CN114807192A

CN114807192A - BSR sequencing-based identification of tomato irregular fruit cracking key gene SlGH9-15 and application

Info

Publication number: CN114807192A
Application number: CN202210449783.6A
Authority: CN
Inventors: 蒋芳玲; 林昊维; 吴震; 周蓉; 吴文丽; 仲钊江; 杨帆
Original assignee: Nanjing Agricultural University
Current assignee: Nanjing Agricultural University
Priority date: 2022-04-26
Filing date: 2022-04-26
Publication date: 2022-07-29
Anticipated expiration: 2042-04-26
Also published as: CN114807192B

Abstract

The invention discloses a key gene for regulating and controlling irregular tomato fruit cracking and an identification method and application thereof. The invention obtains the key gene for regulating and controlling irregular tomato fruit cracking through BSR sequencing and first identificationSlGH9‑15(Solyc09g010210)The gene is involved in the cellulose synthesis process, plays an important role in the biosynthesis and remodeling of cell walls, and is a downstream gene for regulating and controlling dehiscence fruit. The research combines high-throughput sequencing and molecular biology technology, breaks through fruit crackingThe bottleneck of prevention and control provides valuable gene resources for genetic improvement of the tomato dehiscence-resistant character, and promotes the application of basic research results, so that the method has important scientific significance and application prospect.

Description

BSR sequencing-based identification of tomato irregular fruit cracking key gene SlGH9-15 and application

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a key regulatory gene for irregular tomato fruit cracking, and an identification method and application thereof.

Background

The tomato fruits have serious fruit cracking problems in the growth, development and post-harvest fresh-keeping circulation processes, which affect the appearance quality, shorten the shelf life, even infect mildew and hinder the production and sale of the fruits. The fruit cracking can be relieved to a certain extent by adopting measures such as balanced water and fertilizer in the production, but the problem is difficult to solve fundamentally (Richard, 1994; Dorais et al, 2004).

Tomato dehiscence is mainly divided into ring dehiscence, longitudinal dehiscence, irregular dehiscence, cuticle dehiscence, mixed dehiscence, etc. (Khadivi-Khub, 2015). The mechanism of dehiscence is relatively slow to study. Reynard (1951) believes that tomato fruit dehiscence is controlled by 2 recessive genes. Hudson (1956) believes that fruit longitudinal and ring dehiscence are inherited relatively independently, but that there may be modifier genes associated with each other. Lihujia (2016) detected 5 QTLs associated with tomato cracking resistance index located on

chromosomes

1, 2 and 3, respectively, from the crack resistant processing tomato '14803'. Capel et al (2017) performed multi-environmental QTL analysis by constructing tomato RIL populations and found that the major QTL (Ck3) located on chromosome 3 was associated with dehiscence, including expansin and bi-component signal transduction pathway homologous protein genes. Cui et al (2017) found that ER4.1 gene regulates tomato net dehiscence through fine localization. Jiang et al (2019) found that simultaneous inhibition of cell wall degradation related genes PG and EXP1 alleviated tomato fruit cracking. Zhuyu et al (2020) located dehiscence-resistant gene Cr3a within about 349kb, and included genes involved in carbohydrate synthesis and hormone signal transduction. And et al (2021) found that MAPKKK gene on chromosome 2 affects tomato irregular fruit cracking by BSA sequencing and QTL mapping. The tomato fruit cracking character belongs to quantitative character, and is commonly controlled by a plurality of genes, so the influence of environmental conditions is large. Different researchers have different results of tomato crack resistance inheritance due to different selected materials. Therefore, the current understanding of the mechanisms of crazing resistance is still unclear.

Fruit cracking is caused by the imbalance of the growth of the peel and the pulp tissue, and is the reaction of fruits to the incoordination of the inside and outside growth under the adverse conditions. In the long evolution process, plants form a set of mechanisms for responding to adversity stress, including three stages of sensing and transmitting adversity signals, and recognizing and transducing adversity signals by corresponding receptors, and the process is regulated and controlled by a complex signal pathway (Liu et al, 2014). Is there a similar signal pathway or regulatory network in the dehiscence fruit? Xue et al (2020) found that hormone-cell wall-reactive oxygen co-regulates tomato cuticle cleavage, mainly including auxin and ethylene signaling pathways, hydrogen peroxide pathway and cell wall polysaccharide metabolic pathway, by combination of lncRNA and mRNA transcriptome analysis. Chen et al (2019) revealed, by transcriptome analysis, that starch metabolism and cell wall polysaccharide metabolic pathways regulate ripening and dehiscence of jujube fruits. Michail et al (2021) found that water-induced cherry dehiscence was associated with processes such as cell wall pectin metabolism, abscisic acid and ethylene signaling pathways, defense responses, etc. by combined metabolome and transcriptome analysis. Previous studies have also found that genes PG and EXP1 associated with inhibition of cell wall degradation inhibit tomato dehiscence by affecting the pericarp cell wall and the thickness of the waxy layer (Jiang et al, 2019). Whether other genes jointly form a gene network to regulate the tomato fruit cracking character still needs to be further researched.

Disclosure of Invention

The purpose of the invention is as follows: the cellulase gene SlGH9-15(Solyc09g010210) identified by the invention is related to the biosynthesis and remodeling process of cellulose which is the main component of cell walls. Whereas the pericarp cell wall composition and biochemical modification affect the occurrence of dehiscence by changing the hardness of the fruit and the mechanical properties of the pericarp (e.g., elasticity, plasticity, toughness) (Balbont i n et al, 2013; Considine and Brown, 1981). And the promoter region of the gene contains a plurality of response elements related to plant hormones (abscisic acid, ethylene and auxin) and abiotic stress (drought and anaerobism). It shows that in the late mature period of fruit, the gene is induced by some plant hormone and abiotic stress to regulate fruit cracking by affecting the change of cellulose content and structure of cell wall. The invention utilizes modern high-throughput sequencing and molecular technology, breaks through the bottleneck of fruit cracking prevention and control, provides valuable gene resources for genetic improvement of tomato fruit cracking resistance traits, and promotes basic research achievements to be applied, thereby having important scientific significance and application prospect.

The technical scheme is as follows: in order to solve the technical problem, the invention provides a key gene for regulating and controlling irregular tomato fruit cracking, and the gene sequence is shown as SEQ ID NO. 1.

The invention relates to a method for identifying a key regulatory gene for irregular fruit cracking of tomatoes, which comprises the following steps:

1)F ₂ constructing generation segregation population: hybridizing tomato dehiscent fruit variety as female parent and easily dehisced fruit variety as male parent to obtain F ₁ Preparing F through selfing ₂ Separating the population;

2) pool-mixing and re-sequencing extreme characters: from F ₂ Respectively selecting extremely-resistant single fruit plants and extremely-resistant single fruit plants from the generation segregation population to form a fruit cracking resistant pool and an easily-cracked fruit pool, mixing leaf RNA of the single fruit plants in each pool in equal amount to obtain RNA of two parent mixing pools and RNA of two extreme character filial generation mixing pools respectively, constructing a library and performing high-throughput sequencing; and (3) performing the regression fitting of the loess on the delta SNP-index and the delta InDel-index on each chromosome by adopting a loess regression fitting method to obtain an associated region related to the irregular fruit cracking character of the tomato. Meanwhile, RNA sequencing provides the expression level of transcripts among different groups (a crack-resistant variety and a crack-prone variety, a crack-resistant mixed pool and a crack-prone mixed pool and the like);

3) analysis of gene expression level: according to the positioning results of SNP and InDel correlation regions, the differential expression genes of a cracking resistant pool, a cracking easy pool, a cracking resistant mixed pool and a cracking easy mixed pool are combined, crossed genes in the two groups of sequencing pools are analyzed, and 4 genes with SNP are screened out in total, namely Solyc09g010060 (kinesin-like protein), Solyc09g010080 (beta fructofuranosidase), Solyc09g010210 (beta-1, 4-endoglucanase) and Solyc09g010230(B3 protein). And the parental 'NT 91' and 'NT 189' were used for qRT-PCR analysis by saturation irrigation of 30h red ripe fruits. As a result, 1 gene with a significant difference in expression level is screened, and is a cellulase gene SlGH9-15(Solyc09g010210), which is considered as a key gene for regulating irregular tomato fruit cracking.

4) Gene family analysis and physiological analysis: carrying out whole genome identification and biogenesis analysis on a gene family GH9 in which the screened dehiscent fruit character candidate gene SlGH9-15 is positioned, wherein the whole genome identification and biogenesis analysis mainly comprises promoter cis-acting element analysis and expression pattern analysis; meanwhile, the cellulase activity and the cellulose content of the parent fruits are measured and analyzed in the green ripening stage, the color conversion stage and the red ripening stage.

Wherein, the female parent in the step 1) is tomato dehiscence-resistant germplasm 'NT 91' and the male parent dehiscence-susceptible germplasm 'NT 189'.

Wherein the related region in the step 2) is a region of 2.88 to 3.94Mb of chromosome 9 or a region of 53.02 to 53.04Mb of chromosome 11.

The invention also comprises the application of the key gene for regulating and controlling irregular fruit cracking of the tomato in the directional identification or cultivation of tomato varieties with fruit cracking resistance.

The invention also discloses a method for cultivating the tomato with crack resistance, and a method for over-expressing or knocking out the gene in the tomato.

Wherein the method comprises editing or mutagenesis of the gene.

Has the beneficial effects that: compared with the prior art, the invention has the following advantages and effects. The invention firstly identifies and obtains the key gene Solyc09g010210 for regulating irregular fruit cracking of tomatoes, the gene is involved in the synthesis process of cellulose, plays an important role in cell wall biosynthesis and remodeling and is the key gene for regulating fruit cracking. The research utilizes modern molecular design breeding means, breaks through the bottleneck of fruit cracking prevention and control, provides valuable gene resources for genetic improvement of tomato fruit cracking resistance characters, and promotes basic research achievements to be applied, so that the research has important scientific significance and application prospect.

Drawings

FIG. 1: f ₂ A population dehiscence character frequency distribution histogram;

FIG. 2: the distribution map of the Δ SNP-index and Δ InDel-index on the whole genome;

FIG. 3: annotation of classification statistical chart of BSR associated gene GO;

FIG. 4: annotation of classification map by BSR associated gene COG;

FIG. 5: a BSR associated gene KEGG pathway enrichment scatter diagram;

FIG. 6: analyzing the expression quantity of the differentially expressed genes in the related region;

FIG. 7: analyzing a cis-acting element of a SlGH9 promoter;

FIG. 8: analysis of SlGH9 expression pattern, wherein: a is a heat map of expression quantity of SlGH9s gene in BSR-Seq; b is a block diagram of expression quantity of SlGH9s genes in different fruit development stages in tomato cultivation 'M82'; c is a line graph of expression quantity of SlGH9s genes in different fruit development periods in the cultivated tomato MicroTom;

FIG. 9: the difference bar chart of the cellulase activity (A) and cellulose content (B) of the parent and the female parent under different fruit development stages.

Detailed Description

Embodiments of the present invention will be described in detail below with reference to examples, but those skilled in the art will appreciate that the following examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention. The examples, in which specific conditions are not specified, were carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are conventional products which are commercially available, and are not indicated by manufacturers.

Example 1:

1. materials and methods

1.1 test materials and F ₂ Construction of the population

(1) At the early stage, a Tomato irregular dehiscent variety 'NT 189' (LA2661, from Tomato germplasm Center Tomato Genetics Resource Center) and a dehiscent variety 'NT 91' (Tomato Cherry Chocalate, from Davis university, Calif.) were screened out by investigating the dehiscence rate of more than 500 Tomato germplasm resources, and the average dehiscence rate in the red stage was 91.02% and 9.25%, respectively. The two materials are selfed and screened for more than 6 generations, and the fruit cracking property is stable.

(2) Hybridizing by using tomato dehiscent fruit germplasm 'NT 91' as female parent and easy dehiscent fruit germplasm 'NT 189' as male parent to obtain F ₁ Preparation of F by inbreeding ₂ And (4) separating the populations.

(3) Wait for F ₂ When the group seedlings grow to five leaves and one heart, fresh leaves are taken and frozen in liquid nitrogen for RNA extraction.

(4) Wait for F ₂ And (4) performing a fruit cracking phenotype investigation when the fruit of the 3 rd ear of the population is red and ripe, counting the fruit cracking number of a single plant and the total number of fruits, calculating the fruit cracking rate, and analyzing by using SPSS staticistics 25.

To F ₂ Frequency statistics and a single sample K-S (Kolmogorov-Smirnov) test were carried out on the fruit cracking rate of 350 individual plants of the population. As shown in FIG. 1, F ₂ The average value of the fruit cracking rate of the population is 44.76 percent, and the standard deviation is 21.73 percent. The single sample K-S test shows that the progressive significance (double tail) of the tomato is 0.200 and is greater than the significance level by 0.05, the sample is considered to be subjected to normal distribution by accepting the original hypothesis, and the result shows that the tomato irregular fruit cracking character accords with the characteristic of quantitative character.

2. Pool-mixing heavy sequencing for extreme character (BSR-Seq)

The BSR-Seq can efficiently, quickly and inexpensively locate the candidate interval of the target gene and provide the expression data information of the gene by constructing a mixed population. From 350 strains F in this experiment ₂ Selecting 22 extremely-crack-resistant fruit single plants from the group to form a crack-resistant fruit pool, selecting 22 extremely-crack-resistant fruit single plants to form an easily-cracked fruit pool, mixing leaf RNA of the single plants in each pool in equal amount to form two gene pools with extreme characters, sending RNA samples of parents and the mixed pools to Nanjing Chihui-Chiyowa Biotechnology Limited, mechanically breaking and constructing a library, and sequencing by using an Illumina HiSeq platform. According to the positioning result of Clean Reads in a reference genome SL4.0, Picard is used for carrying out pre-treatment such as deduplication, GATK is used for carrying out local duplication comparison, base quality value correction and the like so as to ensure the accuracy of the SNP obtained by detection, the GATK is used for carrying out SNP detection, and a final SNP site set is obtained through filtration. For in the same chromosomeAnd (3) performing Loess regression fitting on the delta (SNP-index) above, and selecting a region above the 99% quantile threshold as a related region related to the dehiscent fruit trait.

The BSR-Seq analysis result of the invention: the reference genome for sequencing is SL4.0, 57.19Gb data volume is obtained altogether, and Q30 reaches more than 92.71%. The comparison efficiency of the sample and the reference genome is more than 93.65%, and the genome coverage is 14.78-20.35X.

116,160 SNP sites and 41,591 Small InDel sites were detected together between each sample. The standard-meeting SNP and Small InDel locus obtained by screening among the mixed pools are 10533 and 1059 respectively, cover 12 chromosomes and are dense on chromosome 9 and chromosome 11.

The SNP _ index is mainly used for searching for a significant difference of genotype frequencies among the mixed pools and is counted by the SNP _ index of a recessive base in the recessive mixed pool. The stronger the association between the marker SNP and the trait, the closer the SNP _ index is to 1.

The calculation method is as follows (briefly):

Δ (SNP _ index) ((Mindex (recessive character)) -Windex (dominant character))

Wherein:

windex ═ frequency of recessive genotypes in the dominant mixed pool/(frequency of recessive genotypes in the dominant mixed pool + frequency of dominant genotypes in the dominant mixed pool).

Mindex is the recessive genotype frequency in the recessive mixed pool/(recessive genotype frequency in the recessive mixed pool + dominant genotype frequency in the recessive mixed pool).

The SNP marker [ Delta ] (SNP _ index) on the same chromosome was subjected to regression fitting of loess, and a region not less than the threshold of 99% quantile was selected as a region related to the dehiscence fruit trait.

Distribution of the Δ (SNP _ index) values among pools across the whole genome was analyzed using the loess regression fitting method (fig. 2). Regions above the 99% threshold line (red) were selected as candidate regions associated with the irregular dehiscence trait (table 1). 1SNP site controlling the dehiscence trait was detected in the region of 2.88 to 3.94Mb (SL4.0ch09: 2878842 to 3938673bp) of chromosome 9, which was designated qCR9, and the region was 1.06Mb in size and contained 122 genes. Similarly, 1 Small InDel locus controlling the dehiscence trait was detected in the region of 3.43 to 3.52Mb (SL4.0ch09: 3431664 to 3517695bp) of chromosome 9, which was designated as qCR9.1, and had a size of 0.09Mb and contained 17 genes. 1 Small InDel locus controlling the fruit cracking trait was detected in the 53.02-53.04 Mb (SL4.0ch05: 53015753-53037210 bp) region of chromosome 11, which was designated as qCR11, and the region was 0.02Mb in size and contained 5 genes.

TABLE 1 statistics of SNP and Small InDel associated regions

The genes in the 2 relevant regions are compared with functional databases such as NR, SwissProt, GO, COG, KEGG, Pfam and the like through BLAST to obtain the annotated classification statistical results of the genes (Table 2). GO analysis was performed on 127 genes within the SNP and Small InDel association regions (fig. 3). The differential gene is found to participate in the biological processes of nitric oxide mediated signal transduction, iron ion homeostasis, positive regulation of fatty acid biosynthesis, cellulose microfibril construction and the like. COG analysis (figure 4) indicates that candidate genes are mainly involved in post-transcriptional modification, sugar transport and metabolism, secondary metabolite biosynthetic transport and metabolism, amino acid transport and metabolism, cell wall biogenesis, inorganic ion transport and metabolism, and intracellular transport and secretion. KEGG pathway analysis (fig. 5) found that candidate genes were significantly enriched in pathways such as starch and sucrose metabolism, galactose metabolism, biosynthesis of cutin wax and suberin, glycan biosynthesis, and pantothenic acid and coa biosynthesis. The results indicate that irregular dehiscence of tomato may be associated with cell wall construction, cuticle synthesis, certain biochemical processes (sugar, acid anabolism) and signal transduction pathways.

TABLE 2 BSR-associated Gene Annotation statistics

3. Analysis of gene expression level: trizol reagent (Betack Biotech) was usedTechnical limited, beijing, china) and the quality and concentration of RNA was determined using a Q6000 nucleic acid concentration detector (Quawell, usa). RNA was reverse transcribed into cDNA using a 5 × All-In-One RT Master Mix reverse transcription kit (Abm, Canada). By using

The qPCR Master Mix kit (Toroivd, uk) was used to perform real-time fluorescent quantitative experiments (qPCR) on a quant 3 real-time fluorescent quantitative PCR instrument (Applied Biosystems, usa). The total qPCR reaction system was 20. mu.L, consisting of 10. mu.L of 1 × SYBR Green qPCR Master Mix, 2. mu.L of cDNA (diluted 10-fold), 0.8. mu.L of each of the upstream and downstream primers, 6.4. mu.L of ddH 2O. The qPCR reaction procedure was: pre-denaturation at 95 ℃ for 1min, denaturation at 95 ℃ for 10s, annealing at 60 ℃ for 30s, and 40 cycles. The dissolution curve program was: 95 ℃ for 15s, 60 ℃ for 1min and 95 ℃ for 1 s.

Using Actin as an internal reference, the primer sequences were all from the qPrimer DB database (Lu et al, 2018), and the primers were synthesized by south beijing smith gold biotechnology limited (south beijing, china). By using 2 ^-ΔΔCt Method (Livak)&

Schmitgen, 2001) calculated the relative expression. And (5) carrying out t test on the expression quantity result by using SPSS staticiscs 25, and counting the difference significance of the expression quantity of each gene between parents.

According to the positioning results of SNP and InDel correlation regions (Table 3), the genes with different expressions of the cracking resistant pool, the cracking easy pool, the cracking resistant mixed pool and the cracking easy mixed pool are combined, the genes which are crossed in the two groups of sequencing pools are analyzed, and 4 genes with SNP are screened out in total, namely Solyc09g010060 (kinesin-like protein), Solyc09g010080 (beta-fructofuranosidase), Solyc09g010210 (beta-1, 4-endoglucanase) and Solyc09g010230(B3 protein). And the parental 'NT 91' and 'NT 189' were used for qRT-PCR analysis by saturation irrigation of 30h red ripe fruits. As a result, 1 gene with a significant difference in expression level is screened, and is a cellulase gene SlGH9-15(Solyc09g010210), which is presumed to be a key gene for regulating irregular tomato fruit cracking.

TABLE 3 partial genes in candidate regions of relevance

4. Gene family analysis and physiological analysis: and carrying out whole genome identification and biogenesis analysis on the GH9 gene family in which the selected dehiscent fruit trait candidate gene SlGH9-15 is located. Promoter cis-acting elements and expression patterns of the tomato GH9 gene family were analyzed (fig. 7, 8). The SlGH9-15 promoter is found to have plant hormone (auxin, ethylene, abscisic acid, methyl jasmonate and salicylic acid) response related elements, anaerobic induction related elements, drought induction related elements and flavonoid biosynthesis gene regulation related elements, and the SlGH9-15 gene can play an important role in certain stress and hormone response processes by participating in a specific regulation path, so that fruit cracking is influenced. Expression pattern analysis shows that the expression level of SlGH9-15 is obviously increased in the fruit color transition stage to the red ripe stage, and the expression level in the easy-to-crack variety is obviously higher than that in the crack-resistant variety. It is shown that at the late fruit ripening stage, SlGH9-15 is highly expressed in split tomato, causing fruit dehiscence. Meanwhile, cellulase activity and cellulose content in the fruits of the male parent 'NT 189' and the female parent 'NT 91' were measured in different development stages of the fruits. It was found that cellulase activity increased and cellulose content decreased as the fruit matured. In three fruit development periods, the cellulase activity of the crack-resistant tomatoes is lower than that of the crack-prone tomatoes, the cellulose content of the crack-resistant tomatoes is higher than that of the crack-prone tomatoes, and the difference is obvious. The cellulase activity of the irregular cracked tomato is 213, 433 and 911 mu mol.h in green ripening period, color conversion period and red ripening period respectively ^-1 ·g ^-1 Which are respectively 1.58 times, 1.17 times and 2.14 times of the crack-resistant tomatoes. And the activity of the easy-cracking tomato cellulase is increased to 2 times of the color conversion period in the red-maturing period. Regarding the cellulose content, the crack resistant tomatoes have the green stage, color conversion stage and red stage of 45, 36 and 31 mg-g respectively ^-1 Is 1.41 times, 1.57 times and 2.07 times of the cracked tomato respectively. The cellulose content of the cracked tomato is rapidly reduced from the color conversion period to the red ripening period, and the cellulose degradation of the cracked tomato mainly occurs before the color conversion period. The obvious difference of the cellulase activity and the cellulose content between the tomato easy to crack and the tomato resistant to crack shows that the higher cellulase activity in the tomato easy to crack can enhance the degradation capability and reduce the cellulose content, thereby accelerating the disintegration of fruit cell walls and finally promoting the fruit cracking.

Therefore, the invention identifies a tomato irregular fruit cracking related gene (Solyc09g010210, GH9-15, endo-1,4-beta-glucanase) which belongs to GH9 gene family and is involved in plant cell wall biosynthesis and construction and response of hormone, biological and abiotic stress.

Sequence listing

<110> Nanjing university of agriculture

<120> identification of tomato irregular fruit cracking key gene SlGH9-15 based on BSR sequencing and application

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 5296

<212> DNA

<213> tomato irregular fruit cracking Key gene Solyc09g010210(Lycopersicon esculentum)

<400> 1

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atacctccat cattttcctc ttccttctct tcaactcctt ttcatgttca ttcggagggg 120

gtcatgatta tcatgacgcc ctccgaaaaa gcatcctgtt ctacgaagga caacgatccg 180

gaaaattacc gccggatcaa cgtatcaaat ggcgtagaga ctccgcatta cacgacggtg 240

cttccgccgg agtaagtagt ataataacaa tcaaaataca tccgtaattt tttgattttc 300

caaacatttt acctggatcg aatttttttt ttcgttacag gttgatttga caggaggcta 360

ttacgatgcc ggagataatg tgaaatttgt ttttccgatg gcgtttacga cgacattgtt 420

atcgtggagt ataattgatt ttaaaaggaa tatagggaat gaattgggta atgcagtgaa 480

ggcggtgaaa tggggaactg attttctgtt gaaagctacg gcgagagatg gagtgatata 540

tgtacaagtt ggtgatgcgt tttcagatca cagttgttgg gagagaccag aagatatgga 600

tacattaaga actgtttata aaattgatgc gaataatccc ggttccgatg tcgccggtga 660

aatcgctgct gcattagctg ctgcatccat tgttttccgt tcactggatt cttcctactc 720

aaatctactg cttgatcgcg ctgttaaagt aaggaaaaaa tatattttat gcttttaata 780

attgactttg ctttgtgaat aaacgaataa taagtaacat gtttttggtg cattggtttt 840

tctttttttt cttttacatt ttcgaataat tatgtattaa aataatttag aaaatccagg 900

attggtttga ttcactctgt aaagtctaaa aagtaaaatg ctttgggcag tgttgtgtta 960

gtgtcttttt ttctctgctg gttgaactgg cattaaataa gtgttgttga ctgcatttgc 1020

ttattagtta tacatatata tattaatatt gtatataaaa gaaattattt tttctaatat 1080

agcctacgaa acgatccgtt aagttactgt ctagtatgga catataaagt ttgttttgca 1140

tgtgaacaag gacttgttgg gtggggtggg gtgggtgggt gggattatta actctaaaca 1200

aaatcttgct attagaatca tgaagattga tttacacatg catcttaatc tatatatgtg 1260

aacatttaaa tttgatgaac atatttatgt atagtcacaa aaataggttt gtctaaccaa 1320

ttatgtaaag ttctaaaatg tcactgtcat ctagcagaaa aaagctaaaa ataaaaaaaa 1380

agaatatgac cgttgtaata aaatcagtct tttggttgct tttgtaggtt ttcgattttg 1440

ccaatagaca tagaggtgca tacagctcca gcctacactc tgctgtttgc cctttctatt 1500

gtgactttaa tggttatcag gtaaactctt gtataatata taaacgtgtc cttaatctag 1560

tcttaactta tatttatatt ctcctacata tgtattcttt aattttgtat atgtataaat 1620

agacacttaa agttgaataa gtagatacac aagtcttgcg tgacatgata tatgtatgag 1680

acatgcgcat atctcgcata atagataaat atgtccttaa tatgatctta gcttgtatct 1740

acaccctcct atatatgtat ccttaaattt tgtatatgta atagtagtca cttaaagttg 1800

aataagtaga acacaggtcc cgcatgacat gatatatgta ggagctatgt gtttatctag 1860

cataacacat tactgtgttc ttaaactggt atatccttca atactgcgca cacgaaagtt 1920

gacacttaaa tgtgtgtaaa gtagaataag tagaaacaca cacacatata gggcacattt 1980

gtttattttg cataatacat aaacgtgttc ttaaattggt gttagctgat atctatatcc 2040

tttaatttta cgcccacata agtcgacact taaatatgta taaaattaag aaagtaagca 2100

cacaacttac atgacgtaat atatatatga cgcacgtgtc tacttattcc gagtcaaaat 2160

tagtaatgtt tgacataaaa tttgggtgag aatgtacagg atgaattgct ttggggtgca 2220

gcatggttac ataaagcaac aagaagaagg caatatagag agtacatagt gaaaaatgaa 2280

gtaattttaa gagcaggaga tacaattaat gaatttggtt gggacaacaa acatgctggt 2340

attaatgtcc ttatttccaa ggttagtaat ttaattaatt aatttatcta tggataagga 2400

gacattatta gtacttaatt aattgtatta cttttttttt ctcattacat aataatctgt 2460

ccttgtctca tttacaactt agatatattc ttgtgatatc tattcttgac ttgttcagtc 2520

tgccaatcac atgctgtaga tagatagata tagttatcca tatcagaatg tccatttagt 2580

gggcccactc ttaatttgaa aattaacgta aaactgcttc atcttaaaat attaatcaaa 2640

tgattcactg aataaatatc gaaaggaaaa agagtaggtt caatcataac aagtaactaa 2700

gcacattgat attttttccc tcttaaaaca aatttcaaaa ctctaatcat cattaatttt 2760

gtgtatgtgt gtaattattc cacatgacag agttttggtt attatgggct ccatgtgtgt 2820

ttggtgttca aacttaactt atttgcatca acatacacat acataaaatt aaaattataa 2880

aatgaaaaaa agaaggtgaa agctgtatat agaagtgctc catgtgaagt gtgtggacaa 2940

aaaatgatac aaaattaaaa tgagacaaaa aaaaagtgag gttggtaata gtccttcatg 3000

aagtaataaa gagcaagaga agtgacttta ctagtggtaa gcatgtgcaa aaagacagtt 3060

gcttatctat atatatatat acatactttc atacggagaa agttagagtt atgatacaag 3120

tggacctctc gattgaagtg ttgattatga attatcaatt atcgttcagg tttaatagtg 3180

gagtcagaat ttttttacaa aaaagttcta tattatataa gaaataattt gaatcatgta 3240

taaatagtat aagttggctc tgacctgtct tcgtcgcaag tttatgtaat gtaatttaac 3300

ttgaaacaaa ttttaagaaa gaaagactaa tttctgaaac tatcaagact acggagtaat 3360

attttgacta agttcatttc attaattaaa ttattactaa atatagtaaa gtatacgaca 3420

gaaaaaataa atgagtcgta tatattaaaa cattgcgagt aatattttat gtaagtcccc 3480

caaccctcaa tcttatattg ttcggactct tcaaagatat taatggatgt gtgtcgaatt 3540

tttcaagagt aatatatatt ttgaaaaatc tgacaaaata aaacatcgaa actgaagact 3600

ctgacaagtt acatttgtgc ttagattctc cttatctcat ggaattatac taatctcaac 3660

tagttggaat ataaagtttt aatatttcaa ataatttttt tattttattt ttgacaggaa 3720

gtgttaatgg gaaaagcacc agatctaaaa tcatttcaag taaatgcaga tgcattcatt 3780

tgttcaatat tacctggaat ttctcatccc caagtccaat attctccagg taaattggga 3840

atagttaatt aattaataat aaaccattta ttaaattaaa ttactaatgt ttttttgttt 3900

gtcattttat tgttgtttgt gtgtgaaggt ggactcattg tcaaacctgg ggtttgtaac 3960

atgcagcatg tgacatcttt gtccttctta ctcttaactt attctaatta tcttagtcat 4020

gccaatcatg ttgtgccatg tggttccatg acagccaccc ctgccctcct caaacacatt 4080

gccaaacgtc aggtactact ctcttcgtct taatttatct gatacagttt aactatatac 4140

aaagtttaag taatgtaaaa aacaactttt acagaggaaa atatttttca gtttttccat 4200

gtttgatcgg tcaaaacgtt aaagaaatga ggaaaacaag ttacacaagt gatattctgg 4260

atatattgtc ttctctctac tcactcgatc cctcaaccta agtcattctc gaacccccac 4320

tccccatctc atctcgttta cttactctat agaaattaga acgcaagtaa agaaatctac 4380

tttttgcgtg tgtgtgttta gtatagagga aaatgttttt caattttttc atatttgatc 4440

ggtcaaaacg ttaaagaaat gaggaaattt ttgacttttc tagaaagaaa aaaaaagtag 4500

gaaaaacaag ttacacaagt gatattctgg atatattgtc ttctccacac tcactcgatc 4560

cctcaacctt tattcatcct cgaaccccca ctccccatct catctcgttt acttagaaca 4620

caagtaaaga aatctactta ttttttcatg aaaacataat gatttttttt ttgaaacaga 4680

ctaaagagtg gttatgaatt gttgtaggtg gattatattc tgggagataa tcctcaaaga 4740

atgtcatata tggtagggta tggtccacat tacccacaaa ggattcacca taggggtagc 4800

tctgtgccat ctgtggccac acattcagca cgtattggtt gcaaagaggg atctcgatac 4860

tttttttcac caaacccaaa cccaaatcga ttaattggtg ctgttgttgg agggccaaat 4920

ttaacagact cgttcccaga cgccagaccc tattttcaag aatctgagcc cacaacatat 4980

gttaatgcac cattagtggg cctattggct tactttgcag cccattctaa ttgatataaa 5040

catgtgtaaa gagagaatgt agtggtgtgc aaaggccacc ctctctatta ttgtgttgtt 5100

gttgtctaat aggactaatg ttgttgtttt ttaatcccac tatatatata tatattatat 5160

taatacaaaa aaagaatatc ttatcccatc ttttgtctaa gaaaaagaaa gatatctaat 5220

gaacaaggga tttgtacttt tgaaattgta gtggaagttg tttttatctt attatacatg 5280

aaaattgttt tgaata 5296

Claims

1. A tomato irregular fruit cracking key regulatory gene is characterized in that the gene sequence is shown as SEQ ID NO. 1.

2. The method for identifying the key regulatory gene for irregular fruit cracking of tomatoes is characterized by comprising the following steps of:

1）F ₂ constructing generation segregation population: hybridizing tomato dehiscent fruit variety as female parent and easily dehisced fruit variety as male parent to obtain F ₁ Preparing F through selfing ₂ Separating the population;

2) pool-mixing re-sequencing for extreme characters: from F ₂ Respectively selecting extremely-resistant single fruit plants and extremely-resistant single fruit plants from the generation segregation population to form a fruit cracking resistant pool and an easily-cracked fruit pool, mixing leaf RNA of the single fruit plants in each pool in equal amount to obtain RNA of two parent mixing pools and RNA of two extreme character filial generation mixing pools respectively, constructing a library and performing high-throughput sequencing; performing the regression fitting of the loess on the delta SNP-index and the delta InDel-index on each chromosome by adopting a loess regression fitting method to obtain an associated region related to the irregular fruit cracking character of the tomato;

3) analysis of gene expression level: analyzing the genes crossed in the two groups of sequencing pools according to the positioning results of the SNP and InDel association areas and by combining the differential expression genes of the cracking resistant pool, the cracking easy pool, the cracking resistant mixed pool and the cracking easy mixed pool, and screening 4 genes with the SNP; through further bioinformatics analysis, a gene with obvious expression quantity difference is screened out and is a cellulase geneSlGH9-15 (Solyc09g010210)Namely, the key gene for regulating and controlling irregular tomato fruit cracking。

3. The method for identifying the key regulatory gene for irregular dehiscent fruit of tomato as claimed in claim 2, wherein in the step 1), the female parent is tomato dehiscent fruit resistant germplasm 'NT 91' and the male parent is dehiscent fruit germplasm 'NT 189'.

4. The method for identifying the key regulatory gene for irregular fruit cracking of tomato as claimed in claim 2, wherein the associated region in step 2) is the region of 2.88-3.94 Mb of chromosome 9 or the region of 53.02-53.04 Mb of chromosome 11.

5. The use of the key gene for regulating tomato irregular fruit cracking as claimed in claim 1 in the targeted identification or cultivation of tomato varieties with fruit cracking resistance.

6. A method for breeding tomato with crack resistance, which comprises overexpressing or knocking out the gene of claim 1 in tomato.

7. The method of claim 7, further comprising editing or mutagenesis of the gene of claim 1.