width of the improved tobacco variety 'K326', converting 3 SNP loci into KASP markers, and providing corresponding KASP detection primers, wherein the primers are as follows:

KASP primer for detecting AX-117602251 site:

forward primer 1 FAM:

GAAGGTGACCAAGTTCATGCTACTTCCGCCAATCAATCCCAC；

forward primer 1 HEX:

GAAGGTCGGAGTCAACGGATTACTTCCGCCAATCAATCCCAA；

reverse primer 1: CGCGAGTGGTTTCGTGACTTTGTCCCATCT;

KASP primer for detecting AX-117602267 site:

forward primer 2 FAM:

GAAGGTGACCAAGTTCATGCTTAACACTAGGGATTCGTGACGGCA；

forward primer 2 HEX:

GAAGGTCGGAGTCAACGGATTTAACACTAGGGATTCGTGACGGCG；

and a reverse primer 2: GAGGGTTGAATATAATGAGCCC, respectively;

KASP primer for detecting AX-117598761 site:

forward primer 3 FAM:

GAAGGTGACCAAGTTCATGCTGGAGTCCTAGCTACAGCATCTCATG, respectively; forward primer 3 HEX:

GAAGGTCGGAGTCAACGGATTGGAGTCCTAGCTACAGCATCTCATC, respectively; reverse primer 3: GTAGTAAACAACAAACAAGGTC are provided.

The method for screening the group of SNP loci for assisting in selecting the leaf width of the improved tobacco variety 'K326' comprises the following steps:

(1) conventionally planting a constructed tobacco variety K326 chromosome segment replacement line material related to the tobacco leaf width character in a field, measuring the tobacco leaf width phenotype in a mature period, selecting 5-7 replacement line materials with the tobacco leaf width obviously larger than that of K326, selecting 5-7 replacement line materials with the tobacco leaf width unchanged or smaller than that of the K326 variety, sampling the selected wide leaf group and narrow leaf group materials and leaves, extracting DNA, and then carrying out genotyping by utilizing a customized 430K SNP chip developed by Zheng State tobacco institute;

(2) processing the genotyping results of the 430K SNP chips of the materials, firstly removing SNP sites without polymorphism and low-quality SNP sites, only keeping homozygous SNP sites with polymorphism, then sequentially comparing the genotypes of each material and each SNP site of a contrast material K326, converting according to the difference of the genotypes of each material and the contrast material K326, marking as 'XX' when the genotype is the same as that of the K326, and recording as 'YY' when the genotype is different from that of the K326;

(3) combining the genotype type and the phenotype of each SNP locus, if the genotype is XX, the phenotype is BLG and is marked as BLG-XX, 4 combination types are totally included, namely BLG-XX, BLG-YY, NLG-XX and NLG-YY, respectively, counting the number of materials contained in each combination type, and then performing significance test on the correlation between the genotype and the phenotype by using a Fisher exact test method;

(4) the SNP loci which are obviously related to the tobacco leaf width traits are obtained, the SNP loci which are positioned in the exon regions of the genes are focused on by combining tobacco reference genome annotation information, and the SNP loci which have no influence or little influence on other traits and have the genotype variation are screened out by combining bioinformatics means such as GO annotation and KEGG annotation, and the SNP loci and the corresponding genotypes are determined.

The invention has the beneficial effects that:

1. the invention provides a group of SNP loci for effectively improving the leaf width character of a tobacco variety K326 and corresponding genotypes. A material G3 is obtained by screening the chromosome fragment substitution line material constructed by screening, and field planting experiments show that the width of each leaf position of the plant line is obviously improved compared with that of K326, and other characters are not changed or are not changed obviously, so that the method has important significance for directionally improving the leaf width character of tobacco varieties.

2. The SNP locus corresponding to the tobacco leaf width character, the KASP marker corresponding to the SNP locus and the detection primer can improve the selection efficiency in the oriented improvement of the K326 leaf width character and accelerate the new variety breeding process.

Drawings

FIG. 1 shows leaf width phenotypes of respective individuals numbered G1 to G11 (A: comparison of leaf width traits at respective leaf positions of test materials; B: control K326 individual phenotype; C: G3 individual phenotype);

FIG. 2 shows SNP sites related to the leaf width trait of tobacco (■ SSR molecular markers related to the leaf width trait of tobacco have been reported;

SNP sites located in intron regions of genes;

SNP site located in exon region of gene)

FIG. 3 shows the phenotype of the entire plant (without G10) in the field maturation period of materials Nos. G1-G11.

FIG. 4 shows the genotype of 3 SNP sites (AX-117602251, AX-117602267, and AX-117598761) and the difference in leaf width at the upper part of the corresponding material (Hap-CCAAGG indicates that the genotypes of the 3 SNP sites are sequentially "CC", "AA", and "GG"; Hap-AAGGCC indicates that the genotypes of the 3 SNP sites are sequentially "AA", "GG", and "CC", and respectively correspond to the narrow leaf material and the wide leaf material);

FIG. 5 shows the result of the KASP primer availability test at SNP site AX-117598761.

Detailed Description

Example 1

(1) The method uses a tobacco variety K326 as a receptor parent and a variety Samsun as a donor parent, and generates BC through backcross for 4 times and selfing for 3 times ₄ F ₃ Seeds were subsequently grown at the flat dam base of the institute for tobacco science, Guizhou province. Screening to obtain 5 strains with significantly increased leaf width, 5 strains with reduced or unchanged leaf width compared with K326, and K326 to obtain 11 materials (numbers G1-G11, as shown in FIG. 1). Randomly selecting 5 plants of each material to measure the leaf width phenotype, simultaneously taking corresponding leaves, and mixing the samples to extract DNA. The Zhengzhou tobacco institute utilized a 430K SNP chip for genotyping.

(2) Utilizing a tobacco 430K SNP chip to perform gene typing on 11 module materials, obtaining 203429 effective SNP sites through preliminary data quality control, and removing SNP sites without genetic polymorphism and heterozygosity and 3123 remained SNP sites by combining a data-imputation method. For further analysis of these SNP sites, the genotype of the control material K326 at each SNP site was designated as "XX", and the letter X represents one of the four base types "A (adenine)", "T (thymine)", "G (guanine)", and "C (cytosine)" at the deoxynucleotide. Other materials the genotype SNP site that differs from K326 is designated "YY" and the letter Y indicates a certain base type that differs from the letter X.

Accordingly, 11 modular materials were sequentially combined at 3123 SNP sites in the order of G1 to G11, resulting in 23 combination types (Table 1). Combining with the results of field test on the leaf width traits of the modular materials, selecting and using a Fisher's exact test method, counting the association between the genotype combination of each SNP site and the leaf width traits, and displaying that the association between the 11 th, 18 th and 19 th genotype combinations and the leaf width traits of tobacco reaches a significant or extremely significant level (table 1), thereby obtaining a total of 167 SNP sites related to the leaf width traits, namely association SNPs.

TABLE 1 analysis of genotype combination types and association with leaf width traits for polymorphic SNP loci

*: significant levels were 0.05 x: the significance level was 0.01. P <0.05, P <0.01.

According to the physical position of the 430K SNP chip probe sequence of tobacco provided by Zhengzhou academy on the Honghuadajinyuan reference genome (Jianiangpeng, Nicotiana tabacum, 2022), 59 associated SNP loci are found to be distributed in 20 Scaffolds, the other 108 associated SNP loci are centrally located in 5 reference genome chromosomes such as Chr04, Chr07, Chr08, Chr09 and Chr18, and the number of the associated SNP loci corresponding to each chromosome is 7, 3, 1, 71 and 26. And (3) dividing 5 chromosomes according to the interval length of 100Kb, counting the number of related SNP loci contained in each interval, and screening the related SNP loci positioned in the exon or intron region of the gene according to the distribution of the genes of the intervals. The results showed that 19 of the 108 related SNP sites distributed in 5 chromosomes are located in 17 genes, and are concentrated on three chromosomes of Chr04, Chr09 and Chr18, and 11 of the sites are located in Exon (Exon) regions of the genes (FIG. 2). In addition, 5 of the related SNP sites distributed in the Scaffold region were found to be located in the exonic region of the gene. Accordingly, it was considered that the genes containing the SNP sites related to the 16 exon regions were important candidate related genes for the tobacco leaf width trait, and they were named as TWL-1 to TWL-16, respectively, based on the physical positions of the reference genome of Honghuadajinyuan (Table 2). Meanwhile, the research result is compared and analyzed with early tobacco leaf width related QTLs or molecular markers, and the related SNP sites in the chromosome Chr09 are mainly located between two published SSR molecular markers of the LG14 linkage group PT6039 and PT6047, while the related SNP sites on the chromosome Chr18 are overlapped with the published SSR molecular marker PT52809 on the LG1 linkage group.

TABLE 2 tobacco leaf Width trait candidate related genes

By comparing the field performance and the quality evaluation results of the materials such as G1-G11, the results show that 4 materials such as G6, G7, G8 and G11 have significant increase in the width of the tobacco leaf compared with the control K326, but the leaf length is shortened, the leaf thickness is thinned, the content is reduced, and the like (figure 3). Analysis shows that the gene types of candidate SNP loci on chromosomes of G3 materials, namely Chr04, Chr18 and the like are consistent with K326, and single nucleotide mutations occur in exon regions of genes, namely TWL-7, TWL-9 and the like, on Chr09, so that the leaf width traits of the G3 materials are improved, but the resistance and quality traits are equivalent, and the result also accords with the analysis results of various gene function annotations and metabolic pathways.

The genotype of SNP sites (AX-117602251, AX-117602267 and AX-117598761) of exon regions of TWL-7 to TWL-9 genes of G1 and G11 materials respectively is analyzed, and the result shows that the genotype of each NLG material is sequentially 'CCAAGG' at 3 SNP sites, while the genotype is changed into 'AAGGCC' in BLG materials (FIG. 4). According to the tobacco reference genome annotation information, the KASP molecular marker can be developed according to the upstream and downstream sequences of 3 SNP loci such as AX-117602251, AX-117602267 and AX-117598761 aiming at the problem of oriented improvement of the leaf width character of a flue-cured tobacco variety K326, so that the selection breeding is assisted, and the breeding efficiency is improved. Specifically, the method comprises the steps of carrying out continuous backcross by using K326 as a female parent and using a tobacco germplasm material with 3 SNP locus gene haplotypes such as AX-117602251, AX-117602267 and AX-117598761 as an "AAGGCC" as a male parent, carrying out genotype selection by combining SNP chips or developed KASP markers, and restoring the genotypes of SNP loci other than AX-117602251, AX-117602267 and AX-117598761 to be the same as K326, while the genotypes of 3 SNP loci such as AX-117602251, AX-117602267 and AX-117598761 are "AAGGCC".

Example 2: a method for genotyping tobacco germplasm resources (KASP marker for genotyping selection), comprising the steps of:

(1) 3 SNP sites such as AX-117602251, AX-117602267, AX-117598761 and the like and DNA single-stranded sequences of 50nt at upstream and downstream are respectively shown as SEQ ID 1, SEQ ID2 and SEQ ID 3, and a KASP primer is designed by combining a tobacco safflower large gold reference genome (V1.2) and entrusted to Shanghai biological engineering technology service company Limited to synthesize.

The KASP detection of the 3 SNP sites adopts the following primer sequences:

detecting an AX-117602251 site KASP primer:

forward primer 1 FAM:

GAAGGTGACCAAGTTCATGCTACTTCCGCCAATCAATCCCAC；

forward primer 1 HEX:

GAAGGTCGGAGTCAACGGATTACTTCCGCCAATCAATCCCAA；

reverse primer 1: CGCGAGTGGTTTCGTGACTTTGTCCCATCT, respectively;

KASP primer for detecting AX-117602267 site:

forward primer 2 FAM:

GAAGGTGACCAAGTTCATGCTTAACACTAGGGATTCGTGACGGCA；

forward primer 2 HEX:

GAAGGTCGGAGTCAACGGATTTAACACTAGGGATTCGTGACGGCG；

reverse primer 2: GAGGGTTGAATATAATGAGCCC, respectively;

KASP primer for detecting AX-117598761 site:

forward primer 3 FAM:

GAAGGTGACCAAGTTCATGCTGGAGTCCTAGCTACAGCATCTCATG；

forward primer 3 HEX:

GAAGGTCGGAGTCAACGGATTGGAGTCCTAGCTACAGCATCTCATC；

reverse primer 3: GTAGTAAACAACAAACAAGGTC are provided.

(2) Selecting G1-G11 materials, selecting 22 materials of agave tongue tobacco, Anshun small hanging branches, earflap rotary cutter small willow leaves, Ludian tobacco caps, Zhenfeng willow leaf tobacco, Zhenfeng small willow leaves, Muslim willow leaves, Virginia indica, Rijiahe, dense leaf No. 1, 68-39 and the like in a germplasm resource bank of the Guizhou province tobacco science research institute, accelerating germination and extracting DNA.

(3) The availability of the KASP primers for the above 3 SNP sites was verified using the KASP technology platform (LGC Co., UK). Specific PCR reaction systems and experimental procedures were referenced to technical documentation provided by LGC corporation. The KASP fluorescence detector is in the model of FLUOstar Omega SNP. Genotyping results were analyzed using KlusterCaller software. The results show that the designed KASP primers of 3 SNP sites are all available, and the genotyping results of materials such as G1-G11 are consistent with the results of 430K SNP chip of Zhengzhou hospital, wherein the KASP experimental verification result of SNP marker AX-117598761 is shown in figure 5.

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Claims (4)

1. A group of SNP loci for assisting in selecting leaf width of improved tobacco variety 'K326', which is characterized in that: the SNP sites are 3 sites of tobacco 430K SNP chip probes with the serial numbers of AX-117602251, AX-117602267 and AX-117598761, and the DNA sequences of the 3 SNP sites and the DNA single-stranded sequences of 50nt at the upstream and the downstream are respectively shown as SEQ ID 1, SEQ ID2 and SEQ ID 3.

2. The panel of SNP sites assisting in the selection of improved tobacco variety 'K326' leaf width according to claim 1, wherein: the genotype of the AX-117602251, AX-117602267 and AX-117598761 loci is selected as AAGGCC.

3. The method for selecting the set of SNP sites for aiding selection of leaf width of improved tobacco variety 'K326' according to claim 1, wherein: for selection of 3 SNP sites AX-117602251, AX-117602267 and AX-117598761 for conversion to KASP markers, corresponding KASP detection primers were provided, said primers were as follows:

KASP primer for detecting AX-117602251 site:

forward primer 1 FAM:

GAAGGTGACCAAGTTCATGCTACTTCCGCCAATCAATCCCAC；

forward primer 1 HEX:

GAAGGTCGGAGTCAACGGATTACTTCCGCCAATCAATCCCAA；

reverse primer 1: CGCGAGTGGTTTCGTGACTTTGTCCCATCT, respectively;

KASP primer for detecting AX-117602267 site:

forward primer 2 FAM:

GAAGGTGACCAAGTTCATGCTTAACACTAGGGATTCGTGACGGCA；

forward primer 2 HEX:

GAAGGTCGGAGTCAACGGATTTAACACTAGGGATTCGTGACGGCG；

reverse primer 2: GAGGGTTGAATATAATGAGCCC, respectively;

KASP primer for detecting AX-117598761 site:

forward primer 3 FAM:

GAAGGTGACCAAGTTCATGCTGGAGTCCTAGCTACAGCATCTCATG；

forward primer 3 HEX:

GAAGGTCGGAGTCAACGGATTGGAGTCCTAGCTACAGCATCTCATC；

and a reverse primer 3: GTAGTAAACAACAAACAAGGTC are provided.

4. The method for screening a set of SNP sites for aiding in the selection of leaf width of improved tobacco variety 'K326' according to claim 1, wherein: comprises the following steps:

(1) conventionally planting a constructed tobacco variety K326 chromosome segment replacement line material related to the tobacco leaf width character in a field, measuring the tobacco leaf width phenotype in a mature period, selecting 5-7 replacement line materials with the tobacco leaf width obviously larger than that of K326, selecting 5-7 replacement line materials with the tobacco leaf width unchanged or smaller than that of the K326 variety, sampling the selected wide leaf group and narrow leaf group materials and leaves, extracting DNA, and then carrying out genotyping by utilizing a customized 430K SNP chip developed by Zheng State tobacco institute;

(2) processing the genotyping results of the 430K SNP chips of the materials, firstly removing SNP sites without polymorphism and low-quality SNP sites, only keeping homozygous SNP sites with polymorphism, then sequentially comparing the genotypes of each material and each SNP site of a contrast material K326, converting according to the difference of the genotypes of each material and the contrast material K326, marking as 'XX' when the genotype is the same as that of the K326, and recording as 'YY' when the genotype is different from that of the K326;

(3) combining the genotype type and the phenotype of each SNP locus, if the genotype is XX, the phenotype is BLG and is marked as BLG-XX, 4 combination types are totally included, namely BLG-XX, BLG-YY, NLG-XX and NLG-YY, respectively, counting the number of materials contained in each combination type, and then performing significance test on the correlation between the genotype and the phenotype by using a Fisher exact test method;

(4) the SNP loci which are obviously related to the tobacco leaf width traits are obtained, the SNP loci which are positioned in the exon regions of the genes are focused on by combining tobacco reference genome annotation information, and the SNP loci which have no influence or little influence on other traits and have the genotype variation are screened out by combining bioinformatics means such as GO annotation and KEGG annotation, and the SNP loci and the corresponding genotypes are determined.

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