CN110257548A - Method based on SSR marker and qPCR detection loquat aneuploid Molecu- lar karyotyping - Google Patents
Method based on SSR marker and qPCR detection loquat aneuploid Molecu- lar karyotyping Download PDFInfo
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Abstract
The present invention provides a kind of method based on SSR marker and qPCR detection loquat aneuploid Molecu- lar karyotyping, and the 17 pairs of SSR primers obtained according to screening can be used for detecting the entire molecule caryogram of loquat aneuploid in conjunction with qPCR technology.The present invention does not need additional reference gene, can be suitable for heterozygosis and homozygous genotype simultaneously, not limited by material and chromosome morphology, at low cost, the time is short, applied widely.
Description
Technical field
The present invention relates to the identifications of loquat karyotype, and in particular to one kind detects loquat based on SSR marker and qPCR
The method of aneuploid Molecu- lar karyotyping.
Background technique
Loquat (Eriobotrya japonica Lindl) is rosaceae (Rosaceae) Maloideae Eriobotrya,
There is extensive plantation in Subtropic of China area and Asia other countries.Loquat originates from China, has cultivation in more than 2,000 years
History is trained, germ plasm resource is abundant.Loquat category subtropical evergreen fruit trees, how spherical in shape mature loquat is or oval, soft
Succulence, is rich in glutamic acid, phenolic substances (especially flavone compound), and the nutritional ingredients such as vitamin and carotenoid are
Important health fruit.Loquat except fresh food in addition to or processed can, jam, fruit cream, jelly and fruit wine good raw material.
Aneuploid (aneuploid) refers to that the genome relative to normal individual (euploid) increases, reduces one
Or the bion of several chromosome.Aneuploid usually with specific to the unique of aneuploid type and usually harmful table
Type is related, and trisomy 21 syndrome and cancer are all typical aneuploids.And in plant, guava, in pears and loquat plant
Aneuploid it is related with the selection of dwarfing rootstock.In aneuploid research, the identification of aneuploid and its entire molecule core
The building of type is vital.Currently, loquat not yet carries out genome sequencing, and loquat field of molecular breeding is made to face bottle
Neck, and the building of genetic map depends on aneuploid material.Loquat aneuploid Molecu- lar karyotyping is configured to its point
Sub- breeding provides more possible, the also aneuploid molecular breeding plan further to explore other species of aneuploidy and enhancing
Provide reliable strategy.Currently, studies on loquat germplasm includes diploid, triploid, tetraploid, and there is a small amount of aneuploid to see
Report.Aneuploid material is mainly derived from the hybridization between Different Ploidy, especially hybridizes with triploid.Loquat aneuploid
Have in the establishment of the corresponding relationship of the determination of the physical location of its gene and molecular labeling, gene transfer, linkage group and chromosome
There is unrivaled advantage.
The detection of aneuploid is mainly based upon the detection of chromosome-specific marker's abundance (quantity).The side detected at present
Method mainly has: chromosome morphology observation, in situ hybridization (ISH), quantitative fluorescent PCR (QF-PCR), high-resolution solubility curve
(HRM), multiple links probe amplification technology (MLPA), high-resolution microarray comparative genomic hybridization hybrid technology (CGH), mononucleotide
Polymorphism microarray analysis technology (SNP array).Chromosome observation is that the form based on different chromosomes is variant；It is in situ miscellaneous
Friendship is then using the distinctive large fragment DNA sequence of chromosome or repetitive sequence as probe；QF-PCR is the peak value based on heterozygous sites
Difference carries out Genotyping；Target sequence in MLPA reaction needs to design long probe and short probe simultaneously, long probe prepare compared with
It is complicated；HRM is different based on mononucleotide melting temperature and forms the gene analysis technique of different shape melting curve；CGH is
Using metaphase chromosome as hybridization target, related gene copy number is reacted with target spot fluorescence intensity；SNP array is based on high density
SNP microarray feature.First two method is observed dependent on more complicated chromosome sectioning and chromosome morphology, is easier to occur
Error；QF-PCR and HRM is confined to heterozygous genotypes when detecting；MLPA and CGH is upper more complex in probe preparation；It is highdensity
SNP higher cost.
Summary of the invention
The method based on SSR marker and qPCR detection aneuploid Molecu- lar karyotyping that the object of the present invention is to provide a kind of, is used
In quick differentiation, identify aneuploid Molecu- lar karyotyping, this method is in other methods compared to also relatively inexpensive.
To achieve the above object, technical solution of the present invention is as follows:
A method of loquat aneuploid Molecu- lar karyotyping is detected based on SSR marker and qPCR, is used to identify loquat
17 pairs of primers of aneuploid Molecu- lar karyotyping are to be screened by loquat high density linkage map to be specifically shown in the following table 1:
1 17 pairs of primers of table
| Primer numbers | Upstream primer | Downstream primer |
| LG1 | GAAGAAGCAAAACCCGAAGA | TTGTTCTCCTCGCCACCTT |
| LG2 | ATTGGCATTGCTTCTCACC | TGCAACAACAATTCCCTTCA |
| LG3 | GCGCTGAAAAAGGTCAGTTT | CAAGGATGCGCATGTATTTG |
| LG4 | TGGCTAAATACTCTTCTCGAAAACAA | GTGATTATTATAGATACCAAGCCTCTC |
| LG5 | GAGTTGGCAGAAAGAAACCA | CTGGGTGAAGACGAGATGCT |
| LG6 | CTGCCCTCAAGGAGAATGTC | ACAGGTGCAGCAAAGGCTAT |
| LG7 | ATCTCCTGCTGTGCTGGTCT | TCACCAAACACCAATCAACAA |
| LG8 | CATTATCCATTTGATTAAACTACACG | GGTAGAAAGAGAAGGAAAGTGGG |
| LG9 | CTGACACCCACTACGATTCAAGA | AAACGAGCTTGGTACGGATTACA |
| LG10 | GATGCGGTTTGACTTGCTTC | GTTTCTCCAGCTCCCATAGATTGC |
| LG11 | GAGGAAGTAACCGCATCAGC | TCTAAGGGCAGGCAGATCAC |
| LG12 | GCGTGAGTTGAGCAAGATGG | TAGAAGCAATAAGGTGGAGTGGT |
| LG13 | CTCATCAGTCTCACTGACTGTGTG | AGGGTCAGGGTCAGTCAGG |
| LG14 | AGACTGCTGAGGGAATCCATAA | TTCCGAGTCAAATGGGGC |
| LG15 | ATTCATTGCACCGACTACCGATT | AGTGGCGTAGTGGGAAGGG |
| LG16 | CAATGGCGTCTGTGTCACTC | GTTTACGACGGGTAAGGTGATGTC |
| LG17 | CAGCAGGAAACACAGAAAAACAG | ATATCGAGCAATCAAGGAAGCAG |
The above-mentioned method based on SSR marker and qPCR detection detection loquat aneuploid Molecu- lar karyotyping, specifically includes as follows
Step:
(1) detected material and its male parent and maternal tender leaf are taken；
(2) genomic DNA is extracted, DNA mass is detected；
(3) qPCR detection is carried out using genomic DNA in above-mentioned SSR primer pair (2), data processing confirmation detects non-whole
Times body Molecu- lar karyotyping.
Above-mentioned data processing the following steps are included:
(1) Exponential growth stage by the amplification curve of qPCR in PCR amplification chooses bottom, middle part, 3, top circulation threshold
It is worth (CT)；
(2) amount (the Δ Rn of the PCR product of each pair of SSR primer is checked respectively in 3 CT values1、ΔRn2、ΔRn3), and find out
Δ Rn average value ((the Δ Rn of each pair of SSR primer1+ΔRn2+ΔRn3)/3)；
(3) the corresponding SSR primer of each linkage group is each other as control, with the Δ Rn average value pair of one pair of them SSR primer
The Δ Rn average value of whole SSR primers is uniformed；
(4) the Δ Rn mean value after whole SSR primer uniforms in male parent and female parent is uniformed again as denominator
And as control, the Δ Rn in detected material is uniformed divided by the Δ Rn value of control, and with compare, according to than
Compared with difference combination chromosome sectioning result judge chromosome number and positioning dyeing body it is abnormal where linkage group.
The Δ Rn ratio situation of change of above-mentioned detected material and its Molecu- lar karyotyping theory corresponding relationship are as follows: 1 (2n, 3n,
4n caryogram), 0.5 (2n-1 caryogram), 1.5 (2n+1 caryogram), 0.67 (3n-1 caryogram), 1.33 (3n+1 caryogram), 0.75 (4n-1 core
Type) and 1.25 (4n+1 caryogram).
The utility model has the advantages that
The present invention provides a kind of method based on SSR marker and qPCR detection loquat aneuploid Molecu- lar karyotyping, with screening
The 17 pairs of SSR primers obtained, in conjunction with qPCR technology, for detecting the entire molecule caryogram of loquat aneuploid.The present invention only needs
17 pairs of SSR primers, do not need a large amount of SSR marker, do not need additional reference gene, can be suitable for heterozygosis and homozygous base simultaneously
It because of type, is not limited by material and chromosome morphology, at low cost, the time is short, applied widely.
Detailed description of the invention
Fig. 1 is qPCR amplification curve；
Fig. 2 is 1 chromosome sectioning figure of embodiment.
Specific embodiment
The present invention is specifically described below by specific embodiment, it is pointed out here that following embodiment is served only for this hair
It is bright to be further described, it should not be understood as limiting the scope of the invention, the person skilled in the art of this field can root
Some nonessential modifications and adaptations are made to the present invention according to foregoing invention content.Loquat Cultivars used in the embodiment of the present invention
" H39 " is the Seedling tree of ' non-unclear state is beautiful ', and " A313 ", " A322 " are the triploid Seedling trees of ' big five-pointed star ' (referring to Xu
Wave etc., the ssr analysis of " big five-pointed star " natural triploid loquats strain, Southwest University's journal natural science edition, in March, 2015, the
The phase of volume 37 the 3rd).
Embodiment 1
1, experimental procedure:
(1) take detected material (Seedling tree ' A313-1 ' of the Seedling tree ' H39 ' of ' non-unclear state beautiful ', A313 ',
Seedling tree ' A322-1 ', ' A322-2 ', ' A322-3 ' of A322 ') and control material (include detected material male parent and mother
Originally tender leaf)；
(2) loquat genomic DNA is extracted, detects DNA mass using 1% Ago-Gel, and be diluted to after surveying concentration
50ng/μL；
(3) the 17 pairs of SSR primers (table 1) obtained using screening carry out qPCR detection to loquat genomic DNA in (3).
2, data processing
(1) pass through qPCR (2 × NovoStart SYBR qPCR SuperMix Plus (Novoprotein
Scientific Inc), 95 DEG C of 1min, 40 circulation (95 DEG C of 20s, 65 DEG C of 1min)) amplification curve (as shown in Figure 1) exist
The Exponential growth stage of PCR amplification chooses 3 CT value (bottom (CT1), middle part (CT2), top (CT3))；
(2) amount (the Δ Rn of the PCR product of each pair of SSR primer is checked respectively in 3 CT values1、ΔRn2、ΔRn3), and find out
Δ Rn average value ((the Δ Rn of each pair of SSR primer1+ΔRn2+ΔRn3)/3)；
(3) the corresponding SSR primer of each linkage group is average with the Δ Rn of SSR primer NZmsCO754252 each other as control
Value uniforms the Δ Rn average value of whole SSR primers；
Δ Rn mean value after (4) 17 pairs of SSR primers uniform in male parent and female parent is uniformed again as denominator
And as control, the Δ Rn in detected material is uniformed divided by the Δ Rn value of control, and with compare, according to than
Compared with difference combination chromosome sectioning result judge chromosome number and positioning dyeing body it is abnormal where linkage group.Detected material
The Δ Rn ratio situation of change of material and its Molecu- lar karyotyping theory corresponding relationship are as follows: 1 (2n, 3n, 4n caryogram), 0.5 (2n-1 core
Type), 1.5 (2n+1 caryogram), 0.67 (3n-1 caryogram), 1.33 (3n+1 caryogram), 0.75 (4n-1 caryogram) and 1.25 (4n+1 cores
Type).
Specific detection to detected material ' H39 ', ' A313-1 ', ' A322-1 ', ' A322-2 ', ' A322-3 ' and control
As a result 2,3,4 be see the table below.
2 H39 testing result of table
3 A313-1 testing result of table
4 A322-1, A322-2, A322-3 testing result of table
The results show that the Δ of linkage group LG3, LG8, LG10, LG16, LG17 of the Seedling tree ' H39 ' of ' non-unclear state is beautiful '
Rn value obviously becomes larger, mean ratio 1.41, is greater than 1.33, less than 1.50.Chromosome sectioning is (referring to Dang Jiangbo et
Al., Identification and characterization of a loquat aneuploid with novel leaf
Phenotypes, HortScience, 2019 year, the of page -808 of page 804 of volume 54): ' H39 ' is aneuploid (2n+5=39),
QPCR is consistent with chromosome sectioning testing result.Conclusion: increased 5 chromosome be located at LG3, LG8, LG10, LG16,
LG17.' A313-1 ', ' A322-1 ', ' A322-2 ', ' A322-3 ' chromosome sectioning result see Fig. 2, ' A313-1 ' be 40
Chromosome, ' A322-1 ' are 70 chromosomes, and ' A322-2 ' is 69 chromosomes, and ' A322-3 ' is 73 chromosomes, qPCR inspection
It is consistent with chromosome sectioning result to survey result.Similarly, the Seedling tree ' A313-1 ' of A313 ' is aneuploid (2n+6=
40), increased 6 chromosome is located at LG1, LG4, LG7, LG11, LG12, LG14；Seedling tree ' the A322- of ' A322 '
1 ' is aneuploid (4n+2=70), and increased 2 chromosome is located at LG2, LG4；The Seedling tree ' A322-2 ' of ' A322 '
For aneuploid (4n+1=69), increased 1 chromosome is located at LG12；The Seedling tree ' A322-3 ' of A322 ' is non-multiple
Body (4n+5=73), increased 5 chromosome are located at LG1, LG5, LG7, LG13, LG14.
Claims (4)
1. a kind of method based on SSR marker and qPCR detection loquat aneuploid Molecu- lar karyotyping, is used to identify that loquat is non-
17 pairs of primers of euploid Molecu- lar karyotyping are to be screened by loquat SSR high density linkage map to be specifically shown in the following table 1:
1 17 pairs of primers of table
。
2. the method as described in claim 1, which is characterized in that specifically comprise the following steps:
(1) tender leaf of detected material and its control material is taken；
(2) genomic DNA is extracted, DNA mass is detected；
(3) genomic DNA carries out qPCR detection in the SSR primer pair (2) obtained using screening, and data processing confirms detected Pi
Rake aneuploid Molecu- lar karyotyping.
3. the method as described in claim 1, which is characterized in that the data processing the following steps are included:
(1) Exponential growth stage by the amplification curve of qPCR in PCR amplification chooses bottom, middle part, 3, top CT value；
(2) the amount Δ Rn of the PCR product of each pair of SSR primer is checked respectively in 3 CT values1、ΔRn2、ΔRn3, and find out each pair of
Δ Rn average value (the Δ Rn of SSR primer1+ΔRn2+ΔRn3)/3；
(3) the corresponding SSR primer of each linkage group is each other as control, with the Δ Rn average value of one pair of them SSR primer to whole
The Δ Rn average value of SSR primer is uniformed；
(4) Δ Rn average value of the whole SSR primer in control material is uniformed as denominator again and as control,
Δ Rn in detected material is uniformed divided by the Δ Rn value of control, and with compare, according to the difference knot after relatively
Close the linkage group that chromosome sectioning result judges chromosome number and the abnormal place of positioning dyeing body.
4. the method as described in claim 1, which is characterized in that the Δ Rn ratio situation of change and its point of the detected material
Daughter nucleus type theory corresponding relationship is as follows: 1 is 2n, 3n, 4n caryogram；0.5 is 2n-1 caryogram；1.5 be 2n+1 caryogram；0.67 is 3n-1
Caryogram；1.33 be 3n+1 caryogram；0.75 is 4n-1 caryogram；1.25 be 4n+1 caryogram.
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| CN112941228A (en) * | 2021-04-06 | 2021-06-11 | 西南大学 | Primer for polyploid loquat pulp color genotyping and genotyping method thereof |
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