CN105803083A - Molecular marker of pear fruit single fruit weight main-effect QTL (quantitative trait loci) site MSF, and application thereof - Google Patents
Molecular marker of pear fruit single fruit weight main-effect QTL (quantitative trait loci) site MSF, and application thereof Download PDFInfo
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Abstract
The invention belongs to the field of pear molecule breeding, and provides a molecular marker of a pear fruit single fruit weight main-effect QTL (quantitative trait loci) site, and application thereof. A starkrimson and Wanxiu pear combined genetic linkage map is built by SSR (simple sequence repeat) marker; the pear fruit single fruit weight phenotype identification is combined; in addition, an interval mapping method is used for QTL analysis; the existence of the main-effect QTL site is detected at the eleventh linkage group of the combined linkage map; the contribution rate of the main-effect QTL site is 51.9 percent. The SSR marker tightly linked with the main-effect QTL site is QS1122-135, and the genetic distance is 4.37cm. The obtained pear fruit single fruit weight main-effect QTL molecular marker can be used for early selection on the basis of fruit characters of single fruit weight; the important theoretical and practice guidance significance is realized for improving the breeding efficiency.
Description
Technical field
The invention belongs to pears molecular genetic breeding field, be specifically related to a kind of pear fruit single fruit weight main effect QTL position
The point molecular marker of MSF and screening technique thereof and application.
Background technology
Pears (Pyrus L.) are the industrial crops that China is important, and cultivated area and yield all occupy first place in the world.Pears
Being of high nutritive value of fruit, crisp succulence, sour and sweet palatability, and there is certain medical value, disappeared by vast
Expense person likes.Single fruit weight is the key factor of pear fruit exterior quality, and fruit is big or little directly determines pears product
The yield planted and economic benefit.Therefore, pear fruit single fruit weight is one of Pear varieties Major Economic.Cultivation is met
The single fruit weight closing different consumer demand is one of pears breeding objective.
Pears are perennial woody plants, and the generation cycle is long, and result is late.In traditional breeding method, it is to utilize fruit phenotype
Character pair pears genotype selects, and this method is not only affected by pears child the phase, and is vulnerable to extraneous ring
The left and right in border.Along with the development of molecular breeding technology, build high density genetic linkage maps also by molecular marker
Fruit single fruit weight carries out QTL (Quantitative trait loci, quantitative trait locus) analyze and be positioned to
For reality, also accuracy and predictability for improving the selection of destination number character excellent genes are laid a good foundation.Profit
By Molecular mapping quantitative trait QTL, its essence is exactly between analyzing molecules labelling and objective trait QTL
Exchange rate, determine the particular location of QTL.Based on obtain marker genetype separate with quantitative trait it
Between relation, can directly determine the site of character of controlling the size, exploitation can be applicable to molecular marker assisted selection and educates
The technology planted, this has achieved successfully application on many important crops.
QTL Position Research about pears quantitative trait at present still belongs to the starting stage, for disease, growth traits
Research with fruit parts character QTL location and molecular marker has been reported.But for pear fruit single fruit weight
QTL location is only limited to ' BAYUEHONG ' this parent, and its practicality is still limited by ' BAYUEHONG ' and ' Dangshan
Suli pear ' F1Hybrid Population.Therefore, carry out different groups genetic linkage maps and build, and carry out pears single fruit
The QTL location of weight is the necessary means widening molecular marker range of application.The present invention use ' red eggplant pears ' and
The filial generation F of ' elegant pears in evening '1The QTL location carrying out genetic linkage maps structure and single fruit weight for colony is
The raising of single fruit weight molecular marker assisted selection breeding technique and supplementing.
Summary of the invention
It is an object of the invention to provide a kind of pear fruit single fruit weight main effect QTL site MSF molecular marker and
The primer pair of this molecular marker.
It is a further object of the present invention to provide the molecular marker of a kind of pear fruit single fruit weight main effect QTL site MSF
Method.
It is yet another object of the invention to provide the molecular marker of a kind of pear fruit single fruit weight main effect QTL site MSF
Screening technique.
Another object of the present invention is to provide the molecular marker of pear fruit single fruit weight main effect QTL site MSF at pears
Application in pears molecular breeding.
The purpose of the present invention can be achieved through the following technical solutions:
A kind of molecular marker QS1122-135 of pear fruit single fruit weight main effect QTL site MSF, this molecule mark
Note QS1122-135 forward primer sequence as shown in SEQ ID NO.1, reverse primer sequences such as SEQ ID
Shown in NO.2.
Above-mentioned molecular marker QS1122-135 is to carry out with the genomic DNA of Pear varieties show in evening pears for substrate
The DNA fragmentation of a length of 135bp of PCR amplification gained, this molecular marker QS1122-135 Yu MSF it
Between the genetic distance be 4.37cm.
A kind of primer pair of the molecular marker QS1122-135 of pear fruit single fruit weight main effect QTL site MSF,
The forward primer sequence of described primer pair as shown in SEQ ID NO.1, reverse primer sequences such as SEQ ID NO.2
Shown in.
A kind of molecule labelling method identifying pear fruit single fruit weight main effect QTL site MSF, described method be with
Pears genomic DNA, as template, carries out PCR with the primer of above-mentioned molecular marker QS1122-135 to for primer
Amplification, amplified production carries out electrophoresis detection, as amplified the DNA fragmentation of a length of 135bp, then indicates pears
Fruit single fruit weight main effect QTL site MSF exists.
A kind of screening technique of the molecular marker QS1122-135 of pear fruit single fruit weight main effect QTL site MSF,
Comprise the following steps:
(1) Pear varieties ' red eggplant pears ' and ' elegant pears in evening ' hybridization are utilized, it is thus achieved that F1For colony's individual plant；
(2) two parents of SSR molecular marker methods analyst and F thereof are utilized1Colony, statistical analysis is between parent
There is the site of the polymorphism hereditary form in progeny population, it is thus achieved that polymorphism mark site, then it is entered
Row linkage analysis, builds ' red eggplant pears ' and ' elegant pears in evening ' associating genetic linkage maps, wherein to polymorphism mark
Note site carries out using Jionmap4.0 mapping software during linkage analysis；
(3) F to ' red eggplant pears ' and ' elegant pears in evening ' filial generation1The fruit single fruit weight of colony's individual plant is measured,
Utilize the Interval Mapping (MapQTL5.0 mapping software) of MapQTL5.0 mapping software, by F1Colony is every
' red eggplant pears ' and ' elegant pears in evening ' that the single fruit weight of individual individual plant builds with step (2) combine genetic linkage maps
In molecular marker carry out chain and qtl analysis, with LOD value >=3 as standard, more than 3 explanation existence one
Individual QTL site, examines in the 11st linkage group of ' red eggplant pears ' and ' elegant pears in evening ' associating genetic linkage maps
Measuring QTL site MSF of single fruit weight, its LOD value is 8.78, and contribution rate is 51.9%, is main effect QTL；
(4) region, main effect QTL site obtained according to step (3), selects little with its linkage distance
In the SSR molecular marker of 5cm, wherein molecular marker QS1122-135 can be to F1Colony's fruit single fruit principal characteristic
Shape carries out good typing, and therefore, the present invention selects QS1122-135 as pear fruit single fruit weight main effect QTL
The molecular marker of site MSF.The a length of 135bp of described molecular marker QS1122-135, with MSF it
Between the genetic distance be 4.37cm, this molecular marker can be as the labelling of pear fruit single fruit weight；Described molecule
The forward primer sequence of labelling QS1122-135 as shown in SEQ ID NO.1, reverse primer sequences such as SEQ ID
Shown in NO.2.
The sieve of the molecular marker QS1122-135 according to above-mentioned pear fruit single fruit weight main effect QTL site MSF
Choosing method, described main effect QTL site MSF is positioned at ' red eggplant pears ' and ' elegant pears in evening ' associating genetic map
In 11st linkage group, this site contribution rate is 51.9%.
The molecular marker QS1122-135 of above-mentioned pear fruit single fruit weight main effect QTL site MSF is at pears molecule
Application in breeding.
The screening side of the molecular marker QS1122-135 of above-mentioned pear fruit single fruit weight main effect QTL site MSF
Method application in pears molecular breeding.
Beneficial effects of the present invention:
(1) present invention uses two parents ' red eggplant pears ' and ' elegant pears in evening ' fruit single fruit Traits change are big,
' red eggplant pears ' mean fruit weight 200g, ' elegant pears in evening ' mean fruit weight about 400g, its filial generation fruit
Real single fruit weight character has carried out good separation, therefore, it can be ensured that pear fruit single fruit weight character QTL site is examined
The accuracy surveyed and repeatability.
(2) present invention employs SSR marker method and carry out pear fruit single fruit weight character QTL location with chain
The screening of labelling, SSR marker operates more preferable, technology the easiest, repeated in molecular mark
Require low, accuracy is high, is codominance and anchor marker, be more prone in molecular mark application.
(3) present invention determines ' red eggplant pears ' and ' elegant pears in evening ' hybrid Population F1For individual plant pear fruit list
The main effect QTL site of fruit weight, higher to this quantitative trait contribution rate, the contribution rate in site is 51.9%, because of
This, the accuracy judged single fruit weight based on this site screening linkage molecule labelling is preferable, especially in single fruit weight pole
Big and minimum between differentiate accuracy higher.
(4) distance of the most universally recognized linked marker and objective trait being applied to molecule assisted Selection needs
≤ 5.0cm, the genetic distance between molecular marker QS1122-135 and the main effect QTL site of the present invention is
4.37cm, chain very tight, it is effectively increased and utilizes Marker-assisted selection pear fruit single fruit weight main effect QTL position
The accuracy selected and the Breeding Efficiency of pears large fruit kind, therefore, the pear fruit single fruit weight main effect of present invention exploitation
QTL site and molecular marker have good using value, can accelerate the improvement to pear fruit single fruit weight character.
Accompanying drawing explanation
Fig. 1 is pears single fruit weight QTL site MSF at ' red eggplant pears ' and ' elegant pears in evening ' associating genetic linkage maps the
Position in 11 linkage groups.
Wherein, what LG represented is linkage group, and the digitized representation after LG is linkage group number；Linkage group is upper left
The numeral of side is the genetic distance between labelling, and unit is cm, and what right side represented is the name of SSR molecular marker
Claim；The solid rectangle instruction interval MSF of QTL mapping on the right side of linkage group.
Fig. 2 is that the primer of molecular marker QS1122-135 is to ' red eggplant pears ' and ' elegant pears in evening ' hybrid Population F1Generation
The electrophoresis detection figure of PCR amplification.Wherein, the band of arrow indication i.e. QS1122-135, filial generation can amplify
This band show as big fruit.
Fig. 3 is that molecular marker QS1122-135 is at ' red eggplant pears ' and ' elegant pears in evening ' hybridization F1For colony fruit
(in figure, each vertical bar band represents 1 strain F to the typing effect of the character of real single fruit weight1For individual plant, vertical bar band from
The F that the left-to-right fruit single fruit weight of expression successively arranges from small to large1For individual plant).Wherein, vertical black band band represents
The F of amplified band without 135bp1Individual plant；Vertical white band band indicates the F of 135bp band1Individual plant；Article two, black
Vertical bar band in the middle of color vertical line represents the F of single fruit weight type placed in the middle1For individual plant.
Detailed description of the invention
Embodiment 1: pear fruit single fruit weight main effect QTL site MSF's and molecular marker QS1122-135 thereof
Obtain
(1) two Pear varieties ' red eggplant pears ' (maternal) and ' elegant pears in evening ' (male parent) cross combination are utilized,
Obtain 81 strain F1Colony；
(2) with two parents of SSR molecular marker methods analyst and F thereof1Colony, statistical analysis has between parent
There is the site of the polymorphism hereditary form in progeny population, be finally obtained 207 polymorphism mark sites.
Then utilize Jionmap4.0 mapping software that polymorphism mark site is carried out linkage analysis, construct Yi Zhangbao
' red eggplant pears ' and ' elegant pears in evening ' associating genetic linkage maps containing 187 SSR marker.Wherein, SSR
The method of the test operation procedure reference Yomamoto T. of labelling etc. (Euphytica, 2002,124:129-137),
The primer is by the synthesis of Jin Weizhi biotechnology company.SSR marker is examined with 8% polyacrylamide highly basic argentation
Survey.
(3) by F1The separation type of each labelling of colony according to " CP " pattern change into lm × ll, nn × np,
Hk × hk, ef × eg, ab × cd 5 kinds separate type, without amplified band or expand unsharp labelling and be designated as "-",
Statistics meets SSR marker at F1Mask data in mapping population, through X 2 test, the most first removes and partially divides
From labelling, choose LOD=4.0-10.0, maximum recombination fraction r=0.4, build heredity with Kosambi mapping function
Linkage map, is then inserted into the labelling partially separated one by one in collection of illustrative plates, does not change the position relationship of other labelling,
Otherwise remove.With the Genetic linkage map that MapChart2.0 Software on Drawing is final.
(4) in the fruit maturation phase, ' red eggplant pears ' and ' elegant pears in evening ' filial generation F is measured1For fruit single fruit weight.
According to arranging the fruit single fruit weight data investigated, MapQTL 5.0 software is used to pass through permutation test
(Permutation Test) (number of times > 1000) determine threshold value, use Interval mapping (Interval Mapping)
Detect the marker site of LOD peak value.
(5) result shows, in the 11st linkage group of ' red eggplant pears ' and ' elegant pears in evening ' associating genetic linkage maps
QTL site MSF (Fig. 1) of single fruit weight being detected, LOD value is 8.78, and contribution rate is 51.90%.Should
QTL site is main effect QTL.
Wherein, SSR molecular marker method is:
With pears genomic DNA as template, carry out PCR with the primer of molecular marker QS1122-135 to for primer
Amplification, amplified production, after 8% polyacrylamide gel electrophoresis separates, can amplify the one of a length of 135bp
Individual band, shows that the molecular marker chain with QTL site MSF phase exists, MSF and molecular marker
Linkage distance between QS1122-135 is 4.37cm.
The forward primer sequence (SEQ ID NO.1) of the primer pair of described molecular marker QS1122-135 is:
CGCCTTCTCGATCTCTTC；Reverse primer sequences (SEQ ID NO.2) is:
CGGTTGCCAGATTCCATA；
Concrete PCR amplification system is (20 μ L): 10 × Buffer (Mg2+Plus) 2 μ L, 2.5mmol/L dNTP
1.6 μ L, 10mmol/L forward primer 0.6 μ L, 10mmol/L reverse primer 0.6 μ L, Taq DNA are polymerized
Enzyme 0.5U, DNA profiling 50ng, distilled water add to cumulative volume be 20 μ L.
The response procedures of PCR amplification is: 94 DEG C of denaturations 5min；94 DEG C of degeneration 30s, 55 DEG C of annealing 30s,
72 DEG C extend 45s, totally 32 circulations；72 DEG C extend 10min.
Embodiment 2: the molecular marker QS1122-135 of present invention application test in pears molecular breeding
(1) with ' red eggplant pears ' for maternal, ' elegant pears in evening ' are that male parent builds F1For hybrid Population, it is thus achieved that 81
Strain F1Colony's individual plant；
(2) to the 81 strain F obtained1Individual plant carries out QS1122-135 marker detection, method particularly includes: with
Pears genomic DNA is template, carries out PCR amplification with the primer of molecular marker QS1122-135 to for primer,
Amplified production separates (result such as Fig. 2) by 8% polyacrylamide gel electrophoresis；According to molecular marker after electrophoresis
The presence or absence of QS1122-135 determines whether there is corresponding labelling, if there is labelling, this individual plant fruit is described
Real single fruit weight is high, there is not explanation low, simultaneously by actually measured fruit single fruit weight result and Markers for Detection
Result is verified mutually.
Wherein, the forward primer sequence (SEQ ID NO.1) of the primer pair of described molecular marker QS1122-135
For: CGCCTTCTCGATCTCTTC；Reverse primer sequences (SEQ ID NO.2) is:
CGGTTGCCAGATTCCATA；
PCR amplification system is (20 μ L): 10 × Buffer (Mg2+Plus) 2 μ L, 2.5mmol/L dNTP 1.6 μ L,
10mmol/L forward primer 0.6 μ L, 10mmol/L reverse primer 0.6 μ L, Taq archaeal dna polymerase 0.5U,
DNA profiling 50ng, distilled water add to cumulative volume be 20 μ L；
The response procedures of PCR amplification is: 94 DEG C of denaturations 5min；94 DEG C of degeneration 30s, 55 DEG C of annealing 30s,
72 DEG C extend 45s, totally 32 circulations；72 DEG C extend 10min.
Result shows: the 81 strain F obtained in ' red eggplant pears ' and ' elegant pears in evening ' hybridization1In colony, single fruit weight exists
In the 30 strain small fruit type individual plants of 45-175 gram, the DNA cloning result except 5 strains detects a length of 135bp's
Outward, other 25 strain individual plant is all not detected by this band to amplified band (QS1122-135)；Single fruit weight is at 242-584
Gram 29 strain large fruit individual plants in be not detected by the amplified band (QS1122-135) of a length of 135bp except 5 strains
Outward, other 24 strain individual plant all detects that this band, detection accuracy reach 83.3%, and therefore, detection is accurately
Rate is higher.It can thus be seen that have preferably with molecular marker QS1122-135 prediction pear fruit single fruit weight
Prediction effect.
Claims (8)
1. the molecular marker QS1122-135 of a pear fruit single fruit weight main effect QTL site MSF, it is characterised in that
The forward primer sequence of described molecular marker QS1122-135 as shown in SEQ ID NO.1, reverse primer sequences
As shown in SEQ ID NO.2.
Molecular marker QS1122-135 the most according to claim 1, it is characterised in that described molecular marker
QS1122-135 is to be that substrate carries out PCR and expands gained with the genomic DNA of Pear varieties ' evening elegant pears '
The DNA fragmentation of a length of 135bp, the genetic distance between this molecular marker QS1122-135 and MSF is
4.37cm。
3. the primer pair of the molecular marker QS1122-135 described in a claim 1, it is characterised in that described in draw
The forward primer sequence of thing pair is as shown in SEQ ID NO.1, and reverse primer sequences is as shown in SEQ ID NO.2.
4. the molecule labelling method identifying pear fruit single fruit weight main effect QTL site MSF, it is characterised in that
Described method is using pears genomic DNA as template, with the molecular marker described in claim 3
The primer of QS1122-135 carries out PCR amplification to for primer, and amplified production carries out electrophoresis detection, as amplified
The DNA fragmentation of a length of 135bp, then mark pear fruit single fruit weight main effect QTL site MSF exists.
5. the screening technique of the molecular marker QS1122-135 described in a claim 1, it is characterised in that include
Following steps:
(1) Pear varieties ' red eggplant pears ' and ' elegant pears in evening ' hybridization is utilized to obtain F1For colony's individual plant；
(2) two parents of SSR molecular marker methods analyst and F thereof are utilized1Colony, has many between statistical analysis parent
State property site hereditary form in progeny population, it is thus achieved that polymorphism mark site, then carries out chain point to it
Analysis, builds ' red eggplant pears ' and ' elegant pears in evening ' associating genetic linkage maps；
(3) F to ' red eggplant pears ' and ' elegant pears in evening ' filial generation1The fruit single fruit weight of colony's individual plant is measured,
Utilize the Interval Mapping of MapQTL5.0 mapping software, by F1The single fruit weight of each individual plant of colony and step (2)
Molecular marker in ' red eggplant pears ' and ' elegant pears in evening ' the associating genetic linkage maps built carries out chain and QTL
Analyze, with LOD value >=3 as standard, there are QTL site more than 3 explanations, at ' red eggplant pears ' and
QTL site MSF of single fruit weight is detected in 11st linkage group of ' elegant pears in evening ' associating genetic linkage maps,
Its LOD value is 8.78, is main effect QTL；
(4) region, main effect QTL site obtained according to step (3), selects the SSR molecule chain with it
It is labeled as QS1122-135, a length of 135bp of described molecular marker QS1122-135, and between MSF
The genetic distance is 4.37cm, and this molecular marker can be as the labelling of pear fruit single fruit weight；Described molecular marker
The forward primer sequence of QS1122-135 as shown in SEQ ID NO.1, reverse primer sequences such as SEQ ID NO.2
Shown in.
The screening technique of molecular marker QS1122-135 the most according to claim 5, it is characterised in that described
Main effect QTL site MSF is positioned in 11 linkage groups of ' red eggplant pears ' and ' elegant pears in evening ' associating collection of illustrative plates, should
Site contribution rate is 51.9%.
7. the application in pears molecular breeding of the molecular marker QS1122-135 described in claim 1.
8. the application in pears molecular breeding of the screening technique described in claim 5.
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| CN107164368A (en) * | 2017-07-19 | 2017-09-15 | 中国农业科学院郑州果树研究所 | The molecular labeling Marker37152 in the operatic circle heart size main effect QTL site and its primer pair and application |
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| CN115261504A (en) * | 2022-08-11 | 2022-11-01 | 青岛农业大学 | Molecular marker associated with pear pollen abortion character and screening method thereof |
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| CN105803083B (en) | 2019-03-12 |
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