CN113462810A - SSR marker for identifying citrus varieties and application thereof - Google Patents

SSR marker for identifying citrus varieties and application thereof Download PDF

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CN113462810A
CN113462810A CN202110938018.6A CN202110938018A CN113462810A CN 113462810 A CN113462810 A CN 113462810A CN 202110938018 A CN202110938018 A CN 202110938018A CN 113462810 A CN113462810 A CN 113462810A
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CN113462810B (en
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宋放
蒋迎春
吴黎明
羊林鑫
王志静
何利刚
王策
马小方
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Institute of Fruit and Tea of Hubei Academy of Agricultural Sciences
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Abstract

The invention relates to an SSR marker for identifying citrus varieties, which is CHR2-2 and/or CHR8-4, wherein the reference sequence of CHR2-2 is shown as SEQ ID NO. 1, and the reference sequence of CHR8-4 is shown as SEQ ID NO. 2. The invention also provides a method for identifying the citrus varieties and the filial generation thereof by using the SSR markers. The SSR markers CHR2-2 and CHR8-4 of the invention have polymorphism to common citrus main cultivars. The two SSR markers have good complementarity, and after combination, the two SSR markers have completely different amplification characteristic peaks in almost all common main citrus cultivars, so that almost all common main citrus cultivars can be distinguished, and 99.23% of true offspring can be identified.

Description

SSR marker for identifying citrus varieties and application thereof
Technical Field
The invention relates to the field of citrus breeding, in particular to an SSR marker for identifying citrus varieties and application thereof.
Background
China is a large citrus producing country, and the planting area and the total yield of citrus are in the first place in the world. The scale of the citrus industry in China is rapidly developed in recent decades, and the total yield of the citrus in China reaches 4584.5 ten thousand tons by 2019. At present, 1753 citrus variety resources are saved in China, and a large number of new citrus varieties are bred and introduced in recent years.
The multi-embryo property of citrus severely hinders the hybrid breeding work, so that the new hybrid citrus varieties mainly select a few single-embryo varieties as female parents, such as 'krementanus', 'Qing' and 'Wangcang'. This results in a number of new citrus varieties that have small differences in plant morphology and can only be distinguished by fruit traits, which is not conducive to the identification of new varieties of citrus and hybrid populations, and affects citrus variety renewal and industry upgrading.
Therefore, there is a need for new methods and markers for early identification of citrus varieties and hybrid populations.
Disclosure of Invention
In order to solve the problems, the invention provides an SSR marker for identifying citrus varieties, which is CHR2-2 and/or CHR8-4, wherein the reference sequence of CHR2-2 is shown as SEQ ID NO. 1, and the reference sequence of CHR8-4 is shown as SEQ ID NO. 2.
The invention also provides a method for identifying citrus varieties, which is characterized by comprising the step of detecting PCR amplification peaks corresponding to the SSR markers in claim 1.
In a specific embodiment, the primer sequences used for amplification of CHR2-2 are shown in SEQ ID NOS: 3 and 4, and the primer sequences used for amplification of CHR8-4 are shown in SEQ ID NOS: 5 and 6.
In a specific embodiment, the citrus variety is selected from the group consisting of: kremen, w. morkott, xiajin, satsuma orange, liang, chunxiang, seedless local morning, chunshe, del ta xiao orange, Cocktail grapefruit, taluoke blood orange, pretty No. 28, peach leaf orange, garland essence, kumquat No. 2, sudao, koshio, wokan, qing, huiyan, or hybrid progeny of these varieties.
The SSR markers CHR2-2 and CHR8-4 of the invention have polymorphism to common citrus main cultivars. The two varieties have different amplification characteristic peaks in most of different citrus varieties, and after the two varieties are combined, the two varieties have completely different amplification characteristic peaks in almost all common citrus main cultivars, so that almost all common citrus main cultivars can be distinguished.
The invention also provides a method for identifying the citrus hybrid progeny, which comprises the following steps:
s1: extracting the genome DNA of the citrus filial generation to be identified;
s2: obtaining fragments corresponding to SSR markers of citrus filial generations to be identified through PCR amplification, and counting characteristic peaks, wherein the SSR markers are CHR2-2 and/or CHR8-4, the reference sequence of CHR2-2 is shown as SEQ ID NO. 1, and the reference sequence of CHR8-4 is shown as SEQ ID NO. 2;
s3: and comparing the SSR marker characteristic peak of the citrus filial generation to be identified with the SSR marker characteristic peaks of possible parents to determine the paternity relationship.
In a specific embodiment, the primer sequences used for amplification of CHR2-2 are shown in SEQ ID NOS: 3 and 4, and the primer sequences used for amplification of CHR8-4 are shown in SEQ ID NOS: 5 and 6.
In a specific embodiment, the citrus hybrids to be identified are hybrids of one or more of the following varieties: the citrus species is selected from: kremen, w. morkott, xiajin, satsuma orange, liang, chunxiang, seedless local morning, chunshe, del ta xiao orange, Cocktail grapefruit, taluoke blood orange, pretty No. 28, peach leaf orange, garland essence, kumquat No. 2, sudao, koshio, wokan, qing, huiyan, or hybrid progeny of these varieties.
We found in practice that when CHR2-2 and CHR8-4 were used alone to test hybrid progenies of ` Exclusive ` and ` Oreg.I `, 79.23% and 52.3% of true progenies, respectively, could be identified. However, by combining the two, 99.23% of true progeny can be identified, and the complementarity of the two can be seen to be better.
Drawings
FIG. 1 is a ` clear ` plot of the CHR2-2 tex increase;
FIG. 2 is a ` clear ` plot of the CHR8-4 tex increase;
FIG. 3 is a plot of CHR 2-2T peak increase for "Egan No.";
FIG. 4 is a plot of CHR 8-4T peak increase for "Egan number one".
Detailed Description
The principles and features of this invention are described below in conjunction with the following drawings, which are set forth by way of illustration only and are not intended to limit the scope of the invention.
1. Acquisition of SSR molecular markers
The applicant carries out genome re-sequencing on 'Qing' and 'Orangent I', develops the SSR marker in the whole genome range, and designs the primer of the SSR molecular marker according to the gene structure annotation result and the site information.
PCR amplification and polyacrylamide gel electrophoresis detection are carried out by using a primer pair 'Qing' and 'Orangent I', and a molecular marker with good many-to-many polymorphism is obtained by primary screening, wherein the molecular marker comprises CHR2-2 and CHR8-4, and the sequence of the used primer and the sequence of a reference sequence are shown in Table 1.
TABLE 1 primer sequences and corresponding reference sequences for SSR molecular markers CHR2-2 and CHR8-4
Figure BDA0003213586890000031
Figure BDA0003213586890000041
2. Application of SSR (simple sequence repeat) marker in identification of main cultivated varieties
Genomic DNA of 20 major cultivars of citrus, including Kriman, W.Mokott, Xiaqin, satay orange, Shuangliang, Chunxiang, Seeduo, Deltaxiao, Cocktail grapefruit, Taruoke blood orange, pretty No. 28, peach leaf orange, Relianhu, kumquat No. 2, eosin, unknown fire and wogan, was extracted. The extracted DNA was amplified by PCR using the SSR molecular markers CHR2-2 and CHR8-4 obtained by screening, and the PCR system and reaction procedure were as described above. The amplified fragment peaks of the PCR amplified products were then detected by using STR capillary electrophoresis technology, and as shown in Table 2, the 20 detected conventional varieties were distinguished according to the characteristic peak difference combinations of CHR2-2 and CHR 8-4.
Table 218 characteristic peaks of CHR2-2 and CHR8-4 of conventional citrus varieties
Figure BDA0003213586890000042
Figure BDA0003213586890000051
3. Detecting F1 generation of Qingzhu orange and Egyan orange by SSR molecular marker
The primers and STR capillary electrophoresis technique in Table 1 were used to detect the amplified fragment peaks of the ` Severe ` and ` Orangent I ` PCR amplification products.
As shown in FIGS. 1-4 and Table 3, in the CHR2-2 amplification product, 'clear' has characteristic peaks at 350bp and 368bp, while 'Erkan I' has characteristic peaks at 325bp and 394 bp; in the CHR8-4 amplification product, 'clear' has characteristic peaks at 227bp and 251bp, while 'Egao I' has a characteristic peak at 210 bp.
TABLE 3 statistics of characteristic peaks for ' see ' and ' Oreg.I
Marking Clear characteristic peaks 'Hubei orange I' characteristic peak
CHR2-2 350、368 325、394
CHR8-4 227、251 210
Detecting the amplified fragment peak of PCR amplified products of 130 strains of ' clear ' Erkan I ' hybrid filial generation by using STR capillary electrophoresis technology. As shown in table 4, using CHR2-2 to detect that 103 strains (79.23%) in the progeny can simultaneously amplify the characteristic peak of the parent and the mother, while 29 strains can only amplify the characteristic peak of the mother 'clear'; CHR8-4 was used to detect 68 strains (52.31%) in the offspring, which could amplify the characteristic peak of the parent and the mother simultaneously.
The two markers were used together, and 130 strains were identified as ` X ` Oreg.I `, F1A total of 129 strains (99.23%) in filial generations of the generation are identified as true filial generations, and the identification efficiency and the accuracy are very high.
Table 4 detection of 130 hybrid F1 generations using three SSR markers
Figure BDA0003213586890000052
Figure BDA0003213586890000061
Figure BDA0003213586890000071
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.
Sequence listing
<110> research institute for fruit tree tea of academy of agricultural sciences of Hubei province
<120> SSR marker for identifying citrus varieties and application thereof
<160> 6
<170> SIPOSequenceListing 1.0
<210> 1
<211> 402
<212> DNA
<213> Citrus sinensis
<400> 1
ttatcaattt atagatctgc aaaaaactca cttacattca tacacacaca tatacataca 60
tgtataattt cccaaaaaat aattaagact ttaggttatc actctacgtg ttactttaga 120
aaagcaaccg tatatgcggc agctcagaga cgtgtgtcca acataaaact ttggtttaca 180
tgtacattga agatatatat atatatatat atatatatat atatatatat atatatatat 240
tacagagttt aattctatcc cacataagaa taaataaaaa ttaaagtatt ttaataaata 300
ccattgaaat ttggattaat aacagagtaa ccgtaacaat taactttaat agggtgagat 360
ggggtttggt aagtgcatat tttttttttc ttttccaaaa ga 402
<210> 2
<211> 228
<212> DNA
<213> Citrus sinensis
<400> 2
acaagtacgg caaggtcgtc gatattttca tacctagaga tcgaaggtat tgaaattgta 60
gagggaaatt attattattg ttgttattat tattattgtt gttgctgttg ttgttgttgt 120
tgttgttgtt gttgttatca tcatcatctc ctcatgaaac tgtttttgga aactctcttg 180
ggcaaatgaa aactagattt tttagataac aaattttggg cgttgaag 228
<210> 3
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
aaaactcact tacattcata cacac 25
<210> 4
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
gcacttacca aaccccatct c 21
<210> 5
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
acaagtacgg caaggtcgt 19
<210> 6
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 6
ttcaacgccc aaaatttgtt 20

Claims (7)

1. An SSR marker for identifying citrus varieties is characterized by being CHR2-2 and/or CHR8-4, the reference sequence of CHR2-2 is shown as SEQ ID NO. 1, and the reference sequence of CHR8-4 is shown as SEQ ID NO. 2.
2. A method of identifying a citrus variety comprising the step of detecting PCR amplification peaks corresponding to the SSR marker of claim 1.
3. The method of claim 2, wherein the primer sequences for amplifying CHR2-2 are shown in SEQ ID NOs 3 and 4, and the primer sequences for amplifying CHR8-4 are shown in SEQ ID NOs 5 and 6.
4. The method according to claim 3, wherein the citrus variety is selected from the group consisting of: kremen, w. morkott, xiajin, satsuma orange, liang, chunxiang, seedless local morning, chunshe, del ta xiao orange, Cocktail grapefruit, taluoke blood orange, pretty No. 28, peach leaf orange, garland essence, kumquat No. 2, sudao, koshio, wokan, qing, huiyan, or hybrid progeny of these varieties.
5. A method for identifying citrus hybrids, comprising the steps of:
s1: extracting the genome DNA of the citrus filial generation to be identified;
s2: obtaining fragments corresponding to SSR markers of citrus filial generations to be identified through PCR amplification, and counting characteristic peaks, wherein the SSR markers are CHR2-2 and/or CHR8-4, the reference sequence of CHR2-2 is shown as SEQ ID NO. 1, and the reference sequence of CHR8-4 is shown as SEQ ID NO. 2;
s3: and comparing the SSR marker characteristic peak of the citrus filial generation to be identified with the SSR marker characteristic peaks of possible parents to determine the paternity relationship.
6. The method of claim 5, wherein the primer sequences for amplifying CHR2-2 are shown in SEQ ID NO 3 and 4, and the primer sequences for amplifying CHR8-4 are shown in SEQ ID NO 5 and 6.
7. The method according to claim 5, wherein the citrus hybrids to be identified are hybrids of one or more of the following varieties: the citrus species is selected from: kremen, w. morkott, xiajin, satsuma orange, liang, chunxiang, seedless local morning, chunshe, del ta xiao orange, Cocktail grapefruit, taluoke blood orange, pretty No. 28, peach leaf orange, garland essence, kumquat No. 2, sudao, koshio, wokan, qing, huiyan, or hybrid progeny of these varieties.
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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114058728A (en) * 2021-11-22 2022-02-18 华南农业大学 Set of molecular markers for identifying phyllanthus emblica strain and application thereof
CN116042905A (en) * 2023-03-10 2023-05-02 湖北省农业科学院果树茶叶研究所 SV (space velocity) marker for identifying citrus varieties and application thereof

Citations (2)

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Publication number Priority date Publication date Assignee Title
CN102011196A (en) * 2010-10-21 2011-04-13 中国农业科学院柑桔研究所 Citrus breed standard DNA fingerprint spectrum library and constructing method thereof
CN110273018A (en) * 2019-05-29 2019-09-24 广西壮族自治区农业科学院 Fertile mandarin orange SSR molecular marker and its application

Patent Citations (2)

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Publication number Priority date Publication date Assignee Title
CN102011196A (en) * 2010-10-21 2011-04-13 中国农业科学院柑桔研究所 Citrus breed standard DNA fingerprint spectrum library and constructing method thereof
CN110273018A (en) * 2019-05-29 2019-09-24 广西壮族自治区农业科学院 Fertile mandarin orange SSR molecular marker and its application

Non-Patent Citations (1)

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Title
李益等: "柑橘品种鉴定的SSR标记开发和指纹图谱库构建", 《中国农业科学》 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114058728A (en) * 2021-11-22 2022-02-18 华南农业大学 Set of molecular markers for identifying phyllanthus emblica strain and application thereof
CN114058728B (en) * 2021-11-22 2023-08-08 华南农业大学 Molecular marker set for tea branch citrus strain identification and application thereof
CN116042905A (en) * 2023-03-10 2023-05-02 湖北省农业科学院果树茶叶研究所 SV (space velocity) marker for identifying citrus varieties and application thereof

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