AU2020100809A4

AU2020100809A4 - Molecular Marker AhyBscc Closely Linked With The Black Seed Coat Of Peanut And Its Application

Info

Publication number: AU2020100809A4
Application number: AU2020100809A
Authority: AU
Inventors: Lei HOU; Aiqin Li; Changsheng Li; Guanghui Li; Pengcheng Li; Junjie Ma; Xingjun Wang; Han Xia; Chuanzhi ZHAO; Shuzhen ZHAO; Yuhan ZHAO
Original assignee: Biotechnology Research Center of Shandong Academy of Agricultural Sciences
Current assignee: Biotechnology Research Center of Shandong Academy of Agricultural Sciences
Priority date: 2020-05-21
Filing date: 2020-05-21
Publication date: 2020-07-02
Anticipated expiration: 2028-05-21

Abstract

This invention discloses a molecular marker AhyBscc closely linked with the black seed coat of peanut, and a specific primer pair of the molecular marker AhyBscc, as described in SEQ ID No: 1 and SEQ ID No: 2. It also discloses a method for identifying the seed coat color of peanut by using the molecular marker AhyBscc, which comprises the following steps: Step 1: Extract DNA from peanut seed or blade; Step 2: Perform PCR amplification for extracted DNA by using specific primer pair; Step 3: Detect the amplified products by non-denaturing polyacrylamide gel electrophoresis. If the characteristic bands with size of 330bp appear, the next generation of seeds of the peanut material to be tested is determined to have black coat. This method is simple and easy to operate, and it can be used in peanut molecular breeding and quality improvement. It can realize the fine locating, separating and cloning of genes, help to improve the efficiency of screening black peanuts, and quickly provide a large number of germplasm resources of new peanut variety. Drawings A B 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 ) 500 bp 250 bp Peanuts with black seed coat Pink seed coat Figure 3 Figure 4 3/4

Description

Drawings

A B 1 2 3 4 5 6 7 8 9 10 11 12 13 14 1516 17 18 19 20

) 500 bp

250 bp

Peanuts with black seed coat Pink seed coat

Figure 3

Figure 4

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Molecular Marker AhyBscc Closely Linked with the Black Seed Coat

of Peanut and its Application

Technical Field This invention is related to the agricultural biotechnology field, in particular to a molecular marker AhyBscc closely linked with the black seed coat of peanut and its application.

Background Technology Peanut is rich in fatty acids, proteins, trace elements such as calcium, iron, zinc, selenium and vitamin E, as well as functional active substances such as resveratrol, which plays an important role in health care. It is known as "longevity nut" (Zhao et al., 2012). The color of peanut seed coat is mainly pink and red, and there are not many peanut varieties with black (or dark purple) seed coat. The seed coat of black peanut is rich in natural pigments, especially anthocyanin, which has many biological effects such as inhibiting free radicals and antioxidation. The market demand of black peanut is increasing. However, there are few varieties of peanut with black seed coat at present, and their yield is low compared with the conventional varieties, so it is difficult to popularize them widely. Therefore, to increase the yield of black peanut varieties is the fundamental way to meet the market demand.

Like many other plants, the seed coat of peanut is also developed from the integument. Compared with other characters, the color of seed coat can only appear by skipping one generation, which limits the cultivation of black peanut varieties by traditional breeding methods to a large extent. Molecular marker assisted breeding is mainly based on genotype identification. For the character of black seed coat, by cutting off part of the cotyledons of the seed and detecting the genotype of the cotyledons, the color of seed coat of the next generation of harvested materials can be determined in advance, which can accelerate the process of breeding new varieties of black peanut and improve the breeding efficiency.

At home and abroad, most of the studies on black peanut are focused on the extraction of seed coat pigment and the stability of pigment. Recently, Zhao et al. (2019) locked the gene controlling the color of the black seed coat of peanut on Chromosome 10 by QTL-seq method. These studies laid a foundation for the identification of black seed coat genes and molecular breeding of black peanut, and some SSR or SNP markers linked with the black

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seed coat of peanut were also developed in relevant studies. Because of the weak linkage between the markers and seed coat color genes reported in the past, or the tedious detection, it is difficult to apply these markers to peanut genetics and breeding research accurately and quickly.

Invention Summary One of the purposes of this invention is to solve the above problems at least and to provide the advantages to be described later at least.

Another purpose of this invention is to provide a molecular marker AhyBscc closely linked with the black seed coat of peanut, which can identify peanut with black seed coat.

Another purpose of this invention is to provide a method for identifying the color of peanut seed coat by using the molecular marker AhyBscc closely linked with the black seed coat of peanut. This method is simple and easy to operate, shortens the time for identifying peanut with black seed coat, and is conducive to improving breeding efficiency.

Another purpose of this invention is to provide the application of a molecular marker AhyBscc closely linked with the black seed coat of peanut, which is mainly used in molecular breeding and quality improvement of peanut, so as to obtain new germplasm resources quickly.

To achieve the above purpose, this invention provides the following scheme:

This invention provides a molecular marker AhyBscc closely linked with the black seed coat of peanut, and the specific primer pair of the described molecular marker AhyBscc include:

Forward primer: 5'-AAATGAAAAGACATGTAACATATATAGC-3';

Reverse primer: 5'- AGAAAAACATAACAATGAATTTTCCA -3'.

This invention also provides a method for identifying the color of peanut seed coat by using the molecular marker AhyBscc closely linked with the black seed coat of peanut, which comprises the following steps:

Step 1: Extract DNA from peanut seed or blade;

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Step 2: Perform PCR amplification for extracted DNA by using the specific primer pair described in Claim 1;

Step 3: Detect the amplified products obtained by Step 2 by non-denaturing polyacrylamide gel electrophoresis. If the characteristic bands with size of 330bp appear, the next generation of seeds of the peanut material to be tested is determined to have black coat.

Preferably, in Step 2, the total volume of PCR amplification is 25uL: 1 L for DNA template, 0.5L for forward primer and reverse primer, 0.5L for dNTP mix, 2.5uL for lOxTaq Buffer, 2.0L for MgCl2, 0.25L for Taq enzyme, and add water to 25L.

Preferably, the PCR amplification reaction conditions in Step 2 are: pre denaturation at °C for 4 min; 94°C for 30s, 58°C for 30s, 72°C for 25s, 35 cycles; extension at 72°C for min.

Preferably, 8% non-denaturing polyacrylamide gel electrophoresis is used in Step 3.

This invention also provides an application of the molecular marker AhyBscc closely linked with the black seed coat of peanut, the described molecular marker AhyBscc is located in the region of 108.0-112.7Mb of chromosome 10, and the described molecular marker AhyBscc is applied to peanut breeding and peanut quality improvement.

Preferably, it is used to identify peanut with black seed coat.

This invention discloses the following technical effects:

This invention provides a molecular marker AhyBsec which can identify peanut with black seed coat. Since the seed coat of peanut is developed from the integument, compared with other characters, the color of seed coat can only appear by skipping one generation. For example, by taking pink seed coat as the female parent to hybridize with black peanut as the male parent, Fl seed coat is pink (female parent), F2 seed coat is light black (parents intermediate type), and only F3 seed coat appears character segregation. Therefore, there is a great blindness in the screening and cultivation of black peanut by naked eyes, and by using the markers provided by this invention, the seed coat color of the next generation of harvested material can be determined in advance by detecting the DNA of the cotyledons, so as to improve the breeding efficiency. In addition, peanut is allotetraploid, of which the alleles of A and B subgenome are highly homologous, so it is difficult to effectively distinguish A and B subgenome with many markers. In contrast, the molecular marker of this

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invention is selected after many scientific experiments and explorations, so it leads to reliable results and is highly reliable.

This invention also provides a method for identifying peanut with black seed coat by using molecular marker AhyBscc. This method adopts simple PCR marker, of which the technical requirements are simple and easy to operate. The identification can be realized by PCR amplification and electrophoresis, and the requirements for operating the instruments are fairly low. The conventional instruments used in conventional experiments can be used for the testing, which is easily accepted by people.

This invention also provides the application of molecular marker AhyBscc, which can be used in molecular breeding and improving peanut varieties, so as to greatly shorten the breeding time, improve the breeding efficiency, and obtain more and better peanut germplasm resources in a shorter time.

Description of Drawings In order to more clearly explain the embodiments of this invention or the technical proposals in existing technology, the figures to be used in the embodiments will be briefly introduced below. Obviously, the figures in the following description are only some embodiments of this invention. Ordinary technicians in this field can also obtain other figures according to these figures without making creative effort.

Figure 1 The distribution map of candidate SNP loci related to the seed coat of black peanut on chromosome

Figure 2 Verify the linkage region of black seed coat characters by BSR;

Figure3 Verification map of the molecular marker AhyBscc in F2 offspring;

Figure 4 Sampling of peanut seeds and germination test chart of peanut seeds after sampling;

Figure 5 The comparison map between the rapid cultivation of high-yield black peanut varieties based on the breeding scheme of molecular marker assisted recurrent selection and the traditional cultivation of black peanut varieties.

Detailed Description of the Presently Preferred Embodiments A variety of exemplary embodiments of this invention are described in detail, which

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shall not be considered as a limitation of this invention, but shall be understood as a more detailed description of certain aspects, features and embodiments of this invention.

It should be understood that the terms described in this invention are to describe particular embodiments only and are not to limit this invention. In addition, for the numerical range in this invention, it should be understood that each intermediate value between the upper limit and the lower limit of the range is also specifically disclosed. Each smaller range between any stated value or intermediate value within the stated range and any other stated value or intermediate value within the stated range is also included in this invention. The upper and lower limit of these smaller ranges can be included or excluded in these ranges independently.

Unless otherwise stated, all technical and scientific terms used herein have the same meaning commonly understood by the ordinary technicians in the field described in this invention. Although this invention only describes preferred methods and materials, any methods and materials similar to or equivalent to those described herein may also be used in the embodiment or testing of this invention. All references in this specification are incorporated by reference to disclose and describe methods and/or materials related to the references. In case of conflict with any incorporated reference, the content of these instructions shall prevail.

Without deviating from the scope or spirit of this invention, various improvements and changes can be made to the specific embodiments of the specification of this invention, which is obvious to the technicians in this field. Other embodiments obtained from the specification of this invention are obvious to technicians. The specification and embodiment of this application are only exemplary.

As for the words "comprise", "include", "have", "contain" and so on used in this paper, they are all open terms, that is, they mean to comprise but not limited to.

Embodiment 1: location of the genes controlling the color of the black seed coat of peanut and design of the molecular marker AhyBscc closely linked with the black seed coat of peanut

In order to locate the genes controlling the color of the black seed coat of peanut, the inventor hybridized peanut variety with pink seed coat (female parent) and peanut variety with black seed coat (male parent), and constructed 4 segregation populations. successively. It was found that all the F1 hybrid seeds harvested were pink, which was consistent with the

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color of the female parent, and was in accordance with the genetic law that "seed coat is developed from integument, and the color of seed coat is consistent with the maternal genotype". Genetic analysis showed that the seed coat color of peanut was controlled by a single gene.

The whole-genome of KF1 population, including the mixed pool of the extreme materials with black seed coat, the mixed pool of the extreme materials with pink seed coat, the male parent Yuhua 29 and the female parent Weihua 10 were re-sequenced by BSA-seq method. A total of 640758 SNP loci were detected between the parents, covering 20 chromosomes of peanut. By bioinformatics analysis, 1798 candidate SNP loci which may be related to the black color of peanut seed coat were screened out, of which 1317 (73.29%) were located on chromosome 10 (as shown in Figure 1); on the chromosome A10 of peanut, the candidate SNP loci were mainly located in three regions, of which about 1/3 of the associated SNP loci were located in the region of A10:108.0-112.7 Mb. In addition, BSR-seq analysis was carried out on the other three segregation populations in Table 1 (as shown in Figure 2). The results showed that the candidate interval located on chromosome 10 was closely linked with the color character of the black seed coat of peanut.

After the whole-genome sequence in the candidate interval was scanned, 102 SSR loci were found in the candidate interval. The primers for these SSR loci were designed by Primer 3 software. By screening the progeny materials of different populations, it was found that one of the primers was closely linked with the black seed coat character of peanut, and the results were consistent and stable in different populations. We named such marker AhyBsec (as shown in Figure 3).

Embodiment 2: New varieties of high-yield peanut with black seed coat were cultivated quickly by using the molecular marker AhyBscc

2.1 Experimental object

The color of the seed coat of the peanut variety Fenghua No. 1 (HFOO1) is pink. HFOO1 is one of the main high-yield peanut varieties in North China. Take HFOO1 as the research object, modify it genetically, improve its seed coat color to black while keeping its high-yield character.

2.2 Experimental steps

(1) Hybridization

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Hybridize HFOO1, as the female parent (recurrent parent), and the black peanut variety Zhonghua 9, as the male parent.

(2) Identification of the trueness of hybrid F1

Identify the trueness of the harvested F1 hybrid generation by the molecular marker AhyBscc as follows:

Sampling: Harvest all the pods of the maternal plant, dry them and number all the harvested seeds; after that, remove part of the seed coat by a scalpel, then remove part of the cotyledon tissue (about 30 mg) and place them in a 1.5 ml centrifuge tube with magnetic beads. Put the remaining peanut seeds into the cold storage for preservation, and then plant them in the field after detection. The experiment showed that the germination rate of peanut seeds was not affected after removing part of the tissue (as shown in Figure 4).

(3) Extract the DNA of peanut seeds to be tested.

(4) PCR amplification

Carry out molecular marker detection on the parents and all Fl hybrids by using the forward primer (as shown in SEQ ID NO: 1) and reverse primer (as shown in SEQ ID NO: 2) of AhyBscc. According to the results of electrophoresis, the hybrids containing the specific bands of male and female parents are true hybrids. PCR amplification reaction conditions: pre denaturation at 95 °C for 4 min; 94°C for 30s, 58°C for 30s, 72°C for 25s, 35 cycles; extension at 72°C for 5min.

(5) PCR amplification products were detected by 8% non-denaturing polyacrylamide gel (Acr: Bis = 39 : 1) electrophoresis.

(6) Backcross and screening of offspring

A breeding scheme by using molecular marker assisted recurrent selection, two seasons a year. The whole cycle needs about three years of generation-adding (as shown in Figure 5). Specifically, HF001, as the female parent (recurrent), and the true hybrid F1, as the male parent, were hybridized. The above hybridization method was applied. The harvested BClF1 was detected by AhyBsec molecular marker again, and the offspring with the specific band of the male parent was retained. The same method was used for DNA extraction and

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molecular marker detection. BC4F1 generation was obtained by backcross and screening for 4 times successively. After selfing, homozygous progeny was selected for seed test and variety registration.

Compared with the traditional method, this invention has a higher screening efficiency. By using the method of combining marker and backcross recurrent selection provided by this invention, the breeding efficiency can be improved, and the germplasm innovation of black peanut can be realized in about 3 years (as shown in Figure 5).

Since the seed coat of peanut is developed from the integument, and the seed coat color is inherited from the female parent, compared with other characters, the color of seed coat can only appear by skipping one generation. Therefore, there is a great blindness to screen peanut by naked eyes and cultivate black peanut. In contrast, this invention adopts the method of molecular marker screening for identification, which can accurately locate the genes, avoid blindness of screening by naked eyes, greatly shorten the screening time, and lay a foundation for quickly providing a large number of germplasm resources of new varieties.

The embodiments described above only describe the preferred method of this invention, and not limit the scope of this invention. On the premise of not deviating from the design spirit of this invention, all modifications and improvements made by ordinary technicians in this field to the technical proposals of this invention shall fall within the protection scope determined by the Claims of this invention.

Claims

Claims 1. A molecular marker AhyBscc closely linked with the black seed coat of peanut, which is characterized in that the specific primer pair of the described molecular marker AhyBscc include:

Forward primer: 5'-AAATGAAAAGACATGTAACATATATAGC-3';

Reverse primer: 5'- AGAAAAACATAACAATGAATTTTCCA -3'.
2. A method for identifying the color of peanut seed coat by using the molecular marker AhyBscc closely linked with the black seed coat of peanut as described in Claim 1, which is characterized in that the method comprises the following steps:

Step 1: Extract DNA from peanut seed or blade;

Step 2: Perform PCR amplification for extracted DNA by using the specific primer pair described in Claim 1;

Step 3: Detect the amplified products obtained by Step 2 by non-denaturing polyacrylamide gel electrophoresis. If the characteristic bands with size of 330bp appear, the next generation of seeds of the peanut material to be tested is determined to have black coat.
3. A method for identifying the color of peanut seed coat by using the molecular marker AhyBscc closely linked with the black seed coat of peanut as described in Claim 2, which is characterized in that in Step 2, the total volume of PCR amplification is 25 L: 1 L for DNA template, 0.5tL for forward primer and reverse primer, 0.5tL for dNTP mix, 2.5[tL for lOxTaq Buffer, 2.0tL for MgCl2, 0.25tL for Taq enzyme, and add water to 25[L.
4. A method for identifying the color of peanut seed coat by using the molecular marker AhyBscc closely linked with the black seed coat of peanut as described in Claim 2, which is characterized in that the PCR amplification reaction conditions in Step 2 are: pre denaturation at 95 °C for 4 min; 94°C for 30s, 58°C for 30s, 72°C for 25s, 35 cycles; extension at 72°C for 5min.
5. A method of identifying the color of peanut seed coat by using the molecular marker AhyBscc closely linked with the black seed coat of peanut as described in Claim 2, which is characterized in that 8% non-denaturing polyacrylamide gel electrophoresis is used in Step 3.
6. An application of the molecular marker AhyBscc closely linked with the black seed coat of peanut as described in Claim 1, which is characterized in that the described molecular marker AhyBscc is located in the region of 108.0-112.7Mb of chromosome 10, and the described molecular marker AhyBscc is applied to peanut breeding and peanut quality improvement.
7. An application of the molecular marker AhyBscc closely linked with the black seed coat of peanut as described in Claim 6, which is characterized in that it is used to identify the peanut with black seed coat.