CN110229928A - Molecular labeling combination and its application for Eggplant Germplasm Resources identification - Google Patents

Molecular labeling combination and its application for Eggplant Germplasm Resources identification Download PDF

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CN110229928A
CN110229928A CN201910567356.6A CN201910567356A CN110229928A CN 110229928 A CN110229928 A CN 110229928A CN 201910567356 A CN201910567356 A CN 201910567356A CN 110229928 A CN110229928 A CN 110229928A
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CN110229928B (en
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袁欣捷
方荣
陈学军
周坤华
雷刚
黄月琴
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VEGETABLE AND FLOWER INSTITUTE JIANGXI ACADEMY OF AGRICULTURAL SCIENCES
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Abstract

The present invention is provided to the molecular labeling combinations and its application of Eggplant Germplasm Resources identification.Molecular labeling combination is made of 17 SNP and InDel label, for expanding the primer sequence of the molecular labeling combination respectively as shown in SEQ ID NO:1-18.Molecular labeling combination provided by the invention, has expanded eggplant serviceable indicia site range, and the method for combining 12 parts of Eggplant Germplasm Resources of identification using the molecular labeling is easy to operate, and accuracy is high, as a result reliably.The present invention has most important theories and application value to Eggplant Germplasm Resources identification, DNA fingerprinting drafting, kind protection and management etc..

Description

Molecular labeling combination and its application for Eggplant Germplasm Resources identification
Technical field
The present invention relates to molecular biology fields, specifically, being related to the molecular labeling identified for Eggplant Germplasm Resources Combination and its application.
Background technique
Eggplant (Solanum melongena L.) is the important cultivated crop of Solanaceae Solanum, contains saponin, vitamin P, the nutritional ingredients such as calcium, phosphorus, economic value with higher and nutritive value, all over the world extensively plantation, be Asia, in The important vegetable in east, the Near East, southern Europe and Africa.For eggplant originating from Tropical Asian area, ancient Gingko is its domestication ground earliest, Production and consumption big country of the China as eggplant, it is considered to be the second area of origin of eggplant.
The eggplant cultivar genetic background relative narrowness after taming for many years has between many germ plasm resources similar Phenetic Shape and genetic background increase difficulty to the identification of germ plasm resource.The identification method of Eggplant Germplasm Resources is mainly form at present Method identifies difficult and inaccuracy.Molecular labeling has many advantages, such as not high by such environmental effects, accuracy, molecular labeling Development and application be of great significance to the identification of Eggplant Germplasm Resources.Wherein SNP (single nucleotide Polymorphism) and InDel (insertion-deletion) label is wide with distribution, density is high, stability is good, variation is rich The features such as rich, can directly react the difference of plant gene level, be the ideal mark of Eggplant Germplasm Resources identification.
Since eggplant (S.melongena L.) is planted, interior germ plasm resource hereditary difference is smaller, and only voucher genetic fragment is more State site is difficult to successfully identify different resource and kind, and the present invention comprehensively utilizes SNP the and InDel mark group of different genes segment Synthesis function identifies 12 parts of Eggplant Germplasm Resources.
Summary of the invention
The object of the present invention is to provide the molecular labeling identified for Eggplant Germplasm Resources combination and its applications.
In order to achieve the object of the present invention, inventor devises the primer that can be used for eggplant PCR amplification, and provides and can be used for eggplant The SNP and InDel of sub- germplasm identification mark combination, successfully filter out 12 parts of eggplant germplasm money with special haplotype Source.
In a first aspect, the present invention is provided to the molecular labeling combination of Eggplant Germplasm Resources identification, the molecular labeling group It closes by SI-1, SI-5, SI-9, SI-14, SI-15 totally 5 InDel label and SI-2, SI-3, SI-4, SI-6, SI-7, SI- 8, totally 12 SNP markers form by SI-10, SI-11, SI-12, SI-13, SI-16, SI-17.
Label SI-1 contains polymorphic at the nucleotide sequence 100bp as shown in SEQ ID NO:19 in eggplant genome Property be G/- nucleotide sequence；
Label SI-2 contains polymorphic at the nucleotide sequence 437bp as shown in SEQ ID NO:19 in eggplant genome Property be A/C nucleotide sequence；
Label SI-3 contains polymorphic at the nucleotide sequence 100bp as shown in SEQ ID NO:20 in eggplant genome Property be T/A nucleotide sequence；
Label SI-4 contains polymorphic at the nucleotide sequence 141bp as shown in SEQ ID NO:20 in eggplant genome Property be G/A nucleotide sequence；
Label SI-5 contains 365~368bp of nucleotide sequence as shown in SEQ ID NO:20 in eggplant genome Locate the nucleotide sequence that polymorphism is CTCT/-；
Label SI-6 contains polymorphic at the nucleotide sequence 100bp as shown in SEQ ID NO:21 in eggplant genome Property be C/A nucleotide sequence；
Label SI-7 contains polymorphic at the nucleotide sequence 54bp as shown in SEQ ID NO:22 in eggplant genome Property be G/A nucleotide sequence；
Label SI-8 contains polymorphic at the nucleotide sequence 78bp as shown in SEQ ID NO:22 in eggplant genome Property be C/G nucleotide sequence；
Label SI-9 contains in eggplant genome at 28~33bp of nucleotide sequence as shown in SEQ ID NO:23 Polymorphism is the nucleotide sequence of GCTAGA/-；
Label SI-10 contains more at the nucleotide sequence 569bp as shown in SEQ ID NO:23 in eggplant genome State property is the nucleotide sequence of C/T；
Label SI-11 contains more at the nucleotide sequence 100bp as shown in SEQ ID NO:24 in eggplant genome State property is the nucleotide sequence of T/A；
Label SI-12 contains more at the nucleotide sequence 118bp as shown in SEQ ID NO:24 in eggplant genome State property is the nucleotide sequence of C/T；
Label SI-13 contains more at the nucleotide sequence 329bp as shown in SEQ ID NO:24 in eggplant genome State property is the nucleotide sequence of G/C；
Label SI-14 contains more at the nucleotide sequence 116bp as shown in SEQ ID NO:25 in eggplant genome State property is the nucleotide sequence of G/-；
Label SI-15 contains 354~364bp of nucleotide sequence as shown in SEQ ID NO:25 in eggplant genome Locate the nucleotide sequence that polymorphism is AATGTATAACA/-；
Label SI-16 contains more at the nucleotide sequence 100bp as shown in SEQ ID NO:26 in eggplant genome State property is the nucleotide sequence of T/A；
Label SI-17 contains more at the nucleotide sequence 100bp as shown in SEQ ID NO:27 in eggplant genome State property is the nucleotide sequence of T/G；
Wherein, the Eggplant Germplasm Resources be selected from Hunan morning eggplant, kermes eggplant (V06B0149), dog tail eggplant (V06B0458), Black beauty (V06B0541), Vetrioe (V06B0543), red skin cold eggplant (V06B0552), Nanfeng County line eggplant (V06B0556), Shexian County The white eggplant (V06B0978) of circle, rattan bridge purple eggplant (V06B1046), oil tank eggplant (V06B0357), purple circle eggplant (V06B0940), Solanummammosum (V06B1256)。
Second aspect, the present invention is provided to the molecular labeling combination primer, including as follows 1.~9. totally 9 groups draw Object:
1. the primer sequence for expanding label SI-1, SI-2 is as shown in SEQ ID NO:1 and SEQ ID NO:2；
2. the primer sequence for expanding label SI-3, SI-4, SI-5 is as shown in SEQ ID NO:3 and SEQ ID NO:4；
3. the primer sequence for expanding label SI-6 is as shown in SEQ ID NO:5 and SEQ ID NO:6；
4. the primer sequence for expanding label SI-7, SI-8 is as shown in SEQ ID NO:7 and SEQ ID NO:8；
5. the primer sequence for expanding label SI-9, SI-10 is as shown in SEQ ID NO:9 and SEQ ID NO:10；
6. the primer sequence such as SEQ ID NO:11 and SEQ ID NO:12 for expanding label SI-11, SI-12, SI-13 It is shown；Wherein,
7. the primer sequence for expanding label SI-14, SI-15 is as shown in SEQ ID NO:13 and SEQ ID NO:14； Wherein,
8. the primer sequence for expanding label SI-16 is as shown in SEQ ID NO:15 and SEQ ID NO:16；
9. the primer sequence for expanding label SI-17 is as shown in SEQ ID NO:17 and SEQ ID NO:18.
In above-mentioned primer sequence, r indicate degeneracy base A or G, y indicate degeneracy base C or T, d indicate degeneracy bases G, A or T, m indicate that degeneracy base A or C, s indicate that degeneracy bases G or C, w indicate that degeneracy base A or T, k indicate degeneracy bases G or T.
The third aspect, the present invention provide the detection reagent or kit for containing the primer.
Fourth aspect, the present invention provide the molecular labeling combination, the primer or the detection reagent or kit Following any application:
1) it is identified for Eggplant Germplasm Resources；
2) it is constructed for eggplant DNA fingerprinting；
3) it is used for eggplant molecular mark.
5th aspect, the present invention provide a kind of method for identifying Eggplant Germplasm Resources, include the following steps:
(1) using the genomic DNA of eggplant material to be measured as template, PCR amplification is carried out using primer described in claim 2, Obtain PCR product；
(2) PCR product is analyzed.
Step (2) specifically: PCR product is sequenced, sequencing result is compared with " molecular labeling combines XII " Right, " molecular labeling combines XII " is following a1)~a12) totally 12 combinations:
a1)GSI-1ASI-2TSI-3ASI-4(CTCT)SI-5CSI-6GSI-7CSI-8(GCTAGA)SI-9CSI-10TSI-11CSI-12GSI- 13GSI-14-SI-15TSI-16TSI-17；
a2)GSI-1ASI-2ASI-3ASI-4(CTCT)SI-5ASI-6GSI-7CSI-8(GCTAGA)SI-9CSI-10TSI-11CSI-12GSI- 13GSI-14-SI-15TSI-16TSI-17；
a3)-SI-1CSI-2TSI-3GSI-4-SI-5CSI-6GSI-7CSI-8(GCTAGA)SI-9CSI-10ASI-11TSI-12CSI-13GSI-14-SI- 15TSI-16TSI-17；
a4)GSI-1ASI-2ASI-3ASI-4(CTCT)SI-5CSI-6GSI-7CSI-8(GCTAGA)SI-9TSI-10TSI-11CSI-12GSI- 13ASI-14(AATGTATAACA)SI-15TSI-16TSI-17；
a5)-SI-1CSI-2ASI-3ASI-4-SI-5CSI-6GSI-7CSI-8(GCTAGA)SI-9CSI-10TSI-11CSI-12GSI-13GSI-14-SI- 15TSI-16TSI-17；
a6)GSI-1ASI-2ASI-3ASI-4(CTCT)SI-5CSI-6GSI-7CSI-8(GCTAGA)SI-9CSI-10ASI-11TSI-12CSI- 13ASI-14(AATGTATAACA)SI-15TSI-16TSI-17；
a7)GSI-1ASI-2TSI-3GSI-4-SI-5CSI-6GSI-7CSI-8(GCTAGA)SI-9CSI-10WSI-11YSI-12SSI-13GSI-14-SI- 15TSI-16TSI-17；
a8)GSI-1ASI-2ASI-3ASI-4(CTCT)SI-5CSI-6GSI-7CSI-8-SI-9CSI-10TSI-11CSI-12GSI-13GSI-14-SI- 15TSI-16TSI-17；
a9)GSI-1ASI-2ASI-3ASI-4(CTCT)SI-5ASI-6GSI-7CSI-8(GCTAGA)SI-9CSI-10TSI-11CSI-12GSI- 13GSI-14-SI-15TSI-16GSI-17；
a10)GSI-1ASI-2TSI-3GSI-4-SI-5CSI-6GSI-7CSI-8(GCTAGA)SI-9CSI-10TSI-11CSI-12GSI-13GSI-14-SI- 15ASI-16TSI-17；
a11)GSI-1ASI-2TSI-3GSI-4-SI-5CSI-6GSI-7CSI-8(GCTAGA)SI-9CSI-10ASI-11TSI-12CSI-13GSI-14-SI- 15TSI-16TSI-17；
a12)GSI-1ASI-2ASI-3ASI-4(CTCT)SI-5CSI-6GSI-7GSI-8(GCTAGA)SI-9CSI-10TSI-11CSI-12GSI- 13GSI-14-SI-15TSI-16TSI-17；
In above-mentioned molecular labeling combination ,-indicate base deletion；W indicates A and T；Y indicates C and T；S indicates that G and C are (i.e. described The corresponding site of Eggplant Germplasm Resources to be measured is heterozygous sites)；
Wherein, the SI-1 is the site InDel, is located at the nucleotide sequence as shown in SEQ ID NO:19 in eggplant genome The 100th bit base n, the site InDel be G/- it is polymorphic；
The SI-2 is SNP site, the 437th of the nucleotide sequence as shown in SEQ ID NO:19 in eggplant genome Bit base n, the SNP site are that A/C is polymorphic；
The SI-3 is SNP site, the 100th of the nucleotide sequence as shown in SEQ ID NO:20 in eggplant genome Bit base n, the SNP site are that T/A is polymorphic；
The SI-4 is SNP site, the 141st of the nucleotide sequence as shown in SEQ ID NO:20 in eggplant genome Bit base n, the SNP site are that G/A is polymorphic；
The SI-5 is the site InDel, the of the nucleotide sequence as shown in SEQ ID NO:20 in eggplant genome 365~368 bit base nnnn, the site InDel are that CTCT/- is polymorphic；
The SI-6 is SNP site, the 100th of the nucleotide sequence as shown in SEQ ID NO:21 in eggplant genome Bit base n, the SNP site are that C/A is polymorphic；
The SI-7 is SNP site, the 54th of the nucleotide sequence as shown in SEQ ID NO:22 in eggplant genome Bit base n, the SNP site are that G/A is polymorphic；
The SI-8 is SNP site, the 78th of the nucleotide sequence as shown in SEQ ID NO:22 in eggplant genome Bit base n, the SNP site are that C/G is polymorphic；
The SI-9 is the site InDel, the of the nucleotide sequence as shown in SEQ ID NO:23 in eggplant genome 28~33 bit base nnnnnn, the site InDel are that GCTAGA/- is polymorphic；
The SI-10 is SNP site, the of the nucleotide sequence as shown in SEQ ID NO:23 in eggplant genome 569 bit base n, the SNP site are that C/T is polymorphic；
The SI-11 is SNP site, the of the nucleotide sequence as shown in SEQ ID NO:24 in eggplant genome 100 bit base n, the SNP site are that T/A is polymorphic；
The SI-12 is SNP site, the of the nucleotide sequence as shown in SEQ ID NO:24 in eggplant genome 118 bit base n, the SNP site are that C/T is polymorphic；
The SI-13 is SNP site, the of the nucleotide sequence as shown in SEQ ID NO:24 in eggplant genome 329 bit base n, the SNP site are that G/C is polymorphic；
The SI-14 is the site InDel, the of the nucleotide sequence as shown in SEQ ID NO:25 in eggplant genome 116 bit base n, the site InDel are that G/- is polymorphic；
The SI-15 is the site InDel, the of the nucleotide sequence as shown in SEQ ID NO:25 in eggplant genome 354~364 bit base nnnnnnnnnnn, the site InDel are that AATGTATAACA/- is polymorphic；
The SI-16 is SNP site, the of the nucleotide sequence as shown in SEQ ID NO:26 in eggplant genome 100 bit base n, the SNP site are that T/A is polymorphic；
The SI-17 is SNP site, the of the nucleotide sequence as shown in SEQ ID NO:27 in eggplant genome 100 bit base n, the SNP site are that T/G is polymorphic；
Finally, being identified according to comparison result the eggplant material to be measured:
If the molecular labeling group of the eggplant material to be measured is combined into a1) shown in molecular labeling combination, the eggplant to be measured Son is or candidate is Hunan morning eggplant；
If the molecular labeling group of the eggplant material to be measured is combined into a2) shown in molecular labeling combination, the eggplant to be measured Son is or candidate is kermes eggplant (V06B0149)；
If the molecular labeling group of the eggplant material to be measured is combined into a3) shown in molecular labeling combination, the eggplant to be measured Son is or candidate is dog tail eggplant (V06B0458)；
If the molecular labeling group of the eggplant material to be measured is combined into a4) shown in molecular labeling combination, the eggplant to be measured Son is or candidate is black beautiful (V06B0541)；
If the molecular labeling group of the eggplant material to be measured is combined into a5) shown in molecular labeling combination, the eggplant to be measured Son is or candidate is Vetrioe (V06B0543)；
If the molecular labeling group of the eggplant material to be measured is combined into a6) shown in molecular labeling combination, the eggplant to be measured Son is or candidate is that red skin trembles with fear eggplant (V06B0552)；
If the molecular labeling group of the eggplant material to be measured is combined into a7) shown in molecular labeling combination, the eggplant to be measured Son is or candidate is Nanfeng County line eggplant (V06B0556)；
If the molecular labeling group of the eggplant material to be measured is combined into a8) shown in molecular labeling combination, the eggplant to be measured Son is or candidate is the white eggplant (V06B0978) of Shexian County circle；
If the molecular labeling group of the eggplant material to be measured is combined into a9) shown in molecular labeling combination, the eggplant to be measured Son is or candidate is rattan bridge purple eggplant (V06B1046)；
If the molecular labeling group of the eggplant material to be measured is combined into a10) shown in molecular labeling combination, the eggplant to be measured Son is or candidate is oil tank eggplant (V06B0357)；
If the molecular labeling group of the eggplant material to be measured is combined into a11) shown in molecular labeling combination, the eggplant to be measured Son is or candidate is purple circle eggplant (V06B0940)；
If the molecular labeling group of the eggplant material to be measured is combined into a12) shown in molecular labeling combination, the eggplant to be measured Son is or candidate is Solanummammosum (V06B1256).
By above-mentioned technical proposal, the present invention at least have following advantages and the utility model has the advantages that
(1) method identification Eggplant Germplasm Resources provided by the invention are accurately reliable, and the management of Eggplant Germplasm Resources can be improved And protection, effectively make up the deficiency of previous methods.
(2) the molecular labeling combination of identification Eggplant Germplasm Resources provided by the invention simplifies evaluator, operation letter Single, favorable reproducibility is conducive to promote.
(3) present invention has expanded eggplant serviceable indicia site range from gene level, mentions for Eggplant Germplasm Resources identification New approaches and method are supplied.
(4) application of the invention is that the apolegamy of parent in crossbreeding is laid a good foundation, and is germplasm money in breeding and production The accuracy of source selection provides guarantee, identifies Eggplant Germplasm Resources, and DNA fingerprinting is drawn, kind protection and management etc. Aspect has most important theories and application value.
Specific embodiment
The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention..Unless otherwise specified, embodiment According to conventional laboratory conditions, such as Sambrook molecular cloning experiment handbook (Sambrook J&Russell DW, Molecular Cloning:a Laboratory Manual, 2001), or according to the condition of manufacturer's specification suggestion.
Eggplant material used in the following embodiment: Hunan morning eggplant is commercial variety, kermes eggplant (V06B0149), dog tail eggplant (V06B0458), black beautiful (V06B0541), Vetrioe (V06B0543), red skin cold eggplant (V06B0552), Nanfeng County line eggplant (V06B0556), the white eggplant (V06B0978) of Shexian County circle, rattan bridge purple eggplant (V06B1046), oil tank eggplant (V06B0357), purple circle eggplant (V06B0940) and Solanummammosum (V06B1256) derives from national genebank.
Embodiment 1 identifies the foundation and its application of Eggplant Germplasm Resources method using SNP and InDel label combination
1, experimental material
Experimental material include 55 parts of eggplant materials: Hunan morning eggplant, kermes eggplant (V06B0149), dog tail eggplant (V06B0458), Black beauty (V06B0541), Vetrioe (V06B0543), red skin cold eggplant (V06B0552), Nanfeng County line eggplant (V06B0556), Shexian County The white eggplant (V06B0978) of circle, rattan bridge purple eggplant (V06B1046), oil tank eggplant (V06B0357), purple circle eggplant (V06B0940), Solanummammosum (V06B1256), bamboo silk eggplant, six leaf eggplants (V06B0039), white eggplant (V06B0066), purple long eggplant (V06B0092), swamp eel head eggplant (V06B1231), Yongan silver pearl eggplant (V06B0108), white elephant child son (V06B0117), prosperous step purple long eggplant (V06B0150), dirty oil Eggplant (V06B0476), flat eggplant (V06B0841), six leaf eggplants (V06B1027), black Herba Ipomoeae Cairicae (V06B1103), new peace smalt eggplant (V06B1108), 86-1 flower training eggplant (V06B1131), green eggplant (V06B1134), Dubi (V06B1186), newly grow it is abnormal (V06B1187), Suzhou ox horn (V06B1188), rough green eggplant (V06B1199), the white eggplant in Pingxiang (V06B1204), Ningbo rattan eggplant (V06B1257), do lipstick eggplant (V06B1258), Fleralba (V06B1338), Bai Yuanqie (V06B1387), Jiangkou pocket eggplant (V06B0559), Zhucheng bracket eggplant (V06B0677), four leaf eggplants (V06B0774), assist the rough green eggplant (V06B1119) in county, spinosity wild Raw (V06B0140), small green eggplant (V06B1297), green circle eggplant (V06B1363), flower eggplant (V06B1043), Shaoguan open country eggplant, improvement Tuo Tuosijia, improvement Solanum Torvum, red eggplant (Hainan Introducing), the green eggplant (V06B1200) of Brazil, YZCM (V06B1305), the Congo eggplant (V06B0523), eggplant (V06B0846), pearl eggplant (V06B1220), ornamental eggplant (V06B1233), Soclanum gibo are watched (V06B1349) and Melanzana Ebany (V06B1299).
2, design of primers
In order to construct SNP and InDel the label combination that can be used to effectively identify Eggplant Germplasm Resources, the present invention selects at random It is its over-designed primer positioned at the single copy gene of eggplant different chromosomes, by carrying out screening verification to PCR primer, most Determine that 9 groups of primers are used for the identification of Eggplant Germplasm Resources eventually.Primer one is SEQ ID NO:1 and SEQ ID NO:2, primer two are SEQ ID NO:3 and SEQ ID NO:4, primer three are SEQ ID NO:5 and SEQ ID NO:6, and primer four is SEQ ID NO:7 With SEQ ID NO:8, primer five is SEQ ID NO:9 and SEQ ID NO:10, and primer six is SEQ ID NO:11 and SEQ ID NO:12, primer seven are SEQ ID NO:13 and SEQ ID NO:14, and primer eight is SEQ ID NO:15 and SEQ ID NO:16, Primer nine is SEQ ID NO:17 and SEQ ID NO:18.
In above-mentioned primer sequence, r indicate degeneracy base A or G, y indicate degeneracy base C or T, d indicate degeneracy bases G, A or T, m indicate that degeneracy base A or C, s indicate that degeneracy bases G or C, w indicate that degeneracy base A or T, k indicate degeneracy bases G or T.
3, PCR product sequencing and polymorphic site screening
Using the young leaflet tablet of material to be tested as experimental material, DNA of plants is extracted using CTAB method, using DNA of plants as template, With the primer pair in step 2, it carries out PCR amplification.The results show that every part of Eggplant Germplasm Resources available one clearly special Different band.It send PCR product to sequencing, the ropy sequence in both ends is deleted, retain the high sequence of intermediate mass.Utilize MUSCLE Script will be compared respectively for 9 genetic fragments for trying eggplant, screen SNP and InDel polymorphic site therein.
The results show that SNP the and InDel polymorphic site that screening obtains is as follows:
SI-1 and SI-2 is respectively InDel and SNP site, positioned at the 100th of nucleotide sequence shown in SEQ ID NO:19 the Position and the 437th；SI-3, SI-4, SI-5 are respectively SNP site, SNP site and the site InDel, are located at SEQ ID NO: The 100th, the 141st and the 365th~368 of nucleotide sequence shown in 20；SI-6 is SNP site, is located at SEQ ID NO: The 100th of nucleotide sequence shown in 21；SI-7 and SI-8 is SNP site, is located at nucleotide sequence shown in SEQ ID NO:22 The 54th and the 78th；SI-9 and SI-10 is respectively the site InDel and SNP site, is located at nucleosides shown in SEQ ID NO:23 The 28th~33 of acid sequence and the 569th；SI-11, SI-12 and SI-13 are SNP site, are located at SEQ ID NO:24 The 100th, the 118th and the 329th of shown nucleotide sequence；The SI-14 and SI-15 is the site InDel, respectively position The 116th of the nucleotide sequence shown in SEQ ID NO:25 and the 354th~364；SI-16 is SNP site, is located at SEQ ID The 100th of nucleotide sequence shown in NO:26；SI-17 is SNP site, positioned at nucleotide sequence shown in SEQ ID NO:27 100th.
4, the building of Eggplant Germplasm Resources molecular labeling combination
Screen in step 2 17 polymorphic sites are intercepted from 55 parts of materials and comes out and is arranged in order, therefrom 12 special haplotypes are filtered out, 12 molecular labeling combinations are formed:
a1)GSI-1ASI-2TSI-3ASI-4(CTCT)SI-5CSI-6GSI-7CSI-8(GCTAGA)SI-9CSI-10TSI-11CSI-12GSI- 13GSI-14-SI-15TSI-16TSI-17；
a2)GSI-1ASI-2ASI-3ASI-4(CTCT)SI-5ASI-6GSI-7CSI-8(GCTAGA)SI-9CSI-10TSI-11CSI-12GSI- 13GSI-14-SI-15TSI-16TSI-17；
a3)-SI-1CSI-2TSI-3GSI-4-SI-5CSI-6GSI-7CSI-8(GCTAGA)SI-9CSI-10ASI-11TSI-12CSI-13GSI-14-SI- 15TSI-16TSI-17；
a4)GSI-1ASI-2ASI-3ASI-4(CTCT)SI-5CSI-6GSI-7CSI-8(GCTAGA)SI-9TSI-10TSI-11CSI-12GSI- 13ASI-14(AATGTATAACA)SI-15TSI-16TSI-17；
a5)-SI-1CSI-2ASI-3ASI-4-SI-5CSI-6GSI-7CSI-8(GCTAGA)SI-9CSI-10TSI-11CSI-12GSI-13GSI-14-SI- 15TSI-16TSI-17；
a6)GSI-1ASI-2ASI-3ASI-4(CTCT)SI-5CSI-6GSI-7CSI-8(GCTAGA)SI-9CSI-10ASI-11TSI-12CSI- 13ASI-14(AATGTATAACA)SI-15TSI-16TSI-17；
a7)GSI-1ASI-2TSI-3GSI-4-SI-5CSI-6GSI-7CSI-8(GCTAGA)SI-9CSI-10WSI-11YSI-12SSI-13GSI-14-SI- 15TSI-16TSI-17；
a8)GSI-1ASI-2ASI-3ASI-4(CTCT)SI-5CSI-6GSI-7CSI-8-SI-9CSI-10TSI-11CSI-12GSI-13GSI-14-SI- 15TSI-16TSI-17；
a9)GSI-1ASI-2ASI-3ASI-4(CTCT)SI-5ASI-6GSI-7CSI-8(GCTAGA)SI-9CSI-10TSI-11CSI-12GSI- 13GSI-14-SI-15TSI-16GSI-17；
a10)GSI-1ASI-2TSI-3GSI-4-SI-5CSI-6GSI-7CSI-8(GCTAGA)SI-9CSI-10TSI-11CSI-12GSI-13GSI-14-SI- 15ASI-16TSI-17；
a11)GSI-1ASI-2TSI-3GSI-4-SI-5CSI-6GSI-7CSI-8(GCTAGA)SI-9CSI-10ASI-11TSI-12CSI-13GSI-14-SI- 15TSI-16TSI-17；
a12)GSI-1ASI-2ASI-3ASI-4(CTCT)SI-5CSI-6GSI-7GSI-8(GCTAGA)SI-9CSI-10TSI-11CSI-12GSI- 13GSI-14-SI-15TSI-16TSI-17。
Note: "-" indicates base deletion；A, T, C and G indicate to show that simple spike, W, Y and S are indicated in sequencing in sequencing Be shown as being superimposed it is bimodal, wherein W indicate A and T；Y indicates C and T；S indicates G and C.
The combination of above-mentioned molecular labeling is corresponding in turn to Hunan morning eggplant, kermes eggplant (V06B0149), dog tail eggplant (V06B0458), black Beautiful (V06B0541), Vetrioe (V06B0543), red skin cold eggplant (V06B0552), Nanfeng County line eggplant (V06B0556), Shexian County circle White eggplant (V06B0978), rattan bridge purple eggplant (V06B1046), oil tank eggplant (V06B0357), purple circle eggplant (V06B0940) and Solanummammosum (V06B1256)。
5, identification Eggplant Germplasm Resources are combined with molecular labeling
(1) using the genomic DNA of eggplant material to be measured as template, PCR amplification is carried out to it by one~primer of primer nine, PCR product send sequencing；
Pcr amplification reaction system: 2 × Es Taq MasterMix (Dye), 30 μ L, 10 μM of forward primers, reverse primers are each 1.2 μ L, 100ng/ μ L DNA profiling 3.0 μ L, ddH2O polishing is to 60 μ L.Wherein, Es Taq MasterMix is century from health The commercial kit CW0690 of Biotechnology Co., Ltd.
PCR response procedures: 94 DEG C of initial denaturation 2min；94 DEG C of denaturation 30s, 53~60 DEG C of annealing 30s, 72 DEG C of extension 2min, 38 circulations；72 DEG C of extension 5min.
(2) nucleotide sequence shown in the sequence and SEQ ID NO:19 obtained from the amplification sequencing of primer one carries out MUSCLE ratio It is right, wherein corresponding to the 100th of SEQ ID NO:19 and the 437th bit base is the site SI-1 and SI-2；The amplification sequencing of primer two Nucleotide sequence shown in obtained sequence and SEQ ID NO:20 carries out MUSCLE comparison, wherein the of corresponding SEQ ID NO:20 100, the 141st and the 365th~368 bit base are the site SI-3, SI-4 and SI-5；The sequence that the amplification sequencing of primer three obtains MUSCLE comparison is carried out with nucleotide sequence shown in SEQ ID NO:21, wherein the 100th bit base of corresponding SEQ ID NO:21 For the site SI-6；Nucleotide sequence shown in the sequence and SEQ ID NO:22 that the amplification sequencing of primer four obtains carries out MUSCLE pairs Than wherein corresponding to the 54th of SEQ ID NO:22 and the 78th bit base is the site SI-7 and SI-8；The amplification of primer five is sequenced Nucleotide sequence shown in the sequence and SEQ ID NO:23 arrived carries out MUSCLE comparison, wherein the 28th of corresponding SEQ ID NO:23 the ~33 and the 569th bit base are the site SI-9 and SI-10；The sequence and SEQ ID NO:24 institute that the amplification sequencing of primer six obtains Show that nucleotide sequence carries out MUSCLE comparison, wherein the 100th, the 118th and the 329th bit base of corresponding SEQ ID NO:23 For the site SI-11, SI-12 and SI-13；Nucleotide sequence shown in the sequence and SEQ ID NO:25 that the amplification sequencing of primer seven obtains MUSCLE comparison is carried out, wherein corresponding to the 116th of SEQ ID NO:25 and the 354th~364 bit base is SI-14 and SI-15 Site；The sequence that the amplification sequencing of primer eight obtains carries out MUSCLE with nucleotide sequence shown in SEQ ID NO:26 and compares, wherein The 100th bit base of corresponding SEQ ID NO:26 is the site SI-16；The sequence and SEQ ID NO that the amplification sequencing of primer nine obtains: Nucleotide sequence shown in 27 carries out MUSCLE comparison, wherein the 100th bit base of corresponding SEQ ID NO:27 is the site SI-17.
(3) site SI-1~SI-17 in step (2) is arranged in order to the molecule mark for forming each eggplant material to be measured The merging of note group identifies it:
If the molecular labeling group of the eggplant material to be measured is combined into GSI-1ASI-2TSI-3ASI-4(CTCT)SI-5CSI-6GSI-7CSI-8 (GCTAGA)SI-9CSI-10TSI-11CSI-12GSI-13GSI-14-SI-15TSI-16TSI-17Shown in molecular labeling combination, then the eggplant to be measured Son is or candidate is Hunan morning eggplant；
If the molecular labeling group of the eggplant material to be measured is combined into GSI-1ASI-2ASI-3ASI-4(CTCT)SI-5ASI-6GSI-7CSI-8 (GCTAGA)SI-9CSI-10TSI-11CSI-12GSI-13GSI-14-SI-15TSI-16TSI-17Shown in molecular labeling combination, then the eggplant to be measured Son is or candidate is kermes eggplant (V06B0149)；
If the molecular labeling group of the eggplant material to be measured is combined into-SI-1CSI-2TSI-3GSI-4-SI-5CSI-6GSI-7CSI-8 (GCTAGA)SI-9CSI-10ASI-11TSI-12CSI-13GSI-14-SI-15TSI-16TSI-17Shown in molecular labeling combination, then the eggplant to be measured Son is or candidate is dog tail eggplant (V06B0458)；
If the molecular labeling group of the eggplant material to be measured is combined into GSI-1ASI-2ASI-3ASI-4(CTCT)SI-5CSI-6GSI-7CSI-8 (GCTAGA)SI-9TSI-10TSI-11CSI-12GSI-13ASI-14(AATGTATAACA)SI-15TSI-16TSI-17Shown in molecular labeling combination, Then the eggplant to be measured is or candidate is black beautiful (V06B0541)；
If the molecular labeling group of the eggplant material to be measured is combined into-SI-1CSI-2ASI-3ASI-4-SI-5CSI-6GSI-7CSI-8 (GCTAGA)SI-9CSI-10TSI-11CSI-12GSI-13GSI-14-SI-15TSI-16TSI-17Shown in molecular labeling combination, then the eggplant to be measured Son is or candidate is Vetrioe (V06B0543)；
If the molecular labeling group of the eggplant material to be measured is combined into GSI-1ASI-2ASI-3ASI-4(CTCT)SI-5CSI-6GSI-7CSI-8 (GCTAGA)SI-9CSI-10ASI-11TSI-12CSI-13ASI-14(AATGTATAACA)SI-15TSI-16TSI-17Shown in molecular labeling combination, Then the eggplant to be measured is or candidate is that red skin trembles with fear eggplant (V06B0552)；
If the molecular labeling group of the eggplant material to be measured is combined into GSI-1ASI-2TSI-3GSI-4-SI-5CSI-6GSI-7CSI-8 (GCTAGA)SI-9CSI-10WSI-11YSI-12SSI-13GSI-14-SI-15TSI-16TSI-17Shown in molecular labeling combination, then the eggplant to be measured Son is or candidate is Nanfeng County line eggplant (V06B0556)；
If the molecular labeling group of the eggplant material to be measured is combined into GSI-1ASI-2ASI-3ASI-4(CTCT)SI-5CSI-6GSI- 7CSI-8-SI-9CSI-10TSI-11CSI-12GSI-13GSI-14-SI-15TSI-16TSI-17Shown in molecular labeling combination, then the eggplant to be measured is Or candidate is the white eggplant (V06B0978) of Shexian County circle；
If the molecular labeling group of the eggplant material to be measured is combined into GSI-1ASI-2ASI-3ASI-4(CTCT)SI-5ASI-6GSI-7CSI-8 (GCTAGA)SI-9CSI-10TSI-11CSI-12GSI-13GSI-14-SI-15TSI-16GSI-17Shown in molecular labeling combination, then the eggplant to be measured Son is or candidate is rattan bridge purple eggplant (V06B1046)；
If the molecular labeling group of the eggplant material to be measured is combined into GSI-1ASI-2TSI-3GSI-4-SI-5CSI-6GSI-7CSI-8 (GCTAGA)SI-9CSI-10TSI-11CSI-12GSI-13GSI-14-SI-15ASI-16TSI-17Shown in molecular labeling combination, then the eggplant to be measured Son is or candidate is oil tank eggplant (V06B0357)；
If the molecular labeling group of the eggplant material to be measured is combined into GSI-1ASI-2TSI-3GSI-4-SI-5CSI-6GSI-7CSI-8 (GCTAGA)SI-9CSI-10ASI-11TSI-12CSI-13GSI-14-SI-15TSI-16TSI-17Shown in molecular labeling combination, then the eggplant to be measured Son is or candidate is purple circle eggplant (V06B0940)；
If the molecular labeling group of the eggplant material to be measured is combined into GSI-1ASI-2ASI-3ASI-4(CTCT)SI-5CSI-6GSI-7GSI-8 (GCTAGA)SI-9CSI-10TSI-11CSI-12GSI-13GSI-14-SI-15TSI-16TSI-17Shown in molecular labeling combination, then the eggplant to be measured Son is or candidate is Solanummammosum (V06B1256).
Note: "-" indicates base deletion；A, T, C and G indicate to show that simple spike, W, Y and S are indicated in sequencing in sequencing Be shown as being superimposed it is bimodal, wherein W indicate A and T；Y indicates C and T；S indicates G and C.
6, a kind of for identifying the kit of Eggplant Germplasm Resources to be measured
Present invention simultaneously provides a kind of for identifying the kit of Eggplant Germplasm Resources to be measured, including primer pair and records The comparison card of " molecular labeling combines XII "；
9 primer sets, 18 single stranded DNAs point as shown in SEQ ID NO:1~SEQ ID NO:18 for PCR amplification Son forms two-by-two；
" molecular labeling combine XII " is by following a1)~a12) totally 12 kinds of molecular labeling groups be combined into:
a1)GSI-1ASI-2TSI-3ASI-4(CTCT)SI-5CSI-6GSI-7CSI-8(GCTAGA)SI-9CSI-10TSI-11CSI-12GSI- 13GSI-14-SI-15TSI-16TSI-17；
a2)GSI-1ASI-2ASI-3ASI-4(CTCT)SI-5ASI-6GSI-7CSI-8(GCTAGA)SI-9CSI-10TSI-11CSI-12GSI- 13GSI-14-SI-15TSI-16TSI-17；
a3)-SI-1CSI-2TSI-3GSI-4-SI-5CSI-6GSI-7CSI-8(GCTAGA)SI-9CSI-10ASI-11TSI-12CSI-13GSI-14-SI- 15TSI-16TSI-17；
a4)GSI-1ASI-2ASI-3ASI-4(CTCT)SI-5CSI-6GSI-7CSI-8(GCTAGA)SI-9TSI-10TSI-11CSI-12GSI- 13ASI-14(AATGTATAACA)SI-15TSI-16TSI-17；
a5)-SI-1CSI-2ASI-3ASI-4-SI-5CSI-6GSI-7CSI-8(GCTAGA)SI-9CSI-10TSI-11CSI-12GSI-13GSI-14-SI- 15TSI-16TSI-17；
a6)GSI-1ASI-2ASI-3ASI-4(CTCT)SI-5CSI-6GSI-7CSI-8(GCTAGA)SI-9CSI-10ASI-11TSI-12CSI- 13ASI-14(AATGTATAACA)SI-15TSI-16TSI-17；
a7)GSI-1ASI-2TSI-3GSI-4-SI-5CSI-6GSI-7CSI-8(GCTAGA)SI-9CSI-10WSI-11YSI-12SSI-13GSI-14-SI- 15TSI-16TSI-17；
a8)GSI-1ASI-2ASI-3ASI-4(CTCT)SI-5CSI-6GSI-7CSI-8-SI-9CSI-10TSI-11CSI-12GSI-13GSI-14-SI- 15TSI-16TSI-17；
a9)GSI-1ASI-2ASI-3ASI-4(CTCT)SI-5ASI-6GSI-7CSI-8(GCTAGA)SI-9CSI-10TSI-11CSI-12GSI- 13GSI-14-SI-15TSI-16GSI-17；
a10)GSI-1ASI-2TSI-3GSI-4-SI-5CSI-6GSI-7CSI-8(GCTAGA)SI-9CSI-10TSI-11CSI-12GSI-13GSI-14-SI- 15ASI-16TSI-17；
a11)GSI-1ASI-2TSI-3GSI-4-SI-5CSI-6GSI-7CSI-8(GCTAGA)SI-9CSI-10ASI-11TSI-12CSI-13GSI-14-SI- 15TSI-16TSI-17；
a12)GSI-1ASI-2ASI-3ASI-4(CTCT)SI-5CSI-6GSI-7GSI-8(GCTAGA)SI-9CSI-10TSI-11CSI-12GSI- 13GSI-14-SI-15TSI-16TSI-17。
Note: "-" indicates base deletion；W indicates A and T；Y indicates C and T；S indicates G and C.
Eggplant material to be measured is any one of following 12 Eggplant Germplasm Resources: Hunan morning eggplant, kermes eggplant (V06B0149), Dog tail eggplant (V06B0458), black beautiful (V06B0541), Vetrioe (V06B0543), red skin cold eggplant (V06B0552), Nanfeng County Line eggplant (V06B0556), the white eggplant (V06B0978) of Shexian County circle, rattan bridge purple eggplant (V06B1046), oil tank eggplant (V06B0357), purple circle Eggplant (V06B0940) and Solanummammosum (V06B1256).
Embodiment 2 identifies different eggplant materials
1, the white skin eggplant (V06B0101) in eggplant material Quanzhou is identified
The tender leaf of the acquisition white skin eggplant (V06B0101) in eggplant material Quanzhou extracts its genome according to the method for embodiment 1 DNA carries out PCR amplification to it with 9 primer sets being made of SEQ ID NO:1~SEQ ID NO:18 and is sequenced respectively, presses It constructs according to 1 method of embodiment and is combined by SI-1~SI-17 molecular labeling that totally 17 sites form: GSI-1ASI-2TSI-3GSI-4-SI- 5CSI-6GSI-7CSI-8-SI-9CSI-10TSI-11CSI-12GSI-13GSI-14-SI-15TSI-16TSI-17, will be in molecular labeling combination and embodiment 1 " molecular labeling combination XII " comparison card compare, find the molecular labeling combination be not present in the comparison card.
2, the long eggplant of eggplant material stone tone tablet is identified
The tender leaf for acquiring the long eggplant of eggplant material stone tone tablet, extracts its genomic DNA, according to the method for embodiment 1, with by SEQ 9 primer sets of ID NO:1~SEQ ID NO:18 composition carry out PCR amplification to it and are sequenced respectively, according to 1 method of embodiment Building is combined totally by SI-1~SI-17 molecular labeling that 17 sites form: GSI-1ASI-2TSI-3GSI-4-SI-5CSI-6GSI-7CSI-8 (GCTAGA)SI-9CSI-10TSI-11CSI-12GSI-13GSI-14-SI-15TSI-16TSI-17, will be in molecular labeling combination and embodiment 1 " Molecular labeling combination XII " comparison card compares, it is found that molecular labeling combination is not present in the comparison card.
3, the white eggplant in eggplant material Xiaoshan (V06B0885) is identified
The tender leaf for acquiring the white eggplant in eggplant material Xiaoshan (V06B0885), extracts its genomic DNA, according to the side of embodiment 1 Method carries out PCR amplification to it with 9 primer sets being made of SEQ ID NO:1~SEQ ID NO:18 and is sequenced respectively, according to The building of 1 method of embodiment is combined totally by SI-1~SI-17 molecular labeling that 17 sites form :-SI-1CSI-2TSI-3GSI-4-SI- 5CSI-6GSI-7CSI-8-SI-9CSI-10TSI-11CSI-12GSI-13(AATGTATAACA)SI-14-SI-15TSI-16TSI-17, by the molecular labeling group Close in embodiment 1 " molecular labeling combine XII " comparison block compare, find the molecular labeling combine in the comparison card It is not present.
4, the red eggplant in eggplant material Dongyang (V06B1255) is identified
The tender leaf for acquiring the red eggplant in eggplant material Dongyang (V06B1255), extracts its genomic DNA, according to the side of embodiment 1 Method carries out PCR amplification to it with 9 primer sets being made of SEQ ID NO:1~SEQ ID NO:18 and is sequenced respectively, according to The building of 1 method of embodiment is combined totally by SI-1~SI-17 molecular labeling that 17 sites form: GSI-1ASI-2ASI-3ASI-4 (CTCT)SI-5CSI-6GSI-7CSI-8(GCTAGA)SI-9CSI-10TSI-11CSI-12GSI-13GSI-14-SI-15TSI-16TSI-17, by the molecule mark Note combination in embodiment 1 " molecular labeling combine XII " comparison block compare, find the molecular labeling combine in the comparison It is not present in card.
5, big purple circle eggplant (V06B1099) of identification eggplant material
The tender leaf for acquiring big purple circle eggplant (V06B1099) of eggplant material, extracts its genomic DNA, according to the side of embodiment 1 Method carries out PCR amplification to it with 9 primer sets being made of SEQ ID NO:1~SEQ ID NO:18 and is sequenced respectively, according to The building of 1 method of embodiment is combined totally by SI-1~SI-17 molecular labeling that 17 sites form :-SI-1CSI-2ASI-3ASI-4 (CTCT)SI-5CSI-6GSI-7CSI-8(GCTAGA)SI-9CSI-10TSI-11CSI-12GSI-13GSI-14-SI-15TSI-16TSI-17, by the molecule mark Note combination in embodiment 1 " molecular labeling combine XII " comparison block compare, find the molecular labeling combine in the comparison It is not present in card.
Pass through verifying, it was demonstrated that the white skin eggplant (V06B0101) in Quanzhou, the long eggplant of stone tone tablet, the white eggplant in Xiaoshan (V06B0885), Dongyang are red The molecular labeling combination of eggplant (V06B1255) and big purple circle eggplant (V06B1099) this 5 parts of eggplant materials and 12 eggplants in embodiment 1 The molecular labeling combination of sub- germ plasm resource is all different, and shows the specificity and validity of the method for the present invention.
Although above the present invention is described in detail with a general description of the specific embodiments, On the basis of the present invention, it can be modified or is improved, this will be apparent to those skilled in the art.Cause This, these modifications or improvements, fall within the scope of the claimed invention without departing from theon the basis of the spirit of the present invention.
Sequence table
<110>Institute of Vegetables and Flowers, Jiangxi Academy of Agricultural Sciences
<120>for the molecular labeling combination and its application of Eggplant Germplasm Resources identification
<130> KHP191112916.1
<160> 27
<170> SIPOSequenceListing 1.0
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dgmaggtgca tgaacatatc c 21
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<213>artificial sequence (Artificial Sequence)
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gcagctggat ttatyggctc ttg 23
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<213>artificial sequence (Artificial Sequence)
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<213>artificial sequence (Artificial Sequence)
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agctctggag tgtagtgaat tgtcttg 27
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<213>artificial sequence (Artificial Sequence)
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<213>artificial sequence (Artificial Sequence)
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ayccsaaaac yccatcragr ac 22
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<213>artificial sequence (Artificial Sequence)
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tcatcaattc cyaggtcaca caacttc 27
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<213>artificial sequence (Artificial Sequence)
<400> 9
gtgagraaag gttcatggac tg 22
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<213>artificial sequence (Artificial Sequence)
<400> 10
ttccttagaa grttagtgtt ccartag 27
<210> 11
<211> 23
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<213>artificial sequence (Artificial Sequence)
<400> 11
gcttcrttgt actcatggtc ttg 23
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<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 12
tactgtctcc cggcawatct g 21
<210> 13
<211> 25
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<213>artificial sequence (Artificial Sequence)
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ccttgtccta cacacataca cttgc 25
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<213>artificial sequence (Artificial Sequence)
<400> 14
cagkctctkk gacagtwgat wtcttg 26
<210> 15
<211> 26
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<213>artificial sequence (Artificial Sequence)
<400> 15
wgtttctgga gttgwaggag atgttg 26
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<213>artificial sequence (Artificial Sequence)
<400> 17
tgcaaatgct gctgctgatg 20
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<213>artificial sequence (Artificial Sequence)
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ctctcttttg tcaatggctt cttc 24
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<213>eggplant (Solanum melongena)
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taattggatc atgcatgaat ataggctcgc tgatgttgat cgatctgctc gtaagaataa 60
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gtgttttcga ttgattgacg atttcgattt tgtttacagc tggatgattg ggttttatgc 180
agaatataca acaaaaaggg atcgattgag aagcacagga aattgagcac cagctatata 240
gataaagctg gatctccagt ggatatgaag ccggagattc taccaccgct acctccaccg 300
cctccgccgc aggagctcca caacgatttt ttctatctgc cgtcggattc cgttccgaag 360
atccactccg attcaagctg ctccgagcac gtgctctcgc cagagttccc ctgcgagagg 420
gaagtgcaga gcgagcngaa gctaacggat tgg 453
<210> 20
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<213>eggplant (Solanum melongena)
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gtatttcatg tggctacacc gatggatttc gagtccaagg accctgaggt actatagaag 60
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atcactcata tcatagaatg nagtaattaa accaacagtc aggggaatgt tgagcatcat 180
agaatcatgt gctaaagcta acacagtaaa gaggctggtt ttcacttcat ctgctggaac 240
tcttgatgtc caagagaacc agaaactctt ctatgacgag actagctgga gcgacttgga 300
cttcatatat gctaagaaga tgacaggatg ggttcgtttg gctgttgttt ttttaataaa 360
taccnnnnct atgtttaaaa cagagtattt tcatcatatt gcctttgtga atttaatttc 420
c 421
<210> 21
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<213>eggplant (Solanum melongena)
<400> 21
ctgttgttgt gtagttctgg gtggaggagt tgggtaaact gtcttggtac tgtataagtg 60
tatagttttg tgtattgcaa cccttttact ctcctggaan agtgatgaat tttgggtttt 120
tgaagctgct gctttttcct attgttatgt agctctgggt ggattcagga gttaagtgaa 180
cttttttgga agtacttgag 200
<210> 22
<211> 220
<212> DNA
<213>eggplant (Solanum melongena)
<400> 22
ctacagtgat cgttttattc cgagtagatc ttcttctaat tttnctcttt ttgacctccc 60
tttacctnca cgatcatctt catctgagga ttccaccaat gcatatactg cactgcttcg 120
ttcagctcta tttggacccg attgtggatc ggttgtgccg ccggtgacac cggacaagtc 180
gtcgggttta aatgcaagaa atttgcagat ttgtagaccc 220
<210> 23
<211> 608
<212> DNA
<213>eggplant (Solanum melongena)
<400> 23
ggtgaaggca agtggcatct tgttcctnnn nnngcaggta aactaatttt cgatctcatg 60
ttatgtgata tttataataa aaaaataata tatatttttt tgaaaattca catgaaaatg 120
tgcaggtctg aatagatgca gaaaaagttg taggctgagg tggttgaatt atttaaggcc 180
acatatcaag agaggtgact ttgcttcgga tgaagtggat ctcatcttga ggcttcataa 240
gctcttaggc aacaggtgca caaaacaaca aaaacaattt ttagtctcaa taaacatgca 300
tattaagaaa aagtgttaat taaagctttt actggcattt gatccaatat tgttataagt 360
tttattgata tttataaaac gtgttaaaat gcattaatta tcactaagct agtaattgtc 420
tccacaataa tatatttagc gaaattaagt tgctatcgtt aattaattac atttaaagat 480
catttttggt gcaatgatga cgctcacaat tcattcaaaa gtttatggtc gatattagtg 540
ataacactta atatgacang atgccatgca tgctgaatat ataaatgtgt gattatttca 600
actaaaag 608
<210> 24
<211> 400
<212> DNA
<213>eggplant (Solanum melongena)
<400> 24
tataaccaca tttcactata acaagttata ttttctgtgg aacaaatctt ttatgttatg 60
ttatatgatt tgtattacaa catttcgcta tagcaaccan aaaatatttg agacaaanga 120
tgttattgag aggttagtct atagttgcga gtattgctgc aacaattatt agaataacgg 180
ataagtttgg tttcatgtct cttgctactg gagttcttac tgtgaatggt tacaattttg 240
agatcattag tgcttttaaa ccaactataa gtgaactggc cttttgcttc cagttcgctt 300
cagtccggca aatgttagat tgtcaaacnc aaagctgcta ccgggaccac aatcctcgag 360
aagccaagat gagacttcat cttcttctag catgaatgaa 400
<210> 25
<211> 368
<212> DNA
<213>eggplant (Solanum melongena)
<400> 25
ttccctcatg catgttattg gttatgctgg tttttaaatt gcaaattaac gatcgtatta 60
gttttataca tgaataactt atttcaaact agccactaaa tattgtataa cttatncata 120
aatgaattgt ttctttacta gctacctaaa aagttggaaa cttttgtttg ttaatgataa 180
agggaagagt tctgcttaaa agaagacaca cacataaaac aatccataga taatggttaa 240
gcatgcttct agaatttagc aaaagcaaat tttcttgtcc acttttccct caagcacatg 300
tcaatatcat taaaatacat tcaacattat aagtaacttg taatgtttcc cggnnnnnnn 360
nnnnacat 368
<210> 26
<211> 200
<212> DNA
<213>eggplant (Solanum melongena)
<400> 26
aaggtaagac aacatcatac cttgctagcc tattaacctt ctatcttaat acgcgacttc 60
cattcctttc tataattcta agattatttg tacttatccn aatgaatact acttactaaa 120
tctcaaaaga actttacctt caaaacctta cgatatccat ttggtcttcc atgagttttg 180
gtaacaattt caacaaaaaa 200
<210> 27
<211> 200
<212> DNA
<213>eggplant (Solanum melongena)
<400> 27
cactcataca agttcactgt taacttccac gcaagtttaa aggccaaagt caatacttgt 60
taatagtagt agtgtaagta ggccagcgct ctaccaactn agctacacaa ccaaaaaaag 120
ataatgtagt aaatacaaaa tggtctggaa aagaggctgg cttctttttt cctgttgaag 180
cacaagtacg tatgaagctg 200

Claims (6)

1. the molecular labeling combination for Eggplant Germplasm Resources identification, which is characterized in that molecular labeling combination by SI-1, SI-5, SI-9, SI-14, SI-15 totally 5 InDel label and SI-2, SI-3, SI-4, SI-6, SI-7, SI-8, SI-10, Totally 12 SNP markers form by SI-11, SI-12, SI-13, SI-16, SI-17:
Marking SI-1 to contain in eggplant genome, polymorphism is at the nucleotide sequence 100bp as shown in SEQ ID NO:19 The nucleotide sequence of G/-；
Marking SI-2 to contain in eggplant genome, polymorphism is at the nucleotide sequence 437bp as shown in SEQ ID NO:19 The nucleotide sequence of A/C；
Marking SI-3 to contain in eggplant genome, polymorphism is at the nucleotide sequence 100bp as shown in SEQ ID NO:20 The nucleotide sequence of T/A；
Marking SI-4 to contain in eggplant genome, polymorphism is at the nucleotide sequence 141bp as shown in SEQ ID NO:20 The nucleotide sequence of G/A；
Label SI-5 contains more at 365~368bp of nucleotide sequence as shown in SEQ ID NO:20 in eggplant genome State property is the nucleotide sequence of CTCT/-；
Marking SI-6 to contain in eggplant genome, polymorphism is at the nucleotide sequence 100bp as shown in SEQ ID NO:21 The nucleotide sequence of C/A；
Marking SI-7 to contain in eggplant genome, polymorphism is at the nucleotide sequence 54bp as shown in SEQ ID NO:22 The nucleotide sequence of G/A；
Marking SI-8 to contain in eggplant genome, polymorphism is at the nucleotide sequence 78bp as shown in SEQ ID NO:22 The nucleotide sequence of C/G；
Label SI-9 contains polymorphic at 28~33bp of nucleotide sequence as shown in SEQ ID NO:23 in eggplant genome Property be GCTAGA/- nucleotide sequence；
Label SI-10 contains in eggplant genome polymorphism at the nucleotide sequence 569bp as shown in SEQ ID NO:23 For the nucleotide sequence of C/T；
Label SI-11 contains in eggplant genome polymorphism at the nucleotide sequence 100bp as shown in SEQ ID NO:24 For the nucleotide sequence of T/A；
Label SI-12 contains in eggplant genome polymorphism at the nucleotide sequence 118bp as shown in SEQ ID NO:24 For the nucleotide sequence of C/T；
Label SI-13 contains in eggplant genome polymorphism at the nucleotide sequence 329bp as shown in SEQ ID NO:24 For the nucleotide sequence of G/C；
Label SI-14 contains in eggplant genome polymorphism at the nucleotide sequence 116bp as shown in SEQ ID NO:25 For the nucleotide sequence of G/-；
Label SI-15 contains more at 354~364bp of nucleotide sequence as shown in SEQ ID NO:25 in eggplant genome State property is the nucleotide sequence of AATGTATAACA/-；
Label SI-16 contains in eggplant genome polymorphism at the nucleotide sequence 100bp as shown in SEQ ID NO:26 For the nucleotide sequence of T/A；
Label SI-17 contains in eggplant genome polymorphism at the nucleotide sequence 100bp as shown in SEQ ID NO:27 For the nucleotide sequence of T/G；
Wherein, the Eggplant Germplasm Resources be selected from the cold eggplant of Hunan morning eggplant, kermes eggplant, dog tail eggplant, black beauty, Vetrioe, red skin, Nanfeng County line eggplant, the white eggplant of Shexian County circle, rattan bridge purple eggplant, oil tank eggplant, purple circle eggplant, Solanummammosum.
2. the primer for expanding the combination of molecular labeling described in claim 1, which is characterized in that including as follows 1.~9. totally 9 groups Primer:
1. the primer sequence for expanding label SI-1, SI-2 is as shown in SEQ ID NO:1 and SEQ ID NO:2；
2. the primer sequence for expanding label SI-3, SI-4, SI-5 is as shown in SEQ ID NO:3 and SEQ ID NO:4；
3. the primer sequence for expanding label SI-6 is as shown in SEQ ID NO:5 and SEQ ID NO:6；
4. the primer sequence for expanding label SI-7, SI-8 is as shown in SEQ ID NO:7 and SEQ ID NO:8；
5. the primer sequence for expanding label SI-9, SI-10 is as shown in SEQ ID NO:9 and SEQ ID NO:10；
6. the primer sequence such as SEQ ID NO:11 and SEQ ID NO:12 institute for expanding label SI-11, SI-12, SI-13 Show；
7. the primer sequence for expanding label SI-14, SI-15 is as shown in SEQ ID NO:13 and SEQ ID NO:14；
8. the primer sequence for expanding label SI-16 is as shown in SEQ ID NO:15 and SEQ ID NO:16；
9. the primer sequence for expanding label SI-17 is as shown in SEQ ID NO:17 and SEQ ID NO:18.
3. detection reagent or kit containing primer described in claim 2.
4. detection reagent described in the combination of molecular labeling described in claim 1, primer or claim 3 described in claim 2 or Following any application of kit:
1) it is identified for Eggplant Germplasm Resources；
2) it is constructed for eggplant DNA fingerprinting；
3) it is used for eggplant molecular mark.
5. a kind of method for identifying Eggplant Germplasm Resources, which comprises the steps of:
(1) using the genomic DNA of eggplant material to be measured as template, PCR amplification is carried out using primer described in claim 2, is obtained PCR product；
(2) PCR product is analyzed.
6. according to the method described in claim 5, it is characterized in that, step (2) specifically: be sequenced, will survey to PCR product Sequence result is compared with " molecular labeling combines XII ", and " molecular labeling combines XII " is following a1)~a12) totally 12 Combination:
a1)GSI-1ASI-2TSI-3ASI-4(CTCT)SI-5CSI-6GSI-7CSI-8(GCTAGA)SI-9CSI-10TSI-11CSI-12GSI-13GSI-14-SI- 15TSI-16TSI-17；
a2)GSI-1ASI-2ASI-3ASI-4(CTCT)SI-5ASI-6GSI-7CSI-8(GCTAGA)SI-9CSI-10TSI-11CSI-12GSI-13GSI-14-SI- 15TSI-16TSI-17；
a3)-SI-1CSI-2TSI-3GSI-4-SI-5CSI-6GSI-7CSI-8(GCTAGA)SI-9CSI-10ASI-11TSI-12CSI-13GSI-14-SI-15TSI- 16TSI-17；
a4)GSI-1ASI-2ASI-3ASI-4(CTCT)SI-5CSI-6GSI-7CSI-8(GCTAGA)SI-9TSI-10TSI-11CSI-12GSI-13ASI-14 (AATGTATAACA)SI-15TSI-16TSI-17；
a5)-SI-1CSI-2ASI-3ASI-4-SI-5CSI-6GSI-7CSI-8(GCTAGA)SI-9CSI-10TSI-11CSI-12GSI-13GSI-14-SI-15TSI- 16TSI-17；
a6)GSI-1ASI-2ASI-3ASI-4(CTCT)SI-5CSI-6GSI-7CSI-8(GCTAGA)SI-9CSI-10ASI-11TSI-12CSI-13ASI-14 (AATGTATAACA)SI-15TSI-16TSI-17；
a7)GSI-1ASI-2TSI-3GSI-4-SI-5CSI-6GSI-7CSI-8(GCTAGA)SI-9CSI-10WSI-11YSI-12SSI-13GSI-14-SI-15TSI- 16TSI-17；
a8)GSI-1ASI-2ASI-3ASI-4(CTCT)SI-5CSI-6GSI-7CSI-8-SI-9CSI-10TSI-11CSI-12GSI-13GSI-14-SI-15TSI- 16TSI-17；
a9)GSI-1ASI-2ASI-3ASI-4(CTCT)SI-5ASI-6GSI-7CSI-8(GCTAGA)SI-9CSI-10TSI-11CSI-12GSI-13GSI-14-SI- 15TSI-16GSI-17；
a10)GSI-1ASI-2TSI-3GSI-4-SI-5CSI-6GSI-7CSI-8(GCTAGA)SI-9CSI-10TSI-11CSI-12GSI-13GSI-14-SI-15ASI- 16TSI-17；
a11)GSI-1ASI-2TSI-3GSI-4-SI-5CSI-6GSI-7CSI-8(GCTAGA)SI-9CSI-10ASI-11TSI-12CSI-13GSI-14-SI-15TSI- 16TSI-17；
a12)GSI-1ASI-2ASI-3ASI-4(CTCT)SI-5CSI-6GSI-7GSI-8(GCTAGA)SI-9CSI-10TSI-11CSI-12GSI- 13GSI-14-SI-15TSI-16TSI-17；
In above-mentioned molecular labeling combination ,-indicate base deletion；W indicates A and T；Y indicates C and T；S indicates G and C；
Wherein, the SI-1 is the site InDel, the of the nucleotide sequence as shown in SEQ ID NO:19 in eggplant genome 100, which is that G/- is polymorphic；
The SI-2 be SNP site, the 437th of the nucleotide sequence as shown in SEQ ID NO:19 in eggplant genome, The SNP site is that A/C is polymorphic；
The SI-3 be SNP site, the 100th of the nucleotide sequence as shown in SEQ ID NO:20 in eggplant genome, The SNP site is that T/A is polymorphic；
The SI-4 be SNP site, the 141st of the nucleotide sequence as shown in SEQ ID NO:20 in eggplant genome, The SNP site is that G/A is polymorphic；
The SI-5 be the site InDel, in eggplant genome the 365th of the nucleotide sequence as shown in SEQ ID NO:20~ 368, which is that CTCT/- is polymorphic；
The SI-6 be SNP site, the 100th of the nucleotide sequence as shown in SEQ ID NO:21 in eggplant genome, The SNP site is that C/A is polymorphic；
The SI-7 is SNP site, and the 54th of the nucleotide sequence as shown in SEQ ID NO:22 in eggplant genome should SNP site is that G/A is polymorphic；
The SI-8 is SNP site, and the 78th of the nucleotide sequence as shown in SEQ ID NO:22 in eggplant genome should SNP site is that C/G is polymorphic；
The SI-9 be the site InDel, in eggplant genome the 28th of the nucleotide sequence as shown in SEQ ID NO:23~ 33, which is that GCTAGA/- is polymorphic；
The SI-10 be SNP site, the 569th of the nucleotide sequence as shown in SEQ ID NO:23 in eggplant genome, The SNP site is that C/T is polymorphic；
The SI-11 be SNP site, the 100th of the nucleotide sequence as shown in SEQ ID NO:24 in eggplant genome, The SNP site is that T/A is polymorphic；
The SI-12 be SNP site, the 118th of the nucleotide sequence as shown in SEQ ID NO:24 in eggplant genome, The SNP site is that C/T is polymorphic；
The SI-13 be SNP site, the 329th of the nucleotide sequence as shown in SEQ ID NO:24 in eggplant genome, The SNP site is that G/C is polymorphic；
The SI-14 is the site InDel, the 116th of the nucleotide sequence as shown in SEQ ID NO:25 in eggplant genome Position, the site InDel are that G/- is polymorphic；
The SI-15 is the site InDel, the 354th of the nucleotide sequence as shown in SEQ ID NO:25 in eggplant genome ~364, which is that AATGTATAACA/- is polymorphic；
The SI-16 be SNP site, the 100th of the nucleotide sequence as shown in SEQ ID NO:26 in eggplant genome, The SNP site is that T/A is polymorphic；
The SI-17 be SNP site, the 100th of the nucleotide sequence as shown in SEQ ID NO:27 in eggplant genome, The SNP site is that T/G is polymorphic；
Finally, being identified according to comparison result the eggplant material to be measured:
If the molecular labeling group of the eggplant material to be measured is combined into a1) shown in molecular labeling combination, the eggplant to be measured is Or candidate is Hunan morning eggplant；
If the molecular labeling group of the eggplant material to be measured is combined into a2) shown in molecular labeling combination, the eggplant to be measured is Or candidate is kermes eggplant；
If the molecular labeling group of the eggplant material to be measured is combined into a3) shown in molecular labeling combination, the eggplant to be measured is Or candidate is dog tail eggplant；
If the molecular labeling group of the eggplant material to be measured is combined into a4) shown in molecular labeling combination, the eggplant to be measured is Or candidate is black beauty；
If the molecular labeling group of the eggplant material to be measured is combined into a5) shown in molecular labeling combination, the eggplant to be measured is Or candidate is Vetrioe；
If the molecular labeling group of the eggplant material to be measured is combined into a6) shown in molecular labeling combination, the eggplant to be measured is Or candidate is the cold eggplant of red skin；
If the molecular labeling group of the eggplant material to be measured is combined into a7) shown in molecular labeling combination, the eggplant to be measured is Or candidate is Nanfeng County's line eggplant；
If the molecular labeling group of the eggplant material to be measured is combined into a8) shown in molecular labeling combination, the eggplant to be measured is Or candidate is the white eggplant of Shexian County circle；
If the molecular labeling group of the eggplant material to be measured is combined into a9) shown in molecular labeling combination, the eggplant to be measured is Or candidate is rattan bridge purple eggplant；
If the molecular labeling group of the eggplant material to be measured is combined into a10) shown in molecular labeling combination, the eggplant to be measured is Or candidate is oil tank eggplant；
If the molecular labeling group of the eggplant material to be measured is combined into a11) shown in molecular labeling combination, the eggplant to be measured is Or it is candidate for purple circle eggplant；
If the molecular labeling group of the eggplant material to be measured is combined into a12) shown in molecular labeling combination, the eggplant to be measured is Or candidate is Solanummammosum.
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CN113999844A (en) * 2021-11-30 2022-02-01 中国农业科学院蔬菜花卉研究所 Primer pair for identifying dominant parthenocarpic gene of eggplant by using InDel molecular marker and application of primer pair
CN114480704A (en) * 2022-01-07 2022-05-13 中国热带农业科学院南亚热带作物研究所 SNP combined marker for identifying eggplant seed resources
CN114480704B (en) * 2022-01-07 2023-06-30 中国热带农业科学院南亚热带作物研究所 SNP combined marker for eggplant seed resource identification
NL2031136B1 (en) * 2022-03-02 2023-09-08 Inst Vegetables & Flowers Caas Set of kompetitive allele specific pcr (kasp)-based core single nucleotide polymorphism (snp) markers for eggplant and use thereof
CN115927699B (en) * 2022-07-18 2024-01-26 江西省农业科学院蔬菜花卉研究所 Molecular marker for identifying germplasm of pepper with epidemic disease resistance and application thereof
CN115927699A (en) * 2022-07-18 2023-04-07 江西省农业科学院蔬菜花卉研究所 Molecular marker for identifying phytophthora-resistant pepper germplasm and application thereof
CN116426684A (en) * 2023-04-24 2023-07-14 上海市农业科学院 InDel molecular marker closely linked with eggplant peel color traits and application
CN116426684B (en) * 2023-04-24 2024-05-28 上海市农业科学院 InDel molecular marker closely linked with eggplant peel color traits and application

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