CN107586878A - Hexabasic base for detecting broomcorn millet hereditary difference repeats motif molecular labeling - Google Patents

Abstract

The hexabasic base for being used to detect broomcorn millet hereditary difference the invention discloses one group repeats motif molecular labeling, is one group of microsatellite marker with base sequence shown in sequence table SEQ ID No.1~SEQ ID No.4.One group of hexabasic base provided by the invention repeats the hereditary difference that motif molecular labeling can apply to detect broomcorn millet, carry out the assessment of broomcorn millet Genetic Diversity of Germplasm, broomcorn millet crop Study on Evolution and molecular mark, it is reproducible, it is a kind of reliable and effective molecular labeling.

Description

Hexabasic base for detecting broomcorn millet hereditary difference repeats motif molecular labeling

Technical field

The invention belongs to molecular biosciences Genetic Markers field, is related to the molecule mark for detecting broomcorn millet hereditary difference Note, the molecular labeling of motif is particularly repeated containing hexabasic base.

Background technology

Broomcorn millet (Panicum miliaceumL.), it is the cultivation tamed earliest in the world originating from the Chinese Huanghe valley One of crop.Broomcorn millet drought resisting is resistance to lean, and breeding time is short, is the characteristic species in the northern area that cools.Global Temperature increasingly increases at present, It is imperative to cultivate screening drought-resistant crops.Broomcorn millet environmental suitability is strong, gets the genetic background of broomcorn millet clear, is effectively to utilize broomcorn millet Premise.

Molecular labeling can disclose the genetic diversity of broomcorn millet from DNA level, be the key of molecular mark.It is micro- Satellite markers are with the characteristics of simple sequence repeats, including dinucleotides, trinucleotide, tetranucleotide repeat etc..

Although the DNA marker of existing report detection broomcorn millet hereditary difference is more, microsatellite marker is short of and come from more Inter-species.Because broomcorn millet specificity SSR marker is few, and mostly three base repeat units, tetranucleotide repeat primitive is less, hexabasic Base repeats primitive and still lacked, and causes broomcorn millet genetic map construction to be limited, broomcorn millet favorable genes are difficult to by genetic engineering means big Played a role in ancestor's crop genetic improvement.Six base repetitive sequences are excavated therefore, it is necessary to develop, utilize these functional genomes Element, effectively realize the salt tolerance of drought genetic improvement of staple crop.

The content of the invention

The purpose of the present invention is to build one group of hexabasic base for being used to detect broomcorn millet hereditary difference to repeat motif molecular labeling, profit It is auxiliary that the assessment of broomcorn millet Genetic Diversity of Germplasm, broomcorn millet crop Study on Evolution and molecular labeling can be carried out with these molecular labelings Help breeding.

Primer pair for detecting the molecular labeling is provided, is another goal of the invention of the present invention.

The one group of the present invention hexabasic base repetition motif molecular labeling for being used to detect broomcorn millet hereditary difference is with sequence One group of microsatellite marker of base sequence shown in list SEQ ID No.1~SEQ ID No.4, microsatellite marker numbering are respectively RYW1, RYW2, RYW3 and RYW4.

Wherein RYW1 is 636 nucleotides, and the hexabasic base with 6 repetitions repeats motif (TCATCT)₆, RYW2 330 Individual nucleotides, the hexabasic base with 5 repetitions repeat motif (CGAAGC)₅, RYW3 is 562 nucleotides, has 5 repetitions Hexabasic base repeats motif (CTGCAA)₅, RYW4 is 473 nucleotides, and the hexabasic base with 5 repetitions repeats motif (GTGCCG)₅。

And then molecular labeling of the present invention is obtained by respective primer pair amplifies respectively.Wherein, for expanding RYW1 Primer pair there is base sequence shown in sequence table SEQ ID No.5 and SEQ ID No.6, for expanding RYW2 primer pair With the base sequence shown in sequence table SEQ ID No.7 and SEQ ID No.8, the primer pair for expanding RYW3 has sequence Base sequence shown in table SEQ ID No.9 and SEQ ID No.10, the primer pair for expanding RYW4 have sequence table SEQ Base sequence shown in ID No.11 and SEQ ID No.12.

The present invention is built by the nucleotide base repetitive sequence mRNA enriched libraries of broomcorn millet six and containing six nucleotide base weight Complex sequences is sequenced, and has obtained one group 4 six nucleotide base repetitive sequences, and exploitation determines the broomcorn millet six of 4 rich polymorphisms Base repeats motif microsatellite marker RYW1, RYW2, RYW3 and RYW4, and for expanding this 4 hexabasic base repeating group tagmemes The primer special of point.

One group of hexabasic base of the above-mentioned offer of the present invention repeats motif molecular labeling and can apply to detect the hereditary poor of broomcorn millet It is different.

Specifically, one group of the present invention hexabasic base repetition motif molecular labeling can be in broomcorn millet germ plasm resource genetic diversity Property assessment, broomcorn millet crop Study on Evolution, molecular mark etc. are applied.

Motif molecular labeling is repeated with the hexabasic base of the broomcorn millet of one group of 4 rich polymorphism of the present invention and carries out broomcorn millet germplasm The assessment of resource genetic diversity, broomcorn millet crop Study on Evolution and molecular mark, it is reproducible, it is a kind of reliable effective Molecular labeling.

The hexabasic base that the present invention develops repeats motif molecular labeling and is used to detect the broomcorn millet germplasm from planted in different ecological areas When, band is clear, reproducible, and polymorphism is high, is the specific microsatellite marker of broomcorn millet.

The present invention chooses 96 parts of broomcorn millet germplasm resources from different ecological cultivation area, with the above-mentioned four pairs of primers of the present invention Amplification polymorphism, it is found that the number of alleles that average each pair primer detection arrives is 3, Detection results are notable.Wherein, polymorphism is believed Cease content 0.57, diversity indices 0.82, be above previous investigation (highest is respectively 0.49 and 0.53).Illustrate that broomcorn millet is lost Pass diversity to enrich, detection provided by the invention is effective assessment tool with molecular labeling.

Brief description of the drawings

Fig. 1 is the electrophoretogram that motif molecular labeling 6 broomcorn millet kinds of amplification are repeated with one group 4 hexabasic bases of the present invention.

Embodiment

Embodiment 1.

Plant two broomcorn millet kinds of salt-resistance significant difference respectively in greenhouse：(the Unified number of salt-enduring cultivars 287 And the old broom corn millet of salt density value kind (Unified number 00005435) 00002661).

Clip tri-leaf period leaf tissue, extracted with Trizol plus kit (ThermoFisher, USA) kit total RNA。

MRNA is enriched with NEBNext Poly (A) mRNA Magnetic Isolation Module (NEB, E7490), Using mRNA as template, with NEBNext mRNA Library Prep Master Mix Set for Illumina (NEB, E6110 machine library) and on NEBNext Multiplex Oligos for Illumina (NEB, E7500) structure.

Library inserts size is detected in the library prepared with 1.8% agarose gel electrophoresis, uses Library Quantification Kit-Illumina GA Universal (Kapa, KK4824) carry out QPCR and quantified.It is qualified to detect Library carries out the generation of cluster on Illumina cbot, finally with Illumina HiSeq^TM2500 are sequenced.Utilize de Novo assembles transcript, obtains 54880 Unigene altogether.

Utilize MISA (http://pgrc.ipk-gatersleben.de/misa) software is to obtained more than the 1kb of screening Unigene do ssr analysis, compared with the genome sequence of the monocotyledon millet in ncbi database, switchgrass etc. It is right, altogether screening obtain 4 Unigene containing six nucleotide base repetitive sequences, be respectively labeled as RYW1, RYW2, RYW3 and RYW4, it is respectively (TCATCT) to repeat motif₆、(CGAAGC)₅、(CTGCAA)₅、(GTGCCG)₅, its nucleotide sequence such as sequence Shown in table SEQ ID No.1~SEQ ID No.4.

Using Primer5.0 forward and backward primer, design of primers ginseng are designed for above-mentioned 4 six nucleotide base repetitive sequences Number is shown in Table 1, and Services Co., Ltd of Shanghai Sangon Biological Engineering Technology And Service Co., Ltd is sent in primer pair synthesis.

It following present the specific primer sequence in tetra- sites of RYW1, RYW2, RYW3 and RYW4.

RYW1-F：5'-TAACGCTTCACCTTCAGACC-3'.

RYW1-R：5'-TGAGATGGAGTTGGCTGATG-3'.

RYW2-F：5'-TTAGGGCTCTCCTGCATCC-3'.

RYW2-R：5'-CAGCGAGTTCACCGTCAAG-3'.

RYW3-F：5'-GGAGGCGTGACAATAAAAC-3'.

RYW3-R：5'-GGCGTGAGGTGTTGTTTTT-3'.

RYW4-F：5'-AATCCACAACGCACACGAC-3'.

RYW4-R：5'-ATTTGCTCCTCTCGTCGGT-3'.

Embodiment 2.

To be examination material from 6 broomcorn millet kinds (table 2) of planted in different ecological areas, the primer feature of four SSR markers is analyzed (allelic variation size and primer resolution ratio/Rp values).

Described 6 examination assortments are implanted in nutritive cube, in tri-leaf period clip seedling leaves, gene is extracted with modified CTAB method Group DNA.DNA mass, ultraviolet trace dna instrument measure DNA purity and concentration are extracted with the detection of 1% agarose gel electrophoresis.

With LifeEco gene-amplificative instraments (TC-96/G/H (b) c, Hangzhou BIOER Technology Co., Ltd) to above-mentioned DNA sequence dna Enter performing PCR amplification.

PCR reaction systems：10 × buffer (Mg containing 25mmol/L²⁺) 2 μ L, 10mmol/L dNTP 1.8 μ L, 5U/L Primer each 0.6 μ L, ddH before and after Taq polymerase 0.4 μ L, 1mmol/L₂The μ L of 13.6 μ L, 30ng/ μ L DNA profilings of O 1, amount to 20μL。

PCR amplification programs：94℃ 5min；94 DEG C of 45s, different Tm (RYW1-F：55.2 DEG C, RYW1-R：56.2℃； RYW2-F：57.4 DEG C, RYW2-R：57℃；RYW3-F：52.8 DEG C, RYW3- R：55.7℃；RYW4-F：59.9 DEG C, RYW4-R： 56.9 DEG C) annealing 50s, 72 DEG C of 1min, 38 circulations；72℃ 10min.

Pcr amplification product is detected with 8% polyacrylamide gel electrophoresis, cma staining development, reads band (Fig. 1).Deng Position variance-length compares band with 50bp DNA Marker (border biological gene Science and Technology Ltd. of Beijing village ally, Beijing).

Gel-tape expression mark (>50bp) size, adjacent ribbons size difference 25bp.If SSR marker is at least 2 parts Material amplifies different DNA bands, then with polymorphism.DNA bands represent allelic variation, and same strap represents identical equipotential Variation.

Primer resolution ratio Resolving power (Rp) calculate according to Prevost ＆ Wilkinson methods：Rp=∑Ib。 Wherein,IbFor some allelic information amount,pOccur for some allele in material is detected Frequency.

Using 6 broomcorn millet kinds that different ecological cultivation area is derived from table 2, after PCR amplifications, four SSR markers are analyzed Primer feature (allelic variation size and Rp values), the results are shown in Table 3.From table 3 it can be seen that the allelic variation size of four marks Between 100~450bp, Rp values are between 3~5.25, average out to 4.

Rp values are to weigh the index of primer pair different genotype resolving ability, direct correlation label information.Rp values are higher, lose It is abundanter to pass diversity.The Rp values of four broomcorn millet specificity SSR primers of the invention are higher, tell existing between different genotype Difference, can be as the effective detection instrument of hereditary difference between discriminating germ plasm resource.

Embodiment 3.

To be examination material from 96 broomcorn millet kinds (table 4) of planted in different ecological areas, broomcorn millet money is assessed using four pairs of SSR primers The genetic diversity in source.

Material implantation methods, leaf DNA extraction, PCR amplifications and electrophoretic band are read with embodiment 2.

Genetic diversity evaluation index (including observation/effective number of allele, observation/expectation heterozygosity, gene diversity Index, polymorphism information content) calculated with PowerMarker 3.25.

Above-mentioned 96 parts of broomcorn millet germ plasm resources are expanded with four pairs of primers, the hereditary difference between analysis of material, the results are shown in Table 5.

As can be seen from Table 5, four sites detect 12 allelic variations altogether, and the equipotential that average each site primer arrives becomes Different is 3；Polymorphism information content is between 0.48~0.63, average out to 0.57, higher than previous investigation（Up to 0.49）； Gene diversity index is between 0.73~0.89, average out to 0.82, higher than previous investigation（Up to 0.53）.Illustrate 96 parts Abundant genetic diversity between broomcorn millet resource be present, therefore above-mentioned primer can be used for effectively assessing the genetic diversity of broomcorn millet resource Property.

Sequence table

<110>Agricultural University Of Shanxi

<120>Hexabasic base for detecting broomcorn millet hereditary difference repeats motif molecular labeling

<160> 12

<170> SIPOSequenceListing 1.0

<210> 1

<211> 636

<212> DNA

<213>Broomcorn millet (Panicum miliaceum L.)

<400> 1

cagtagaggc agataaaatc acgcttcacc ttcagaccaa cataagtaag aaggccactg 60

ggatccttca aatattcact gtcaggtatg aaaaagccat gcttcctgtg catatggacc 120

atgcaatctt cgatggtgtc atgcttaaga tcgcacatga aacatgacga gatatccaac 180

tcttcaagat catccatgtc atcatctgat ttggatgact gctcatcaac ttgcatgttt 240

gaagtagact catcagccaa ctccatctca tttgggtcaa cttcaaccca ctcttcctcc 300

tcatcttcat cttcatcttc atcttcatct tcatcttcat cctcctctac tgcagatgga 360

actctacgtg gaactcgctc aggaagtggc ttgactatag taattccagc aattgaggca 420

ttgggctcct gggaggctct cataagatgt aatcgtgagt taagatgctg tgcatgagct 480

ttggagctcc tgtactcctt tccacagaga gcacagctgt atagcatagg ggtgcttgca 540

ggcttgttcc cctctgctaa agcagcttgt cttgccaaaa acagagcctc ggtcacacca 600

ggaactccag ccaccttgcg cttgaggttg tagcgg 636

<210> 2

<211> 330

<212> DNA

<213>Broomcorn millet (Panicum miliaceum L.)

<400> 2

tagatcttgc ggatgagcgc ccagcgcagc tcggggctct cctgcatccc cgggtacagc 60

gcccgctgct gccccgcggc gccggaggtc cccgcctcga ggtcgccgcc cttctggtac 120

ccgaacgccg acgacatctc ggcgccgccg gagacgacga cgacgacgag ggccttctcc 180

ctcactgtcc ccgggtcgag cggggctggc gcggctgcga ggaggagagg aggagcccgt 240

gtgcgagggg aggcgacggc ggaggaggcg aggacggcgc tcccaagtgg ggggagaaga 300

cgaagccgaa gccgaagccg aagccgaagc 330

<210> 3

<211> 562

<212> DNA

<213>Broomcorn millet (Panicum miliaceum L.)

<400> 3

cctcgccggc cttgacggtg aactcgctgg gctcgaagac gaggacgcct ccggtggcgc 60

ccagcagcac ctcctgggcc atggcgccgc cggcgagcag ggcgctggcg gccacggcca 120

cggcggcggt gccggcggcc ttgccgagcg aggcgcgcac ggtgaaagca gcagcagagc 180

gcctgctggc aacggcgcgg gctggcctgg cagccgcgaa ggaaggggcg gtgacggcgg 240

cagaggagag ggaggccatt gttaagttgg tggtagactc actagaccga gcaaataaag 300

ctgcaactgc aactgcaact gcaactgcaa agcgagagag cgagcgagcg cgagcgagcg 360

agggagcaag ctatagccta tagctagcca ggccccggca ggtggagagg agggagaaat 420

aggaggaggt ggcaggggag gatgaggcaa cgggtggcca gggagaaatc cttatcccgg 480

cgatccttat ggccgtgctt gcgttgcatg cccacggatg gatagatctc tccactccat 540

tccagatgag atttcccatc ca 562

<210> 4

<211> 473

<212> DNA

<213>Broomcorn millet (Panicum miliaceum L.)

<400> 4

acacgcgggg cgggatcgcc gccgccgaag gacgcaagcc ggtagacctc gtgcagcgcc 60

gcggtggccg cccgcacgtc gccggcctcc ccagccgcca ccgacgagag gagcaaatgc 120

atcagccgca ggccgccacc gccgccagca gcatcatcgg gcgctactct ctggtcgtcc 180

gactccagcc tcgccaccat ccgctccctc gcctggagaa gcctcaccac cctgccacgg 240

tcaccaccat cgcccaccac gtcgtcgaca acaccgtccc gcaccttgcg cttcttgccc 300

gtgccggtgc cggtgccggt gccggtgccg ccattgacgg ctagactcaa cgtcaggttc 360

atgcacgcct tccagcttcc gcggcccaac agacctcagc tcgtggcctc tccaagaatt 420

ctaaggaatc cacaacgcac acgacggcga tggcggcacc agaggagggg gac 473

<210> 5

<211> 20

<212> DNA

<213>Artificial sequence ()

<400> 5

taacgcttca ccttcagacc 20

<210> 6

<211> 20

<212> DNA

<213>Artificial sequence ()

<400> 6

tgagatggag ttggctgatg 20

<210> 7

<211> 19

<212> DNA

<213>Artificial sequence ()

<400> 7

ttagggctct cctgcatcc 19

<210> 8

<211> 19

<212> DNA

<213>Artificial sequence ()

<400> 8

cagcgagttc accgtcaag 19

<210> 9

<211> 19

<212> DNA

<213>Artificial sequence ()

<400> 9

ggaggcgtga caataaaac 19

<210> 10

<211> 19

<212> DNA

<213>Artificial sequence ()

<400> 10

ggcgtgaggt gttgttttt 19

<210> 11

<211> 19

<212> DNA

<213>Artificial sequence ()

<400> 11

aatccacaac gcacacgac 19

<210> 12

<211> 19

<212> DNA

<213>Artificial sequence ()

<400> 12

atttgctcct ctcgtcggt 19

Claims (6)

1. for detect broomcorn millet hereditary difference hexabasic base repeat motif molecular labeling, be with sequence table SEQ ID No.1~ One group of microsatellite marker of base sequence shown in SEQ ID No.4, microsatellite marker numbering be respectively RYW1, RYW2, RYW3 and RYW4。

2. the primer of motif molecular labeling is repeated for the 1 hexabasic base of test right requirement, it is characterized in that for expanding RYW1 Primer pair there is base sequence shown in sequence table SEQ ID No.5 and SEQ ID No.6, for expanding RYW2 primer pair With the base sequence shown in sequence table SEQ ID No.7 and SEQ ID No.8, the primer pair for expanding RYW3 has sequence Base sequence shown in table SEQ ID No.9 and SEQ ID No.10, the primer pair for expanding RYW4 have sequence table SEQ Base sequence shown in ID No.11 and SEQ ID No.12.

3. hexabasic base described in claim 1 repeats application of the motif molecular labeling in broomcorn millet hereditary difference is detected.

4. hexabasic base described in claim 1 repeats motif molecular labeling answering in the assessment of broomcorn millet Genetic Diversity of Germplasm With.

5. hexabasic base described in claim 1 repeats application of the motif molecular labeling in broomcorn millet crop Study on Evolution.

6. hexabasic base described in claim 1 repeats application of the motif molecular labeling in molecular mark.