CN110331227A - The identification method of cotton fiber length main effect QTL qFL-chr.5 a kind of and application - Google Patents
The identification method of cotton fiber length main effect QTL qFL-chr.5 a kind of and application Download PDFInfo
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Abstract
The present invention relates to cotton varieties to cultivate field, specifically, providing identification method and the application of a kind of cotton fiber length main effect QTL qFL-chr.5.Inventor has found cotton fiber length QTL qFL-chr.5 and its SSR molecular marker NAU2000 and BNL3020.Using at least a pair can detect cotton gene group in the corresponding primer of above-mentioned molecular labeling, identification or the auxiliary identification excellent individual of cotton fiber length character, while can construct to obtain cotton fiber length Dominant variety using above-mentioned qFL-chr.5 and SSR molecular marker.The fibre length character of qFL-chr.5 and SSR molecular marker label prediction cotton material, are conducive to the breeding of new cotton variety fiber quality, can effectively accelerate the selection process of high-quality cotton kind, shorten the breeding time limit.
Description
Technical field
The present invention relates to cotton varieties to cultivate field, in particular to a kind of cotton fiber length main effect QTL qFL-
The identification method of chr.5 and application.
Background technique
Cotton is worldwide important industrial crops.Cotton fiber is a kind of excellent natural fiber and textile industry
Leading raw material, accounts for critical role in the world and Chinese national economy.Since the 1980s, Cotton in China thoroughly finishes
Raw cotton relies on the history of import for a long time, and cotton textiles and clothes have become large exporting, accounts for national commodity total export value
20% or so, it plays an important role to the national economic development.Cotton fibre quality is made of many physical characteristics, mainly has fiber long
Degree, fibre strength, fibre fineness and uniformity.Fibre length is to evaluate one of the key index of fiber quality characteristics, it is final
Affect the quality of end product.When other index of quality are identical, fiber is longer, and spinning count is higher, and the intensity of cotton yarn is cured
Greatly.For long fibre because fiber contacts area is larger, cohesive force is strong, and yarn strength is higher.Stronger cotton fiber can spin thinner yarn
Branch.Twisting number needed for longer fibers spinning simultaneously is less, and spinning efficiency is higher.But since fibre length is controlled by multidigit point
Quantitative character, genetic mechanism are complicated, it is often more important that, the important quality indicators such as fibre length, fibre strength and yield and yield
There is significant negative correlation in constituent element, these factors constrain the genetic improvement of cotton fibre quality, cause cotton quality breeding
Though the negative correlation of yield and quality still has by the effort of decades.Fibre length is one for determining cotton fibre quality
Crucial character.The genetic analysis of general cotton fiber strength character shows typical amounts character inheritance mode, vulnerable to environment
Condition influences.In different segregating populations, hereditary pattern is also different.Therefore, always to the improvement of cotton fiber length character
For the difficult point for constructing advantage cotton variety, in the prior art to advantage cotton breeding there are at high cost, difficulty is big, makes slow progress
Problem.
In view of this, the present invention is specifically proposed.
Summary of the invention
The first object of the present invention is to provide the application of cotton fiber length main effect QTL qFL-chr.5 a kind of, be somebody's turn to do
QFL-chr.5 is located in cotton gene group, it is thus possible to the correlative study for cotton fiber length character.
The second object of the present invention is to provide the SSR molecule mark of above-mentioned cotton fiber length main effect QTL qFL-chr.5
Note, SSR molecular marker and qFL-chr.5 close linkage, can accurately detect qFL-chr.5.
The third object of the present invention be to provide primer pair for identifying or assisting identification cotton fiber length character and
Kit realizes the detection of cotton fiber length character.
The fourth object of the present invention is to provide the identification of identification or the auxiliary identification excellent individual of cotton fiber length character
The construction method of method and the cotton variety for being endowed cotton fiber length main effect QTL qFL-chr.5 is cotton fiber length
The identification of character and building Dominant variety provide effective means.
The fifth object of the present invention is to provide the application of SSR molecular marker or primer pair or kit.
In order to realize above-mentioned purpose of the invention, the following technical scheme is adopted:
A kind of application of cotton fiber length main effect QTL qFL-chr.5 in following any one:
A) identify or assist identification cotton fiber length character；
B) cotton breeding；
The qFL-chr.5 is located on No. 5 chromosomes of cotton in BNL3020 and NAU2000 marker interval.
The SSR molecular marker of cotton fiber length main effect QTL qFL-chr.5, including NAU2000 or BNL3020；
The BNL3020 is that the length that BNL3020 corresponding primer amplifies is 195- using cotton gene group as template
The DNA fragmentation of 215bp；
BNL3020 corresponding primer includes:
BNL3020-F:5 '-GATAGACTAAGGGACATATCTTCTGC-3 ' (SEQ ID NO.1)；
BNL3020-R:5 '-TACTTTCTTCCGCAGTCCTT-3 ' (SEQ ID NO.2)；
The NAU2000 is that the length that NAU2000 corresponding primer amplifies is 2995- using cotton gene group as template
The DNA fragmentation of 3005bp；
The corresponding primer of NAU2000 includes:
NAU2000-F:5 '-GAAAATGTTCCCCTCTTGTG-3 ' (SEQ ID NO.3)；
NAU2000-R:5 '-GTCGAAACCAGGGGAAATC-3 ' (SEQ ID NO.4).
For identifying or assisting the primer pair of identification cotton fiber length character, the primer pair include it is following a) or b) in
It is at least one:
A) SEQ ID NO.1 and SEQ ID NO.2；
B) SEQ ID NO.3 and SEQ ID NO.4.
For identifying or assisting the kit of identification cotton fiber length character, contain primer pair as claimed in claim 3.
Further, the kit further includes at least one of dNTP, archaeal dna polymerase or amplification buffer.
The application of above-mentioned SSR molecular marker or primer pair or kit in following any one:
A) identify or assist identification cotton fiber length character；
B) cotton breeding.
A method of identification or the excellent individual of auxiliary identification cotton fiber length character detect cotton gene group to be measured
DNA, if cotton to be measured is or candidate is that cotton fiber is long containing at least one of NAU2000 or BNL3020 molecular labeling
Spend the excellent individual of character.
Further, using cotton genomic dna as template, PCR amplification detects NAU2000 and/or BNL3020；
Preferably, the product of the PCR amplification of BNL3020 molecular labeling is 195-215bp, and primer includes:
BNL3020-F:5 '-GATAGACTAAGGGACATATCTTCTGC-3 ' (SEQ ID NO.1)；
BNL3020-R:5 '-TACTTTCTTCCGCAGTCCTT-3 ' (SEQ ID NO.2)；
Preferably, the product of the PCR amplification of NAU2000 molecular labeling is 2995-3005bp, and primer includes:
NAU2000-F:5 '-GAAAATGTTCCCCTCTTGTG-3 ' (SEQ ID NO.3)；
NAU2000-R:5 '-GTCGAAACCAGGGGAAATC-3 ' (SEQ ID NO.4).
A kind of construction method for the cotton variety being endowed cotton fiber length main effect QTL qFL-chr.5, makes qFL-
Chr.5 is located on No. 5 chromosomes of cotton in BNL3020 and NAU2000 marker interval, obtains being endowed cotton fiber length main effect
The cotton variety of QTL qFL-chr.5.
Further, comprising the following steps:
1) whether there is genetic determinant qFL-chr.5 using above method measurement donor cotton, screening is included
The excellent donor cotton of fibre length character；
2) optionally fibre length trait phenotypes are verified；
3) the selection donor cotton excellent comprising fibre length character, and by itself and receptor cotton hybrid, include to generate
The filial generation material of qFL-chr.5；
4) optionally use filial generation material described in step 3) as raw material, repeat step 1-3) with receptor cotton after
Continuous backcrossing 2-5 times, then be selfed 1-5 times, to generate other progeny populations；
Preferably, the receptor cotton is the kind of Agronomically elite；
Preferably, the donor cotton and/or receptor cotton are cotton culture kind, preferably hirsutum cultivar.
Compared with prior art, the invention has the benefit that
Inventor, which utilizes, to be located on No. 5 chromosomes of cotton, the infiltration derived from the new sea 21 of Xinjiang long wool sea island cotton cultivar
8 pairs of extra large land interspecies variation SSR markers encrypt map in segment, complete the work of cotton fiber length character QTL screening and identification.?
To cotton fiber length main effect QTL qFL-chr.5, the chromosome segment containing fibre length main effect QTL qFL-chr.5 is led
Entering is the big individual plant selfing of IL05-08 target zone heterozygosis, constructs a big BC5F4Secondary segregating population (1248 plants), passes through
Graphing method is replaced, by the preliminary finely positioning of qFL-chr.5 between BNL3020-NAU2000, genetic distance 0.9cM, sieve
Choose close linkage SSR marker therewith.The phenotypic variation that the QTL is explained is up to 17.6%.Multi-environment lower and different generations group
Result of study show qFL-chr.5 performance it is very stable, be the QTL of great application value.For the fibre for improving Upland Cotton
Dimension quality trait is laid a good foundation.
The present invention overcomes the at high cost of quality breeding in the prior art, and difficulty is big, and the defect made slow progress utilizes the present invention
The cotton fiber length main effect QTL site of exploitation and its molecular labeling greatly improve the efficiency of selection of fibre length, excavate out excellent
The fiber quality genetic resources of matter provide the technology of genetic resources and marker assisted selection for the quality breeding of cotton, thus greatly
The big fiber quality for improving Cotton in China kind, can be applied to the production and Quality Detection of good fiber quality cotton variety.
Detailed description of the invention
It, below will be to specific in order to illustrate more clearly of the specific embodiment of the invention or technical solution in the prior art
Embodiment or attached drawing needed to be used in the description of the prior art be briefly described, it should be apparent that, it is described below
Attached drawing is some embodiments of the present invention, for those of ordinary skill in the art, before not making the creative labor
It puts, is also possible to obtain other drawings based on these drawings.
Fig. 1 is finely positioning schematic diagram of the qFL-chr.5 on cotton chromosome in embodiment, wherein " a " indicates recombination
The phenotypic number of body and introgressive line IL05-8 are in 0.05 level without significant difference；" b " indicates in recombinant and new land 36 phenotypic number
In 0.05 level without significant difference；36 genotype in the new land of white area=homozygosis, the new sea 21 of black region=homozygosis, grey area
Domain=recombination generation area；
Fig. 2 is molecular labeling BNL3020 in embodiment through target fragment obtained by primer amplification shown in SEQ ID NO:1 and 2
(shown in arrow), wherein swimming lane 1 is DNA maker, and swimming lane 2 is that new extra large 21 genotype expand banding pattern, and swimming lane 3 is 36 in new land
Genotype expands banding pattern；
Fig. 3 is molecular labeling NAU2000 in embodiment through target fragment obtained by primer amplification shown in SEQ ID NO:3 and 4
(shown in arrow), wherein swimming lane 1 is DNA maker, and swimming lane 2 is that new extra large 21 genotype expand banding pattern, and swimming lane 3 is 36 in new land
Genotype expands banding pattern.
Specific embodiment
Embodiment of the present invention is described in detail below in conjunction with embodiment, but those skilled in the art will
Understand, the following example is merely to illustrate the present invention, and is not construed as limiting the scope of the invention.It is not specified in embodiment specific
Condition person carries out according to conventional conditions or manufacturer's recommended conditions.
Unless otherwise indicated, profession used herein and meaning phase known to scientific term and one skilled in the art
Together.In addition, any method similar to or equal to what is recorded or material can also be applied in the present invention.
A kind of application of cotton fiber length main effect QTL qFL-chr.5 in following any one:
A) identify or assist identification cotton fiber length character；
B) cotton breeding；
Wherein, qFL-chr.5 is located on No. 5 chromosomes of cotton in BNL3020 and NAU2000 marker interval.
Inventor, which utilizes, to be located on No. 5 chromosomes of cotton, the infiltration derived from the new sea 21 of Xinjiang long wool sea island cotton cultivar
8 pairs of extra large land interspecies variation SSR markers encrypt map in segment, complete the work of cotton fiber length character QTL screening and identification.?
To cotton fiber length main effect QTL qFL-chr.5, the chromosome segment containing fibre length main effect QTL qFL-chr.5 is led
Entering is the big individual plant selfing of IL05-08 target zone heterozygosis, constructs a big BC5F4Secondary segregating population (1248 plants), passes through
Graphing method is replaced, by the preliminary finely positioning of qFL-chr.5 between BNL3020-NAU2000, genetic distance 0.9cM, sieve
Choose close linkage SSR marker therewith.The phenotypic variation that the QTL is explained is up to 17.6%.Multi-environment lower and different generations group
Result of study show qFL-chr.5 performance it is very stable, be the QTL of great application value.For the fibre for improving Upland Cotton
Dimension quality trait is laid a good foundation.
Quantitative trait locus (QTL): refer to the character indicated with numerical value for controlling generally contiguous distribution to a certain extent
Genetic loci.
The present invention provide cotton fiber length main effect QTL qFL-chr.5 SSR molecular marker, including NAU2000 or
BNL3020, wherein
BNL3020 is that the length that BNL3020 corresponding primer amplifies is 195-215bp's using cotton gene group as template
DNA fragmentation；
BNL3020 corresponding primer includes:
BNL3020-F:5 '-GATAGACTAAGGGACATATCTTCTGC-3 ' (SEQ ID NO.1)；
BNL3020-R:5 '-TACTTTCTTCCGCAGTCCTT-3 ' (SEQ ID NO.2)；
NAU2000 is that the length that NAU2000 corresponding primer amplifies is 2995-3005bp using cotton gene group as template
DNA fragmentation；
The corresponding primer of NAU2000 includes:
NAU2000-F:5 '-GAAAATGTTCCCCTCTTGTG-3 ' (SEQ ID NO.3)；
NAU2000-R:5 '-GTCGAAACCAGGGGAAATC-3 ' (SEQ ID NO.4).
The BNL3020 and NAU2000 and QTL being located on No. 5 chromosomes of cotton (main effect QTL site is known as qFL-chr.5)
It is chain, and it is this it is chain be close linkage, both identifications one is that can determine the presence of qFL-chr.5；Two label genetic distances
For 0.9cM.2 SSR molecular markers, the fibre length character of label prediction cotton material, are conducive to new cotton variety fabric
The breeding of matter can effectively accelerate the selection process of high-quality cotton kind, shorten the breeding time limit.By big to primer and amplified production
Small restriction can accurately define BNL3020 and NAU2000 molecular labeling.
It should be noted that BNL3020 correspondence is drawn when using the genome in the new sea 21 of Xinjiang long wool island cotton variety as template
The length that object amplifies is the DNA fragmentation of 205bp, and the length that NAU2000 corresponding primer amplifies is the DNA fragmentation of 3000bp.
Linkage inheritance is a kind of phenomenon, refers to 2 or 2 or more non-allelic genes on phase homologous chromosomes in heredity
Combined frequency is greater than the phenomenon that frequency desired by Independent distributive rule.
The abbreviation of SSR:Simple Sequence Repeat, Chinese meaning be simple sequence repeat marker, SSR be with
Round pcr is the DNA molecular marker technology of core, and alternatively referred to as microsatellite sequence marks (Microsatellite
Sequence, MS) or Short tandem repeatSTR label (Short Tandem Repeat, STR).
The present invention is provided to identify or assist identification cotton fiber length character primer pair, primer pair include it is following a)
At least one of or b):
A) SEQ ID NO.1 and SEQ ID NO.2；
B) SEQ ID NO.3 and SEQ ID NO.4.
For example, provided by the present invention for identify or assist identification cotton fiber length character primer pair may include
SEQ ID NO.1 and SEQ ID NO.2, perhaps including SEQ ID NO.3 and SEQ ID NO.4 or including SEQ ID NO.1
With SEQ ID NO.2 and SEQ ID NO.3 and SEQ ID NO.4.
Cotton gene group is detected with the primer pair in a) and/or b), as a result occur the DNA fragmentation of 205bp and/or
The DNA fragmentation of 3000bp shows that the cotton has fibre length excellent shape.
For identifying or assisting the kit of identification cotton fiber length character, which contains above-mentioned primer pair.
Cotton gene group is detected using above-mentioned primer pair or kit, if containing NAU2000 or BNL3020
At least one of, then the cotton variety is or candidate is the excellent individual of cotton fiber length character.To cotton variety screening and
Cultivating has important application value.
In some embodiments, kit further includes at least one of dNTP, archaeal dna polymerase or amplification buffer.
A method of identification or the excellent individual of auxiliary identification cotton fiber length character detect cotton gene group to be measured
DNA, if cotton to be measured is or candidate is that cotton fiber is long containing at least one of NAU2000 or BNL3020 molecular labeling
Spend the excellent individual of character.Detect cotton genomic dna to be measured whether contain method existing for NAU2000 and/or BNL3020 can be with
For but be not limited to PCR amplification or sequencing.This method can be using to NAU2000, BNL3020 or NAU2000 and BNL3020 tri-
Kind scheme is detected, simple and quick accurately to make evaluation to cotton fiber length character, is screened and is cultivated to cotton variety
It is of great significance.
In some embodiments, using cotton genomic dna as template, PCR amplification detects NAU2000 and/or BNL3020
Molecular labeling whether there is, and specifically amplimer and product are as follows:
The product of the PCR amplification of BNL3020 molecular labeling is 195-215bp, and primer includes:
BNL3020-F:5 '-GATAGACTAAGGGACATATCTTCTGC-3 ' (SEQ ID NO.1)；
BNL3020-R:5 '-TACTTTCTTCCGCAGTCCTT-3 ' (SEQ ID NO.2)；
The product of the PCR amplification of NAU2000 molecular labeling is 2995-3005bp, and primer includes:
NAU2000-F:5 '-GAAAATGTTCCCCTCTTGTG-3 ' (SEQ ID NO.3)；
NAU2000-R:5 '-GTCGAAACCAGGGGAAATC-3 ' (SEQ ID NO.4).
A kind of construction method for the cotton variety being endowed cotton fiber length main effect QTL qFL-chr.5, specifically includes:
It is located at qFL-chr.5 on No. 5 chromosomes of cotton in BNL3020 and NAU2000 marker interval, obtains being endowed cotton fiber long
Spend the cotton variety of main effect QTL qFL-chr.5.Using the means of molecular biology and genetic engineering, so that cotton variety is assigned
Cotton fiber length main effect QTL qFL-chr.5 is given, the breeding of cotton fine quality is significantly promoted.
Be preferably carried out in mode some, construction method the following steps are included:
1) adopt whether identification donor cotton with the aforedescribed process has genetic determinant qFL-chr.5, screening is wrapped
The excellent donor cotton of fibre-bearing length character；
2) optionally fibre length trait phenotypes are verified；
3) the selection donor cotton excellent comprising fibre length character, and by itself and receptor cotton hybrid, include to generate
The filial generation material of qFL-chr.5；
4) optionally use filial generation material described in step 3) as raw material, repeat step 1-3) with receptor cotton after
Continuous backcrossing 2-5 times, then be selfed 1-5 times, to generate other progeny populations.
This method by it is above-mentioned it is simple hybridization, backcrossing and selfing process can quickly breeding obtain have comprising fiber it is long
The excellent cotton variety of character is spent, fibre length excellent shape is oriented and is added in target cotton, the breeding time limit is greatly shortened,
Improve the improved certainty of cotton variety.
It should be noted that hybridization refers to the mating of two kinds of mother plants.
F1 hybrid/F1 generation refers to the first generation filial generation of two kinds of non-equal gene plants hybridization.
In F2 hybrid/F2 generation, refers to that F1 generation is selfed generated filial generation.
In being preferably carried out mode, receptor cotton is the kind of Agronomically elite.
" Agronomically elite " refers to genotype as used herein, the optimal representation with many distinguishability shapes,
It is the agronomy attribute possessed by plant known in those skilled in the art conducive to plant production and/or business application.Such as list
Strain knot bell number, single plant seed cotton yield, lint yield per plant, bell weight, clothing part, fibre length, fibre strength, mic value, growth
Rate, drought tolerance, heat-resisting quantity, nitrogen intake, suitable for machine adopt, harvest index, plant height, salt tolerance, early stage seedling vigor and
The lower emergence vigor of damage or crop failure caused by waterlogging stress, vegetative vigor resist withered and yellow characteristic of disease, easy pick up to spend.
In being preferably carried out mode, donor cotton and/or receptor cotton are cotton culture kind, further preferably land
Cotton cultivar.
Donor cotton with cotton fiber length advantageous shape is carried out with the receptor kind with other excellent shapes
Hybridization, can accurately orient and quickly obtain the cotton dominant population with a variety of excellent shapes, have to cotton breeding, agricultural development
There is important Practical significance.
In some embodiments, the new 21 fibre length main effect QTL qFL- of sea of Xinjiang long wool island cotton variety will be contained
Secondary segregating population derived from the chromosome segment introgressive line IL05-08 of chr.5, respectively at plantation BC in 20135F4Group and
2014 kinds of plant BC5F5Group is 2 meters long in Tarim University Aksu proving ground row, and film is 0.75 meter wide, and secondary repetition is planted simultaneously
36 and Xin Hai 21 in new land is planted, 25 cotton bolls of every row material are collected to cotton, weighed, cotton ginning weighs gined cotton weight, delivers
Academy of agricultural sciences, state Cotton Research Institute fiber check and measure Spot detection quality trait.Utilize polymorphism mark qualification result combination WinQTL
Cartographer 2.5 positions mapping software qtl analysis result and displacement mapping results and target QTL is positioned at SSR molecule mark
Remember between BNL3020 and NAU2000.BC is planted in Kurle region within 20155F5Segregating population, again in conjunction with displacement drawing method
Once target QTL is positioned between SSR molecular marker BNL3020 and NAU2000.Show the QTL in different generations and environment
Label is extremely stable, has very high Breeding Application value.
The application of SSR molecular marker or primer pair or kit in following any one:
A) identify or assist identification cotton fiber length character；
B) cotton breeding.
Present invention will be further explained by specific examples below, it should be understood, however, that, these embodiments are only
It is used, is but should not be understood as present invention is limited in any form for being described in more detail.
It should be noted that implementation procedure of the invention is: new sea 21 by the breeding of the one teacher institute of agricultural sciences of north Xinjiang agriculture morning
Ripe, high yield, high-quality long wool cotton variety (Cotton, 2004).In new land 36 by the breeding of cotton institute, Shihezi Univ Early-mid ripening
Upland cotton new varieties (Cotton, 2011).Above-mentioned material is to be widely applied kind in production.Through Jiangxi Province science
After institute, Tarim University introduce, selfing saves breeding for many years, if other colleagues need, Jiangxi Academy of Agricultural Sciences, Tarim Basin
University guarantees that these germplasm materials can be provided in 20 years to studies in China unit from the applying date.
Embodiment
1, material to be tested
Jiangxi Academy of Agricultural Sciences in 2008 is receptor parent, Xinjiang in the Xinjiang Upland Cotton high yield new land of disease-resistant variety 36
Cotton variety new sea 21 in long wool island is donor parents, carries out interspecific hybridization.
2, hybridization, backcross progeny selection
Winter in 2009 plants F1 in Hainan Ya Cheng breeding base, and totally 15 plants, backcrossing obtains BC1F1Seed, it is whole after mature
It is mixed to receive.Summer in 2010 plants BC in Tarim, Xinjiang university, proving ground, Aksu1F1(120 plants) of group continues and circulation parent
This backcrossing obtains BC2F1Seed.Winter in 2010 is in Hainan Ya Cheng breeding base kind BC2F1Group's (250 plants), selects 20 at random
Strain obtains BC with 36 backcrossing, mixed receipts in the new land of recurrent parent3F1Seed.Summer in 2011 tries in Tarim, Xinjiang university Aksu
Test base plantation BC3F1Group's (270 plants), selects 60 plants at random and continues to be returned, and mixed receipts obtain BC4F1Seed, winter rice Hai Nanya
City breeding base kind BC4F1Group's (800 plants), selects 90 plants at random and continues to be returned with recurrent parent, and mixed receipts obtain BC5F1Seed.
Summer in 2012 is in Tarim, Xinjiang university Aksu experimental cultivation BC5F11500 plants of acquisition BC of group5F2Seed.
3, QTL mapping analysis
Winter in 2012 in Hainan base Ya Cheng, plants BC5F3Family 1500, based on the linkage map announced
(Guo et al.2008) selects 660 SSR markers of uniform fold cotton gene group to carry out assisted Selection, new in upland cotton
Filter out chromosome segment introgressive line 187 only containing new extra large 21 introgressed segments of 1-2 sea island cotton in land in 36 backgrounds altogether.Often
Strain acquisition 3-4 piece blade undeployed is put into the centrifuge tube of 1.5ml, and the Extraction buffer of the Fresh of pre-cooling is added
600 μ l, are ground with beveller, using CTAB method (Paterson Plant Molecular Biology Reporter, 1993,
11 (2): 122-127.) DNA is extracted, PCR amplification, 10 μ l of amplification system, 1 μ l of DNA profiling, 94 DEG C of pre- changes are carried out with SSR primer
Property 5min, 94 DEG C of denaturation 0.5min, 57 DEG C of renaturation 0.5min, 72 DEG C of extension 1min, 30 circulation after, 72 DEG C re-extend 10min,
Amplified production is through non denatured polyacrylamine gel electrophoresis: gel strength 8%, and electrophoretic buffer is 0.5 times of TBE, and 220V is permanent
Piezoelectricity is swum 1 hour.Above-mentioned 660 SSR molecular markers identify chromosome segment introgressive line IL05-08 genomic DNA analysis
Has polymorphic SSR marker BNL3452 on to No. 5 chromosomes.Introgressive line is examined according to the identical t testing method of variance
64 separation single plant molecular marker gene type analysis of IL05-08 group are as a result, discovery SSR marker BNL3452 and fibre length
Shape is significantly positively correlated (P=0.0011).Single marking can explain 15.8% phenotypic variation, and synergy gene source is in new sea 21
(21 allele of new sea increase fibre length).
BC derived from the introgressive line target interval heterozygote is planted in experiment station, Aksu of Xinjiang within 20135F4Secondary separation
Group's (1248 plants).Has polymorphism in target fragment to what SSR marker was screened using BNL3452 close-proximity target zone 33
8 SSR markers (table 1), to 1248 single plants carry out recombination single plant genotype identification.Wherein, molecular labeling BNL3020PCR
(swimming lane 1 is DNA maker to amplification, and swimming lane 2 is that new extra large 21 genotype expand banding pattern, and swimming lane 3 is in new land as shown in Figure 2
36 genotype expand banding pattern), (swimming lane 1 is DNA maker, swimming lane 2 to molecular labeling BNL2000PCR amplification as shown in Figure 3
Banding pattern is expanded for new extra large 21 genotype, swimming lane 3 is that 36 genotype expand banding pattern in new land).
Molecular Marker Information in 1 introgressive line IL05-08 target fragment of table
Mapping software is positioned according to WinQTL Cartographer 2.5, using composite interval mapping method (Composite
Interval mapping), LR threshold value is 11.5 (being equivalent to LOD value 2.5), and 1000 test Analysis methods find the QTL's
LOD value is 9.5, and the phenotypic variation of explanation is up to 17.6%, and additive effect value is 0.53, and fibre length synergistic effect direction is from sea
The new extra large 21 target fragment genotype of island cotton variety, are then named as qFL-chr5 (a QTL for fiber length on
chromosome 5).According further to (Aksu Prefecture in 2013, Aksu Prefectures in 2014 and 2015 under continuous three environment
Kurle region) fiber quality testing result, by displacement drawing method by the fibre length QTL qFL- on No. 5 chromosomes
Chr.5 finely positioning is between molecular labeling BNL3020-NAU2000, genetic distance 0.9cM, as a result as shown in Figure 1.
Fig. 1 is the BC by 1248 single plants5F4The linkage map in the bulk segregant analysis data building region QTL.Homozygous recombinant
Offspring in 2013,2014 and 2015 years fibre length character investigation results by qFL-chr.5 finely positioning in SSR molecular marker
Between BNL3020 and NAU2000, recombinant is divided into 14 groups according to marker gene type analysis result.Every group of recombinant number
And it is marked on the right with the phenotypic difference conspicuousness of the fibre length in new land between 36 and introgressive line IL05-8." a " indicates weight
The phenotypic number of group body and introgressive line IL05-8 are in 0.05 level without significant difference；" b " indicates in recombinant and new land 36 phenotype
Value is in 0.05 level without significant difference.36 genotype in the new land of white area=homozygosis, the new sea 21 of black region=homozygosis, grey
Region=recombination generation area.
The QTL is in Aksu Prefecture in 2013, Aksu Prefectures in 2014 and 2015 Kurle region planting environment conditions
Under, averagely increase the fibre length of 1.7mm, 1.6mm and 1.5mm, improved effect in 36 backgrounds respectively in the new land of recurrent parent
Up to the level of signifiance (p < 0.05).Therefore the fibre length point of cotton can effectively be carried out using the molecular marker assisted selection QTL
Sub- breeding.Target QTL and respective markers will have very high utility value in upland cotton fiber quality breeding.
Although illustrate and describing the present invention with specific embodiment, it will be appreciated that without departing substantially from of the invention
Many other change and modification can be made in the case where spirit and scope.It is, therefore, intended that in the following claims
Including belonging to all such changes and modifications in the scope of the invention.
SEQUENCE LISTING
<110>Jiangxi Academy of Agricultural Sciences
Tarim University
<120>identification method of cotton fiber length main effect QTL qFL-chr.5 a kind of and application
<160> 16
<170> PatentIn version 3.5
<210> 1
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<212> DNA
<213>artificial sequence
<400> 1
gatagactaa gggacatatc ttctgc 26
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tactttcttc cgcagtcctt 20
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gaaaatgttc ccctcttgtg 20
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gtcgaaacca ggggaaatc 19
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ggatagcagc agggttaaaa 20
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aggaaagcga agagatcctt 20
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tgtaactgag cagccgtacg 20
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cctagagtga gacgaaaccg 20
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tccctttctc aactctcagg 20
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tccctttctc aactctcagg 20
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taaaagctca gccattagcc 20
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tgtgaccaac ggatttttcc 20
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gttacccttg ttcccatgac 20
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Claims (10)
1. a kind of application of cotton fiber length main effect QTL qFL-chr.5 in following any one:
A) identify or assist identification cotton fiber length character；
B) cotton breeding；
The qFL-chr.5 is located on No. 5 chromosomes of cotton in BNL3020 and NAU2000 marker interval.
2. the SSR molecular marker of cotton fiber length main effect QTL qFL-chr.5, which is characterized in that including NAU2000 or
BNL3020；
The BNL3020 is that the length that BNL3020 corresponding primer amplifies is 195-215bp's using cotton gene group as template
DNA fragmentation；
BNL3020 corresponding primer includes:
BNL3020-F:5 '-GATAGACTAAGGGACATATCTTCTGC-3 ' (SEQ ID NO.1)；
BNL3020-R:5 '-TACTTTCTTCCGCAGTCCTT-3 ' (SEQ ID NO.2)；
The NAU2000 is that the length that NAU2000 corresponding primer amplifies is 2995-3005bp using cotton gene group as template
DNA fragmentation；
The corresponding primer of NAU2000 includes:
NAU2000-F:5 '-GAAAATGTTCCCCTCTTGTG-3 ' (SEQ ID NO.3)；
NAU2000-R:5 '-GTCGAAACCAGGGGAAATC-3 ' (SEQ ID NO.4).
3. for identifying or assisting the primer pair of identification cotton fiber length character, which is characterized in that the primer pair includes such as
It is lower at least one of a) or b):
A) SEQ ID NO.1 and SEQ ID NO.2；
B) SEQ ID NO.3 and SEQ ID NO.4.
4. for identifying or assisting the kit of identification cotton fiber length character, which is characterized in that containing described in claim 3
Primer pair.
5. kit according to claim 4, which is characterized in that the kit further includes dNTP, archaeal dna polymerase or expansion
Increase at least one of buffer.
6. SSR molecular marker as claimed in claim 2 or primer pair as claimed in claim 3 or reagent as claimed in claim 4
Application of the box in following any one:
A) identify or assist identification cotton fiber length character；
B) cotton breeding.
7. a kind of method of identification or the auxiliary identification excellent individual of cotton fiber length character, which is characterized in that detect cotton to be measured
Flower genomic DNA, if cotton to be measured is or candidate is cotton containing at least one of NAU2000 or BNL3020 molecular labeling
The flower excellent individual of fibre length character.
8. the method according to the description of claim 7 is characterized in that PCR amplification detects using cotton genomic dna as template
NAU2000 and/or BNL3020；
Preferably, the product of the PCR amplification of BNL3020 molecular labeling is 195-215bp, and primer includes:
BNL3020-F:5 '-GATAGACTAAGGGACATATCTTCTGC-3 ' (SEQ ID NO.1)；
BNL3020-R:5 '-TACTTTCTTCCGCAGTCCTT-3 ' (SEQ ID NO.2)；
Preferably, the product of the PCR amplification of NAU2000 molecular labeling is 2995-3005bp, and primer includes:
NAU2000-F:5 '-GAAAATGTTCCCCTCTTGTG-3 ' (SEQ ID NO.3)；
NAU2000-R:5 '-GTCGAAACCAGGGGAAATC-3 ' (SEQ ID NO.4).
9. a kind of construction method for the cotton variety for being endowed cotton fiber length main effect QTL qFL-chr.5, which is characterized in that
It is located at qFL-chr.5 on No. 5 chromosomes of cotton in BNL3020 and NAU2000 marker interval, obtains being endowed cotton fiber long
Spend the cotton variety of main effect QTL qFL-chr.5.
10. construction method according to claim 9, which comprises the following steps:
1) whether there is genetic determinant qFL-chr.5 using method of claim 7 measurement donor cotton, screens
To the donor cotton excellent comprising fibre length character；
2) optionally fibre length trait phenotypes are verified；
3) the selection donor cotton excellent comprising fibre length character, and by itself and receptor cotton hybrid, it include qFL- to generate
The filial generation material of chr.5；
4) optionally use filial generation material described in step 3) as raw material, repeat step 1-3) continue back with receptor cotton
It hands over 2-5 times, then is selfed 1-5 times, to generate other progeny populations；
Preferably, the receptor cotton is the kind of Agronomically elite；
Preferably, the donor cotton and/or receptor cotton are cotton culture kind, preferably hirsutum cultivar.
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| CN111793715A (en) * | 2020-08-31 | 2020-10-20 | 中国农业科学院棉花研究所 | Haplotype molecular marker related to length of upland cotton fiber and application thereof |
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