CN108085406A - A kind of identification method in upland cotton precocity molecular breeding correlation SSR marker site - Google Patents

A kind of identification method in upland cotton precocity molecular breeding correlation SSR marker site Download PDF

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CN108085406A
CN108085406A CN201711328014.6A CN201711328014A CN108085406A CN 108085406 A CN108085406 A CN 108085406A CN 201711328014 A CN201711328014 A CN 201711328014A CN 108085406 A CN108085406 A CN 108085406A
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刘国栋
张军
陈鹏云
张传云
王芙蓉
周娟
杜召海
陈煜�
张景霞
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Abstract

The invention discloses a kind of identification methods in upland cotton precocity molecular breeding correlation SSR marker site.No. 37 are ground as female parent with Mid-late ripening cotton variety Shandong cotton, No. 19 are ground as male parent with early-maturity cotton kind Shandong cotton, hybridized for 1 generation, 1 generation acquisition F2 is selfed again, and 1/8 precocious strain and 1/8 late-maturing strain are selected in F2 groups, plants F3, selfing obtains F4, the first time that precocious related SSR marker site is carried out using F2 groups is identified, finally carries out chi-square analysis, primarily determines that by the effective upland cotton precocity molecular breeding correlation SSR marker of seedling flower bud phase character determination.Second of identification in precocious related SSR marker site is carried out using F4 groups, finally carries out chi-square analysis, it is final to determine by the effective upland cotton precocity molecular breeding correlation SSR marker of seedling flower bud phase character determination.The beneficial effects of the invention are as follows provide reliable molecular labeling site for upland cotton precocity molecular breeding to support.

Description

A kind of identification method in upland cotton precocity molecular breeding correlation SSR marker site
Technical field
The invention belongs to technical field of crop propagation, are related to a kind of upland cotton precocity molecular breeding correlation SSR marker site Identification method.
Background technology
Cotton prematurity is an excellent Comprehensive Traits, mainly includes the time of infertility, seedling stage, flower bud phase, flowering and boll-setting period, first The characters such as flower rate before fruit branch position and frost, but these characters are all quantitative characters, are controlled by multiple quantitative character gene locus therefors, are lost Pass complicated mechanism and easily by such environmental effects；Traditional breeding way relies primarily on the experience of breeder, to prematureness correlation Shape is difficult to accurately hold, therefore traditional breeding way lacks foresight, and efficiency is low.
Cotton prematurity is quantitative character, and traditional genetic analysis method is difficult accurate track and localization target gene miscellaneous The flow direction in offspring is handed over, the research for developing into quantitative character of molecular marking technique provides effective tool.Linkage analysis is with closing Connection analysis is the main method of current research Quantitative Trait Genes in Plants type, and breeding time, seedling have been located using linkage analysis Flower rate etc. and the relevant QTL of upland cotton Early mature apricot before phase, flower bud phase, flowering and boll-setting period, the first fruit branch position, the first fruit branch position height and frost (Li C,Wang X,Na D,et al.QTL analysis for early-maturing traits in cotton using two upland cotton(Gossypiumhirsutum L.)crosses[J].Breeding Science, 2013,63(2):154-163.), but QTL Mapping parents are not the larger production kinds of the upper popularizing area of production, and QTL makees Map analysis, which can not detect, all to be existed in structure mapping population parent but not to have discrepant allele, and the QTL navigated to With stronger " group's specificity ", therefore the QTL mapping analysis achievements in research based on linkage analysis are difficult to directly apply to cotton The genetic improvement of flower variety Early mature apricot.It has been obtained and upland cotton precocity correlated traits significantly associated 3 using linkage analysis A SSR sites, wherein CER0098-400 are significantly associated with the time of infertility, and DPL0375-250 and HAU2414-147 begin with fruit branch Section position significantly associates that (Liang Bing etc., what upland cotton economical character and SSR (Simple Sequence Repeats) were marked associate point Analysis, Cotton Science, 26 (5)：387-395), still, do not find to show using the cotton prematurity correlated traits of acquisition for many years The play-by-play that cotton prematurity breeding is instructed in associated molecular labeling site is write, this is because the land obtained using association analysis Cotton prematureness related molecular marker site do not verify its whether with cotton prematurity objective trait close linkage, and these molecules Associated objective trait is marked to be unfavorable for instructing assisted Selection in terms of Mature breeding.
" heredity is got a lift " (genetic hitch-hiking) effect, which refers to work as in group, to be occurred to selecting relevant equipotential During gene substitution, due to selecting the effect of pressure, the gene frequency of favourable gene and its linkage site with selection target Rise, and the gene frequency of unfavorable gene and its linkage site declines, theoretically those bases unrelated with objective trait Because site will not be influenced by M8003 line, Mendel's regulavity of segregation is still conformed in segregating population.It is available based on the principle Upland cotton Mature breeding selection group inversely identifies the compact linkage molecule marker site of upland cotton precocity correlated traits, really real Application of the existing molecular marker assisted selection in the practice of upland cotton Mature breeding.
The content of the invention
It is an object of the invention to provide a kind of identification methods in upland cotton precocity molecular breeding correlation SSR marker site.
The purpose of the present invention can be achieved through the following technical solutions：
1) No. 37 are ground as female parent with Mid-late ripening cotton variety Shandong cotton, No. 19 is ground as male parent with early-maturity cotton kind Shandong cotton, it is miscellaneous It handed over for 1 generation, then is selfed 1 generation acquisition F2, F2 generation by single plant harvest；Foundation seedling flower bud phase character selects about 1/8 precocity in F2 groups Strain and about 1/8 late-maturing strain plant F3, and selfing obtains F4, and per generation is all to mix to receive miscegenation；
2) first time that precocious related SSR marker site is carried out using F2 groups is identified：F2 kinds are planted, and are extracted using CTAB methods DNA with reference to the precocial linkage analysis of upland cotton and association analysis result of study, selects the cotton prematurity identified related SSR primers carry out PCR amplification.Then test of independence is carried out to single SSR marker, as shown in table 1, is carrying out SSR molecule marks When remembering genotyping, the parent Lu Mianyan banding patterns of No. 37 are denoted as " 1 ", the parent Lu Mianyan banding patterns of No. 19 are denoted as " 2 ", two The complementary banding pattern of a parent is denoted as " 3 "；Processing is made choice according to seedling flower bud phase character pair F2 groups, about 1/ is selected in F2 groups 6 precocious single plant individual structure F2 is for cotton prematurity M8003 line group.
The general type of 1 test of independence of table is：
Chi-square analysis is finally carried out, is primarily determined that related by the effective upland cotton precocity molecular breeding of seedling flower bud phase character determination SSR marker.
3) second of identification in precocious related SSR marker site is carried out using F4 groups：F4 kinds are planted, after every plant of extraction DNA, Prematureness correlation SSR primers is recycled to determine the genotype of F4 single plants, processing is made choice according to seedling flower bud phase character pair F4 groups, About 1/3 precocious single plant individual is selected to build F4 for cotton prematurity M8003 line group in F4 groups.Then to single SSR Mark carries out test of independence, as shown in table 2；
The general type of 2 test of independence of table is：
Chi-square analysis is finally carried out, it is final to determine by the effective upland cotton precocity molecular breeding correlation of seedling flower bud phase character determination SSR marker.
The beneficial effects of the invention are as follows provide reliable molecular labeling site for upland cotton precocity molecular breeding to support.
Description of the drawings
Fig. 1 utilizes prematureness M8003 line group identification upland cotton precocity molecular breeding correlation SSR marker site
Specific embodiment
The present invention is described in detail With reference to embodiment.
1 Selection parent of embodiment
Shandong Cotton Research Center grinds No. 37 as female parent, pest-resistant short season cotton Shandong cotton with Mid-late ripening Normal insect-resistant cotton kind Shandong cotton No. 19 are ground as male parent, builds F2 groups and F4 groups respectively, according to seedling flower bud phase (SSP, from the number of days for being seeded into cotton and blooming) property Shape carries out identification selection to the precocious single plant of F2 groups and F4 groups respectively.Parent Lu Mianyan 37 is to grind No. 16 with Shandong cotton to turn Bt The choosing of gene Normal insect-resistant cotton is Shandong S6145 for male parent, and hybridization of female parent is in mid-early maturity excellent strain Shandong 9136 and what is selected turn base Because of Mid-late ripening Mature Cotton New Variety, there is the features such as plant type is compact, and blade is loose, and stalk is tough and tensile, high-output stress-resistance；Parent Lu Mian Grind No. 19 be using the choosing of No. 3 trans Bt gene Normal insect-resistant cottons of nasal mucus cotton be 55 system of Shandong as male parent, 458 system of short season cotton Shandong is hybridization of female parent choosing The short season Mature Cotton New Variety of transgenosis brought out, has the characteristics that prematureness is good, ginning outturn is high.
Embodiment 2
Planted respectively in Linqing experiment station of Shandong Cotton Research Center within (1) 2014 year Shandong cotton grind No. 37 and Shandong cotton grind No. 19 it is each 1 row, Shandong cotton grind No. 37 as female parent, and Shandong cotton grinds No. 19 and generates F1 for the combination of male parent preparing hybrid, mixes and receives after ripe, exists in the winter then Sanya, Hainan plants 2 row F1, and selfing obtains F2, and F2 generations are harvested by single plant, according to seedling flower bud phase (SSP, from being seeded into what cotton bloomed Number of days) character, about 1/8 precocious strain and about 1/8 late-maturing strain are selected in F2 groups, plants F3, selfing obtains F4, often Generation is all mixed receipts miscegenation.Field management carries out according to a conventional method.
(2) prematureness M8003 line group is built：20 rows are planted in Linqing experiment station of Shandong Cotton Research Center within 2015 F2, obtains the F2 groups comprising 477 single plant individuals by often row 22-26 plants, by single plant investigate F2 groups seedling flower bud phase (SSP, from Be seeded into the number of days that cotton blooms) character, according to seedling flower bud phase trait data selected in F2 groups about 1/6 precocious single plant individual F2 is built for cotton prematurity M8003 line group.
20 row F4 are planted within 2016 in Linqing experiment station of Shandong Cotton Research Center, often row 20-24 plants, are obtained comprising 445 The F4 groups of a single plant individual, by seedling flower bud phase (SSP, from the number of days for being seeded into cotton and blooming) character of single plant investigation F4 groups, The precocious single plant individual that about 1/3 is selected in F4 groups according to seedling flower bud phase trait data builds F4 for cotton prematurity M8003 line Group.
(3) molecular marker gene type is identified：In F2 and F4 groups, 1 blade being just unfolded of every plant of acquisition is put into 2.0mL In Eppendorf pipes, steel ball and 700 μ L Extraction buffers (0.35M Glucose, 0.1M Tris-HCl are sequentially added (pH8.0), (V/V) β of 5mM Na-EDTA (pH8.0), 2%PVP, 1%-Me), blade is ground using tissue grinder instrument.Using CTAB methods (PatersonAH, Brubaker CL, Wendel JF.A rapid method for extraction of cotton(Gossypium spp.)genomic DNA suitable for RFLP or PCR analysis[J].Plant Mol Biol Rep,1993,11,2:DNA 122-127) is extracted, PCR expansions are carried out using 24 pairs of cotton prematurity correlation SSR markers Increase, amplification system is 10 μ L, 2 each 0.6 μ L, 10mM dNTPMix0.2 μ of the forward and reverse primer of μ L, SSR (10 μm of ol/L) of DNA profiling 1 μ L of L, 10 × PCR Buffer, 5U Taq archaeal dna polymerases 0.1 μ L, 94 DEG C of pre-degeneration 5min；94 DEG C of denaturation 40s, 55 DEG C are moved back Fiery 45s, 72 DEG C of extension 50s, after 32 cycle；72 DEG C re-extend 5min, and amplified production is through non-denaturing polyacrylamide gel electricity Swimming：Gel strength is 8%, and electrophoretic buffer is 1 times of TBE, and 260V constant pressures electrophoresis to indicator (dimethylbenzene blue or green) is close to offset plate During bottom, terminate electrophoresis." 1 " will be denoted as with No. 37 identical banding patterns of parent Lu Mianyan, it will be identical with parent Lu Mianyan 19 Banding pattern is denoted as " 2 ", and the complementary banding pattern of two parents is denoted as " 3 ", finally obtains 477 F2 groups single plants and 445 F4 groups are single Molecular marker gene type of the strain in 24 SSR markers.
(4) test of independence of single SSR marker：
Whether cotton prematurity character determination between SSR molecular marker two parameters of favourable banding pattern, if independently of each other, representing The Early mature apricot selects and the favourable banding pattern frequency height of SSR marker is unrelated, and Early mature apricot is selected to the favourable band of the SSR molecular marker Type frequency is without influence；If not independently of each other, then it represents that the Early mature apricot selects banding pattern frequency height favourable with SSR marker related, Early mature apricot selection has an impact the favourable banding pattern frequency of the SSR molecular marker.Using X2Carry out the null hypothesis of test of independence It is：H0Two parameters are mutual indepedent, HAIt is relative to each other for two parameters.Work as X2＜ X2 0.05,1When, just receive H0, i.e. two parameter phases It is mutually independent；Work as X2≥X2 0.05,1When, just negate H0, receive HA, i.e. two parameter correlations.It is single SSR marker base as shown in table 3 Because of the general type of type test of independence.
Table 3:The general type of single SSR marker genotype test of independence
Corresponding do not make choice according to the SSR molecular marker genotype of prematureness M8003 line group and group SSR molecular marker genotype data calculates the chi-square value in single SSR marker site by the following formula：
Banding pattern is the theoretical single plant number of " 2 " in precocious Single-plant selection group：E11=R1×C1/N；
Banding pattern is not the theoretical single plant number of " 2 " in precocious Single-plant selection group：E12=R1×C2/N；
Banding pattern is the theoretical single plant number of " 2 " in non-selected group：E21=R2×C1/N；
Banding pattern is not the theoretical single plant number of " 2 " in non-selected group：E22=R2×C2/N；
X2=(∣ a11-E11∣-0.5)2/E11+(∣a12-E12∣-0.5)2/E12+(∣a21-E21∣-0.5)2/E21+(∣a22-E22∣- 0.5)2/E22
It is significantly correlated to judge that single SSR marker site chooses whether with Early mature apricot according to chi-square value：
X2 0.05,1=3.84, X2 0.01,1=6.63
Work as X2≥X2 0.05,1When=3.84, P ＜ 0.05 negate H0, receive HA.Show SSR marker precocity beneficial gene type Whether selection and the selection of corresponding Early mature apricot is significantly correlated, i.e., judges cotton prematurity using SSR marker precocity beneficial gene type Correlated traits has remarkable result.
(5) by the present invention identification obtain 3 SSR marker CGR6185s significantly correlated with cotton seedling flower bud phase character, DPL0041 and DPL0354.Chi-square values of the SSR marker DPL0041 in F2 and F4 groups is respectively 18.64 and 11.48 (X2 0.01,1=6.63), chi-square values of the SSR marker CGR6185 in F2 and F4 groups is respectively 15.47 and 13.22 (X2 0.01,1= 6.63), therefore SSR marker DPL0041 and CGR6185 can lead cotton prematurity breeding in low acute pyogenic infection of finger tip；SSR marker DPL0354 exists Chi-square value in F2 and F4 groups is respectively 0.79 and 5.44 (X2 0.05,1=3.84), therefore the mark is suitble to lead cotton in high acute pyogenic infection of finger tip Flower Mature breeding.
Advantage of the present invention also resides in the upland cotton prematureness related molecular marker using linkage analysis and association analysis acquisition Site is there are poor reliability, the defects of instructing cotton prematurity breeding effect poor.The identification method that the present invention innovates is selected according to breeding Group is selected, selects reverse identification molecular labeling site with reference to seedling flower bud phase Early mature apricot, therefore this method is identified to obtain molecular labeling It may be directly applied to Mature breeding practice；In addition the present invention also identification obtain 3 it is significantly correlated with upland cotton prematurity character SSR marker (CGR6185, DPL0041 and DPL0354) provides reliable molecular labeling position for upland cotton precocity molecular breeding Point is supported.
The above is only the better embodiment to the present invention, not makees limit in any form to the present invention System, any simple modification that every technical spirit according to the invention makes embodiment of above, equivalent variations and modification, Belong in the range of technical solution of the present invention.

Claims (2)

1. a kind of identification method in upland cotton precocity molecular breeding correlation SSR marker site, it is characterised in that according to following steps It carries out：
1) No. 37 are ground as female parent with Mid-late ripening cotton variety Shandong cotton, grinds No. 19 with early-maturity cotton kind Shandong cotton as male parent, hybridization 1 Generation, then 1 generation acquisition F2, F2 generation is selfed by single plant harvest；According to seedling flower bud phase character, the precocious strain of selection 1/8 in F2 groups With 1/8 late-maturing strain, plantation F3, selfing acquisition F4, per generation is all mixed receipts miscegenation；
2) first time that precocious related SSR marker site is carried out using F2 groups is identified：F2 kinds are planted, and DNA is extracted using CTAB methods, With reference to the precocial linkage analysis of upland cotton and association analysis result of study, the cotton prematurity correlation SSR identified is selected Primer carries out PCR amplification, then carries out test of independence to single SSR marker, is carrying out SSR molecular marker genotyping When, the parent Lu Mianyan banding patterns of No. 37 are denoted as 1, the parent Lu Mianyan banding patterns of No. 19 are denoted as 2, the complementary banding pattern of two parents It is denoted as 3；Processing is made choice according to seedling flower bud phase character pair F2 groups, 1/6 precocious single plant individual structure is selected in F2 groups F2 is for cotton prematurity M8003 line group；Chi-square analysis is finally carried out, is primarily determined that by the effective land of seedling flower bud phase character determination Ground cotton precocity molecular breeding correlation SSR marker；
3) second of identification in precocious related SSR marker site is carried out using F4 groups：F4 kinds are planted, after every plant of extraction DNA, then profit The genotype of F4 single plants is determined with prematureness correlation SSR primers, processing is made choice according to seedling flower bud phase character pair F4 groups, in F4 The precocious single plant individual structure F4 of selection 1/3 in group for cotton prematurity M8003 line group, then to single SSR marker into Row test of independence finally carries out chi-square analysis, final to determine to be educated by the effective upland cotton precocity molecule of seedling flower bud phase character determination The related SSR marker of kind.
2. according to a kind of identification method in upland cotton precocity molecular breeding correlation SSR marker site, feature described in claim 1 It is：The seedling flower bud phase is SSP from the number of days for being seeded into cotton and blooming.
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