A gene PeLSRD1 for resistance to basal rot in passion fruit stems and its molecular markers

Abstract

The present invention relates to the field of bioengineering technology, and in particular to a passion fruit stem rot resistance gene PeLSRD1 and a molecular marker thereof, wherein the cDNA nucleotide sequence of the passion fruit stem rot resistance gene PeLSRD1 is shown in SEQ ID NO. 1. The present invention identifies the passion fruit stem rot resistance gene PeLSRD1, and develops a molecular marker closely linked to the gene, which can be efficiently used in passion fruit stem rot resistance breeding.

Description

Technical Field

[0001] The invention belongs to the technical field of bioengineering, and particularly relates to a passion fruit stem basal rot resistance gene PeLSRD1 and a molecular marker thereof. Background Art

[0002] Cultivated passion fruit is an important fruit tree in southern China. It is a herbaceous vine with aromatic fruit, rich in sugars, vitamins, and mineral elements such as calcium, iron, and zinc, and has extremely high nutritional value. With the improvement of people's living standards and changes in consumption concepts in my country, the consumption of passion fruit is growing rapidly, which will stimulate the rapid development of the passion fruit industry. However, the occurrence and spread of passion fruit stem rot caused by fungi such as Fusarium solani seriously affects the yield and quality of passion fruit.

[0003] By using passion fruit germplasm resources to mine resistance genes and develop markers that are closely linked to target genes, direct gene selection can be achieved, which greatly improves breeding efficiency and shortens the breeding period. However, no passion fruit stem rot resistance genes / QTLs have been reported so far. BSA (Bulked segregant analysis) is also known as cluster separation analysis or mixed pool grouping analysis. In 2013, Japanese scholars Takagi et al. (Takagi et al., Plant Journal, 2013) combined BSA with high-throughput sequencing to develop QTL-seq technology. Currently, this technology is widely used in plant gene / QTL positioning, which has the advantages of simplicity, accuracy and efficiency.

[0004] In recent years, the consumption of passion fruit in my country has been growing rapidly, and the area of ​​passion fruit cultivation has expanded rapidly. However, the occurrence and spread of passion fruit stem rot caused by fungi such as Fusarium oxysporum and Fusarium solani seriously affects the yield and quality of passion fruit. Studies on important crops such as rice and corn have shown that the use of molecular breeding technology to cultivate new disease-resistant varieties is the most effective and economical method to prevent and control diseases. At present, there are no reports of passion fruit stem rot resistance genes / QTLs at home and abroad. Summary of the invention

[0005] In view of the above, the present invention identified the passion fruit stem rot resistance gene PeLSRD1 and developed a molecular marker closely linked to it, which can be effectively used in passion fruit stem rot resistance breeding.

[0006] In order to achieve the above object, the technical solution adopted by the present invention is:

[0007] Passiflora stem rot resistance gene PeLSRD1, wherein the cDNA of the passionflower stem rot resistance gene PeLSRD1 is the nucleotide sequence shown in SEQ ID NO.1;

[0008] Specifically:

[0009]

[0010] The present application also provides a molecular marker for the passion fruit stem rot resistance gene PeLSRD1, wherein the upstream primer sequence of the marker is SEQ ID NO.2, specifically: ggtggccttgaaagcatc; the downstream primer sequence is SEQ ID NO.3, specifically: ttgccttcttgtctggca.

[0011] The above molecular markers are used in breeding passion fruit resistant to stem base rot.

[0012] The present application also provides a method for screening passion fruit resistant to stem base rot, which uses the molecular marker PCR described in claim 2 to amplify the genomic DNA of the passion fruit to be tested. If a 196 bp amplified fragment is amplified, it indicates the presence of passion fruit variety resistance to stem base rot.

[0013] The present invention has the following beneficial effects:

[0014] The present invention identifies the passion fruit stem base rot resistance gene PeLSRD1 and develops a molecular marker closely linked to it, which can be efficiently used in passion fruit stem base rot resistance breeding, provides gene resources and technical support for passion fruit disease resistance molecular breeding, and has very broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 QTL-seq was used to locate the passion fruit stem rot resistance gene PeLSRD1;

[0016] Figure 2 It is a candidate gene map for screening PeLSRD1 using RNA-seq;

[0017] Figure 3 This is a diagram showing the effect of PeLSRD1 molecular marker. DETAILED DESCRIPTION

[0018] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the specific embodiments of the present invention are described in detail below in conjunction with the accompanying drawings. In the following description, many specific details are set forth to facilitate a full understanding of the present invention. However, the present invention can be implemented in many other ways different from those described herein, and those skilled in the art can make similar improvements without violating the connotation of the present invention, so the present invention is not limited by the specific implementation disclosed below.

[0019] In this example, the passion fruit stem rot resistance gene PeLSRD1 is obtained by the following steps:

[0020] (1) Isolation and purification of Fusarium solani: A 5 mm × 5 mm tissue block was cut from the edge of a fresh lesion of passion fruit stem base rot (the junction of diseased and healthy parts), soaked in 75% ethanol for 30 seconds, and then sterilized in 0.1% mercuric chloride for 3 minutes. The block was then rinsed with sterile water three times, and the surface moisture was dried with sterile filter paper. The block was placed on a plate of separation medium (PDA containing 150 mg·L-1 streptomycin sulfate) and cultured in the dark at 28°C. The pathogen was purified to obtain a representative strain.

[0021] (2) Identification of passion fruit stem rot resistance: An F2 population was constructed by hybridizing “Jinling purple fruit × yellow fruit native species”. 533 plants from the F2 population were selected to construct the F 2:3 Family groups. In the greenhouse, plant 30 plants of each family of the F3 group of 'Jinling Purple Fruit × Yellow Fruit Native Species', and pay attention to isolation from the outside world. Select healthy plants at the 6-8 leaf stage for disease resistance test, and retain some branches for cuttings before inoculation to obtain offspring seeds. The above-mentioned purified and preserved pathogens were activated and inoculated onto PDA culture medium, cultured at 28°C for 5 days, and punctured with a 1000ul pipette tip to obtain mycelium blocks. Then, the in vitro inoculation method was used for disease resistance identification. The base of the stem was artificially punctured, and the prepared mycelium block was picked up to the punctured site, with the front of the mycelium close to the wound, to identify 533 F produced by 'Jinling Purple Fruit × Yellow Fruit Native Species'. 2:3 Disease resistance of the family.

[0022] (3) DNA pool construction and sequencing: Based on 533 F 2:3 The results of the disease resistance identification of the family screened out 30 extremely resistant and susceptible plants, extracted DNA from each individual, and mixed the DNA in equal amounts to form a disease-resistant pool (H-bulk) and a disease-susceptible pool (L-bulk). The two parents were separately built, and the disease-resistant pool and the disease-susceptible pool were built, and then double-end sequencing was performed using the Illumina NovaSeq6000 platform.

[0023] (4) SNP detection and high-quality SNP screening: After quality control, the sequencing data was compared with the Passiflora reference genome (https: / / db.cngb.org / search / assembly / CNA0017758 / ) to obtain SNPs of the two parents and the two pools. Then, SNPs were further screened according to the following conditions: ① SNP sites with multiple genotypes were filtered out, and only diallel genotype sites were retained; ② SNP sites with mixed pool read support less than 4 were filtered out; ③ SNP sites with homozygous and consistent genotypes between the mixed pools were filtered out; ④ SNP sites with homozygous and consistent genotypes between the two parents were filtered out; ⑤ SNP sites whose recessive mixed pool genotypes were not from the recessive parent were filtered out, and SNP sites whose dominant mixed pool genotypes were not from the dominant parent were filtered out. Finally, 1,827,840 high-quality credible SNP sites were obtained.

[0024] (5) QTL-seq analysis: Figure 1 As shown, the association analysis using the SNP-index method is mainly to find significant differences in genotype frequencies between mixed pools, and Δ(SNP-index) is used for statistics. The stronger the correlation between the marker SNP and the trait, the closer Δ(SNP-index) is to 1. In order to eliminate false positive sites, the ΔSNP-index values ​​of the markers on the same chromosome can be fitted using the position of the marker on the genome. The ΔSNP-index is fitted using the DISTANCE method, and then the region above the threshold is selected as the region associated with the trait based on the association threshold. In order to make full use of the data, the threshold is lowered to find a more likely positioning region. Using 99% of the fitted ΔSNP-index, i.e. 0.31, a passion fruit stem base rot resistance site was detected in the 245,500,000-246,600,000bp interval of chromosome 1 of passion fruit. We named it PeLSRD1, and its cDNA is the nucleotide sequence shown in SEQ ID NO.1;

[0025] Specifically:

[0026]

[0027] (6) Analysis of candidate genes using RNA-seq: Samples were collected 1, 3, and 7 days after the parental Jinling purple-fruit and yellow-fruit native species were inoculated with Fusarium solani. Reagent kit (Invitrogen, USA) was used to extract RNA, and the specific process was carried out according to the instructions. An Illumina PE library was constructed for 2×150bp sequencing, and the transcriptome sequencing was completed using the Illumina NovaSeq6000 sequencing platform. RSEM software was used to quantitatively analyze the overall expression levels of genes / transcripts, respectively, in order to subsequently analyze the differential expression of genes / transcripts between different samples. The quantitative indicator was the TPM value. In the 245,500,000-246,600,000bp range of chromosome 1 of Passiflora, only ZX.01G0130080 had significant differences in expression levels in the three treatment periods. Subsequently, RT-qPCR was used to verify the RNA-seq results. Figure 2 shown.

[0028] In addition, the molecular marker PeLSRD1, a gene that is resistant to stem rot in passion fruit, was developed and verified. Specifically:

[0029] The purple passion fruit genome assembled by Xia et al. (Xia et al., Horticulture Research, 2021) was aligned with the passion fruit reference genome, and 391901 markers were developed based on InDel and PAV. We selected the marker PeJJFB1 near ZX.01G0130080, the upstream primer sequence of which is: ggtggccttgaaagcatc; the downstream primer sequence is: ttgccttcttgtctggca. It was verified in 500 F2 populations, and the results showed that the genotype-phenotype match rate was 98.1%. Therefore, this marker can be used for passion fruit breeding for resistance to stem rot. The primer usage conditions: 20μL reaction system, including 10μL2×Taq PCRMaster Mix, 0.5μLForward primer, 0.5μLReverse primer, 1μL DNA, 8μLddH2O. The reaction conditions were 94°C pre-denaturation for 5 min; (94°C for 30 s, 59°C for 30 s, 72°C for 30 s) for 35 cycles. Then, PCR products were detected by 6% polyacrylamide gel electrophoresis. Figure 3 As shown, the genotype of the passion fruit F2 population was identified using the molecular marker PeJJFB1. In the figure, 1-12 can amplify a 196bp band, representing a disease-resistant genotype; 13-24 can amplify a 171bp band, representing a susceptible genotype.

[0030] The above-mentioned embodiments merely express several implementation modes of the present invention, and the description thereof is relatively specific and detailed, but it should not be understood as limiting the scope of the present invention.

Claims

1. Passiflora stem rot resistance gene PeLSRD1, characterized in that: The cDNA of the passion fruit stem basal rot resistance gene PeLSRD1 is a nucleotide sequence shown in SEQ ID NO.

1.

2. The molecular marker of the passion fruit stem rot resistance gene PeLSRD1 is characterized by: The upstream primer sequence of the marker is SEQ ID NO.2; the downstream primer sequence is SEQ ID NO.

3.

3. Application of molecular markers as claimed in claim 2 in breeding passion fruit resistant to stem base rot.

4. A method for screening passion fruit resistant to stem base rot, characterized in that: The molecular marker PCR described in claim 2 is used to amplify the genomic DNA of the passion fruit to be tested. If a 196 bp amplified fragment is amplified, it indicates the existence of the passion fruit variety's resistance to stem base rot.

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