A molecular marker, primers and their application for identifying the cold resistance of hardy kiwifruit.

Abstract

This invention relates to a molecular marker, primers, and their applications for identifying the cold resistance of *Actinidia arguta*. The molecular marker of this invention is an InDel marker, with its InDel site located at -117 bp of the promoter of the LAR gene (colorless anthocyanin reductase gene) in *Actinidia arguta*. In non-cold-resistant germplasm, a 60 bp insertion exists at this site. The upstream primer sequence of this invention is 5'-CCACCCCTATACGAAGTTTGTAGAC-3', and the downstream primer sequence is 5'-TTAAAAATAAGGCTTTTTAGAGAGA-3'. All samples that amplify a 540 bp product are considered to have strong cold resistance in *Actinidia arguta*. The molecular marker and detection method of this invention are rapid, direct, and highly specific, enabling rapid and effective identification and screening of cold-resistant *Actinidia arguta* strains. The operation is simple, accurate, and can significantly accelerate the breeding process.

Description

Technical Field

[0001] This invention belongs to the field of artificial cultivation and molecular marker technology of hardy kiwifruit, specifically involving a molecular marker, primer, and its application and kit for identifying the cold resistance of hardy kiwifruit. The molecular marker and primer of this invention can be used for the identification of cold resistance traits in hardy kiwifruit and for cold resistance breeding of hardy kiwifruit. Background Technology

[0002] Kiwifruit belongs to the genus *Actinidia* (family Actinidiaceae) and is a perennial dioecious plant. my country is the origin of *Actinidia* species and possesses abundant natural resources; of the 54 existing species, 52 are endemic to my country or centrally distributed there (Huang Hongwen, 2013). Kiwifruit breeding mainly includes seedling selection, hybridization breeding, and mutation breeding. Traditional breeding methods are time-consuming and inefficient, and cannot quickly screen for lines with target traits. With the development of PCR and modern sequencing technologies, molecular marker-assisted selection breeding technology has emerged. Because this method selects the desired traits based on genotype, it has gained popularity among breeders and geneticists.

[0003] For a long time, cultivating varieties with high cold resistance has been one of the important goals of kiwifruit breeding. Low temperature stress is an important abiotic stress, and low temperature freezing damage has a serious impact on the kiwifruit industry. In mild cases, it causes a reduction in yield in the same year, and in severe cases, it leads to crop failure or tree death and orchard destruction (Lin Miaomiao et al., 2020; Qi Xiujuan et al., 2011). Studies have shown that commercially cultivated Chinese kiwifruit (Actinidia chinensis Planch.), including Chinese kiwifruit (original variety) and delicious kiwifruit (variety), can withstand temperatures of around -10℃ in winter on dormant branches. Furthermore, kiwifruit cultivation is closely related to weather conditions. In recent years, extreme low temperatures have occurred frequently, and kiwifruit orchards across the country suffer frost damage every year. In Shaanxi Province, a major kiwifruit producing area, from 1991 to 2016, Baoji City alone reported as many as 13 large-scale frost and late frost damage incidents, affecting a total area of ​​536,200 mu (Li Guangwen et al., 2018). In the winter of 2009, low temperatures caused severe frost damage to the kiwifruit resources preserved in the Zhengzhou Fruit Tree Research Institute's kiwifruit resource nursery. The frequent occurrence of abnormal climate and extreme low temperatures has caused significant losses to the kiwifruit industry. Therefore, it is essential to cultivate highly cold-resistant kiwifruit using molecular breeding methods. Currently, there is still no research on the molecular markers related to cold resistance traits in kiwifruit. Since it takes 3-4 years from hybridization breeding to obtaining seedlings and growing into large seedlings suitable for traditional cold resistance assessment, it is possible to save breeding costs and obtain varieties with the desired cold resistance traits earlier if cold resistance assessment can be conducted during the seedling stage.

[0004] Actinopterygium arguta is a geographically widespread species within the genus Actinopterygium, ranging from 20°N to 50°N. This significant latitudinal difference leads to variations in cold resistance within the species (Cui Zhixue, 1993). Specifically, Actinopterygium arguta distributed in Northeast China can withstand temperatures as low as -40°C, while those distributed in the south can only withstand -15°C. Therefore, developing molecular markers for cold resistance in Actinopterygium arguta has important practical significance and significant application value for assisted breeding of this species. Summary of the Invention

[0005] To address the lack of effective molecular markers in the cold-resistance breeding of hardy kiwifruit, this invention provides a new and effective molecular marker and related primers for identifying the cold resistance of hardy kiwifruit, as well as a method and test kit for identifying the cold resistance of hardy kiwifruit.

[0006] In a first aspect, the present invention provides a molecular marker for identifying the cold resistance of Actinidia arguta, the molecular marker being an InDel marker, the sequence of which is shown in SEQ ID NO:1. The InDel site of the InDel marker is located at -117bp of the promoter of the LAR gene of the colorless anthocyanin reductase gene in Actinidia arguta, and a 60bp insertion exists at this site in non-cold-resistant germplasm.

[0007] Furthermore, the upstream primer sequence of the above molecular marker is 5'-CCACCCCTATACGAAGTTTGTAGAC-3', and the downstream primer sequence is 5'-TTAAAAATAAGGCTTTTTAGAGAGA-3'.

[0008] In addition, the present invention also relates to the application of the above-mentioned molecular marker detection reagents in identifying the cold resistance trait of hardy kiwifruit and in the breeding of hardy kiwifruit varieties.

[0009] Secondly, the present invention provides a test kit for identifying the cold resistance of hardy kiwifruit, the test kit containing the aforementioned molecular marker test reagent.

[0010] Furthermore, the above-mentioned test kit contains the following primer pairs:

[0011] Upstream primer: 5'-CCACCCCTATACGAAGTTTGTAGAC-3',

[0012] Downstream primer: 5'-TTAAAAATAAGGCTTTTTAGAGAGA-3'.

[0013] Thirdly, the present invention provides a method for identifying the cold resistance of hardy kiwifruit, comprising the following steps:

[0014] (1) DNA extraction

[0015] Total DNA was extracted from leaves or branches of the hardy kiwifruit using the CTAB method.

[0016] (2) PCR amplification

[0017] a. The reaction system includes:

[0018] 1 μl of total DNA from leaves or branches of the hardy kiwifruit plant (100 ng / μl).

[0019] Upstream primer 5'-CCACCCCTATACGAAGTTTGTAGAC-3', 1 μl

[0020] Downstream primer 5'-TTAAAAATAAGGCTTTTTAGAGAGA-3', 1 μl

[0021] PCR mix 10μl,

[0022] ddH2O 8μl;

[0023] b. The reaction procedure is as follows:

[0024] 94℃ for 5 minutes,

[0025] 94℃ for 30 seconds, 60℃ for 1 minute, 72℃ for 30 seconds, for a total of 35 cycles.

[0026] 72℃ for 5 minutes;

[0027] (3) Electrophoretic pattern analysis

[0028] The band pattern is interpreted based on the agarose gel electrophoresis amplification results of the sample. If the amplified band of the sample contains a 540bp band, the sample is a cold-resistant variety or strain. If the amplified band of the sample contains only a 600bp band, the sample is a non-cold-resistant variety or strain.

[0029] Furthermore, in the above method for identifying the cold resistance of hardy kiwifruit, the amplification band of the sample containing a 540bp band refers to either a single 540bp band or both 540bp and 600bp bands.

[0030] In addition, the present invention also provides a method for identifying the cold resistance trait of hardy kiwifruit, which includes the step of detecting the aforementioned molecular markers.

[0031] In summary, this invention provides a molecular marker and kit related to the cold resistance of Actinidia arguta, which can be used to identify the cold resistance of Actinidia arguta. It also provides a method for identifying the cold resistance of Actinidia arguta. The molecular markers screened and the related detection methods of this invention are rapid, direct, and highly specific. Using the molecular markers and methods of this invention, cold-resistant strains of Actinidia arguta can be quickly and effectively identified and screened. The operation is simple, the accuracy is high, and it can significantly accelerate the breeding process, thus providing a powerful technical means for cold-resistant breeding of Actinidia arguta. Attached Figure Description

[0032] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0033] Figure 1 Electrophoresis results of LAR promoter amplification for 'KL' and 'RB'.

[0034] Figure 2 The graph shows the relative conductivity results of different genotypes of hardy kiwifruit after being treated at -25℃.

[0035] Figure 3 Agarose gel electrophoresis results of different cold-resistant kiwifruit samples. Detailed Implementation

[0036] To make the objectives and technical solutions of this invention clearer, the invention will be further described in detail below with reference to specific embodiments. Those skilled in the art can easily understand other advantages of this invention from the content disclosed in this specification. This invention can also be implemented or applied through other different specific embodiments, and various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of this invention.

[0037] Before further describing specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terminology used in the embodiments of the present invention is for describing specific embodiments and not for limiting the scope of protection of the present invention.

[0038] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by those skilled in the art. In this invention, unless otherwise specified, all instruments, reagents, and raw materials are commercially available or commonly used in the art. The methods in the following embodiments, unless otherwise specified, are conventional methods in the art.

[0039] Example 1: Obtaining the InDel site for the cold resistance trait in hardy kiwifruit

[0040] This invention uses dormant branches of the cold-resistant 'KL' and cold-resistant 'RB' varieties of hardy kiwifruit as materials. After treatment at -25℃ for 0h, 1h, 4h, and 7h, full-length transcriptomic analysis was performed. Differential expression analysis revealed that the colorless anthocyanin reductase gene LAR was significantly expressed in the cold-resistant material. Then, the LAR promoter sequence was further amplified using chromosome walking, revealing a 60bp InDel at the -117bp position in both the cold-resistant 'KL' and cold-resistant 'RB' LAR promoter sequences.

[0041] Example 2: Primer design and identification of InDel-labeled cold-resistant kiwifruit

[0042] Based on the InDel site discovered in the LAR promoter region, InDel primers were designed and validated in dormant branches of 'KL' and 'RB'.

[0043] The upstream primer is: 5'-CCACCCCTATACGAAGTTTGTAGAC-3'.

[0044] The downstream primer is: 5'-TTAAAAATAAGGCTTTTTAGAGAGA-3'.

[0045] The LAR promoter sequences for 'KL' and 'RB' are shown in SEQ ID NO:1 and SEQ ID NO:2.

[0046] The verification method includes the following steps:

[0047] (1) DNA extraction

[0048] Total DNA was extracted from leaves of Actinidia arguta using the CTAB method;

[0049] (2) PCR amplification

[0050] a. The reaction system includes:

[0051] 1 μl of total DNA from 100 ng / μl of Actinidia arguta leaves.

[0052] Upstream primer 5'-CCACCCCTATACGAAGTTTGTAGAC-3', 1 μl

[0053] Downstream primer 5'-TTAAAAATAAGGCTTTTTAGAGAGA-3', 1 μl

[0054] PCR mix 10μl,

[0055] ddH2O 8μl;

[0056] b. The reaction procedure is as follows:

[0057] 94℃ for 5 minutes,

[0058] 94℃ for 30 seconds, 60℃ for 1 minute, 72℃ for 30 seconds, for a total of 35 cycles.

[0059] 72℃ for 5 minutes;

[0060] (3) Electrophoretic pattern analysis

[0061] The PCR products were detected and interpreted by 1% agarose gel electrophoresis (see attached results). Figure 1 ).

[0062] Example 3: Application of InDel markers in the identification of cold resistance traits in hardy kiwifruit

[0063] (1) The test materials are shown in Table 1 below:

[0064] Table 1. Kiwifruit materials used in the test

[0065]

[0066] (2) Genotyping of 13 known cold-resistant kiwifruit germplasms was performed using the InDel molecular markers and methods described in Example 2 (see Appendix for results of cold resistance identification). Figure 2 Dormant branches were treated at -25℃ for 8 hours, and their relative electrical conductivity was measured. The formula for calculating the relative electrical conductivity was C1 / C2*100%. The results showed that there were 9 hardy kiwi fruit varieties / strains with strong cold resistance, namely CB-15, MDJ, Y-4, GZ, FL, HH, LH, LD, and Kucha, and 4 hardy kiwi fruit varieties / strains without cold resistance, namely NYT, ZJ, RB-4, and HR.

[0067] (3) According to the agarose gel electrophoresis results, if the amplified band contains the same 540bp band as that amplified in KL, then the material being tested exhibits cold resistance; if the amplified band only contains the same 600bp band as that amplified in RB, then the material being tested exhibits non-cold resistance (see attached electrophoresis results). Figure 3 ).

[0068] (4) Appraisal results

[0069] The test results of 15 hardy kiwifruit accessions showed that 10 accessions exhibited the cold-resistant banded genotype (i.e., all contained the same 540bp band as KL), and 5 accessions exhibited the non-banded genotype (containing only the same 600bp band as RB). The phenotypic and genotypic concordance rate of the 15 hardy kiwifruit accessions reached 100%.

[0070] The above results demonstrate that the InDel molecular markers of this invention can rapidly and accurately identify the cold resistance trait of hardy kiwifruit, significantly improve the selection efficiency of cold resistance breeding of hardy kiwifruit, thereby accelerating the breeding process and realizing targeted genetic improvement of cold resistance in hardy kiwifruit.

[0071] The preferred embodiments and examples of the present invention have been described in detail above. However, the present invention is not limited to the above embodiments and examples. Within the scope of knowledge possessed by those skilled in the art, various changes can be made without departing from the concept of the present invention.

Claims

1. A molecular marker for identifying cold hardiness in Actinidia arguta, characterized in that: The molecular marker is an InDel marker, and the sequence is shown as SEQ ID NO:1 and SEQ ID NO:2, and the sequence of the InDel marker in the cold-resistant germplasm is shown as SEQ ID NO:

2.

2. The molecular marker of claim 1, wherein: The sequence of the upstream primer of the molecular marker is 5'-CCACCCCTATACGAAGTTTGTAGAC-3', and the sequence of the downstream primer is 5'-TTAAAAATAAGGCTTTTTAGAGAGA-3'.

3. The use of the reagent for detecting the molecular marker in claim 1 or 2 in identifying the cold resistance trait of Actinidia arguta or breeding a cold-resistant variety of Actinidia arguta.

4. A method for identifying cold hardiness in Actinidia arguta, characterized by: The method comprises the following steps: (1) DNA extraction The total DNA of the leaf or branch of Actinidia arguta is extracted by using the CTAB method; (2) PCR amplification a. The reaction system comprises: 100 ng / μl of total DNA of the leaf or branch of Actinidia arguta 1 μl, the upstream primer 5'-CCACCCCTATACGAAGTTTGTAGAC-3', 1 μl, the downstream primer 5'-TTAAAAATAAGGCTTTTTAGAGAGA-3', 1 μl, PCR mix 10 μl, ddH2O 8 μl; b. The reaction procedure is as follows: 94℃ 5min, 94℃ 30s, 60℃ 1min, 72℃ 30s, for a total of 35 cycles, 72℃ 5min; (3) Electrophoretic pattern analysis The band type is interpreted according to the amplification result of the agarose gel electrophoresis of the sample, if the amplification band of the sample contains a 540 bp band, the sample is a cold-resistant variety or strain, and the amplification band of the sample containing a 540 bp band means that it contains a 540 bp band alone or contains 540 bp and 600 bp bands at the same time, if the amplification band of the sample contains only a 600 bp band, the sample is a cold-intolerant variety or strain.

5. A method of identifying cold hardy traits in Actinidia arguta, characterized by: The method comprises the step of detecting the molecular marker in claim 1, and the sequence of the molecular marker in the cold-intolerant germplasm is shown as SEQ ID NO:2.

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