CN103114140B - Zoysia salt tolerance major gene site qLF-2 molecular marking method - Google Patents
Zoysia salt tolerance major gene site qLF-2 molecular marking method Download PDFInfo
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- CN103114140B CN103114140B CN201310036272.2A CN201310036272A CN103114140B CN 103114140 B CN103114140 B CN 103114140B CN 201310036272 A CN201310036272 A CN 201310036272A CN 103114140 B CN103114140 B CN 103114140B
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- 230000015784 hyperosmotic salinity response Effects 0.000 title claims abstract description 37
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 22
- 238000000034 method Methods 0.000 title claims abstract description 16
- 241001520823 Zoysia Species 0.000 title abstract description 16
- 150000003839 salts Chemical class 0.000 claims abstract description 22
- 244000025254 Cannabis sativa Species 0.000 claims description 28
- 238000000926 separation method Methods 0.000 claims description 3
- 238000013467 fragmentation Methods 0.000 claims description 2
- 238000006062 fragmentation reaction Methods 0.000 claims description 2
- 239000003147 molecular marker Substances 0.000 abstract description 3
- 238000012254 genetic linkage analysis Methods 0.000 abstract 1
- 238000009396 hybridization Methods 0.000 abstract 1
- 239000000463 material Substances 0.000 description 10
- 230000002068 genetic effect Effects 0.000 description 5
- 238000004458 analytical method Methods 0.000 description 4
- 239000003550 marker Substances 0.000 description 4
- 241000196324 Embryophyta Species 0.000 description 3
- 238000009395 breeding Methods 0.000 description 3
- 230000001488 breeding effect Effects 0.000 description 3
- 238000013507 mapping Methods 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 241000981595 Zoysia japonica Species 0.000 description 2
- 238000000137 annealing Methods 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 230000004087 circulation Effects 0.000 description 2
- 238000012136 culture method Methods 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000011156 evaluation Methods 0.000 description 2
- 238000002372 labelling Methods 0.000 description 2
- 238000009938 salting Methods 0.000 description 2
- 239000011780 sodium chloride Substances 0.000 description 2
- 239000002689 soil Substances 0.000 description 2
- 239000002699 waste material Substances 0.000 description 2
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 description 1
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 description 1
- 102100023706 Steroid receptor RNA activator 1 Human genes 0.000 description 1
- 101710187693 Steroid receptor RNA activator 1 Proteins 0.000 description 1
- 239000003513 alkali Substances 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 239000013256 coordination polymer Substances 0.000 description 1
- 238000009402 cross-breeding Methods 0.000 description 1
- 238000013016 damping Methods 0.000 description 1
- 238000004925 denaturation Methods 0.000 description 1
- 230000036425 denaturation Effects 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 238000012252 genetic analysis Methods 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 230000008774 maternal effect Effects 0.000 description 1
- 238000001558 permutation test Methods 0.000 description 1
- 230000008635 plant growth Effects 0.000 description 1
- 230000010152 pollination Effects 0.000 description 1
- 229920002401 polyacrylamide Polymers 0.000 description 1
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- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
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Abstract
The invention provides a zoysia salt tolerance major gene site qLF-2 molecular marking method belonging to the field of molecular genetics. The method comprises the following steps: hybridizing zoysia seed sources Z105 and Z061, and performing genetic linkage analysis on the genotype of a F1 family obtained through the hybridization and a leaf withering rate under salt stress, thus obtaining a zoysia salt tolerance major gene site qLF-2 molecular marker. By using the zoysia salt tolerance major gene site molecular marker to detect whether the major gene site is contained in zoysia and filial generations thereof, the salt resistance level can be predicted, thereby greatly saving the cost and improving the salt tolerance selection efficiency of zoysia.
Description
one, technical field
The invention provides jielu grass salt tolerant major gene loci
qLF-2molecule marking method, belong to molecular genetics field, be exclusively used in the utilization of the seed selection of jielu grass New salt-tolerant cultivar and germ plasm resource.
two, background technology
Salting of soil is a global problem, saltings, whole world area nearly 9.6 * 10
8hm
2, there is saline soil 3.47 * 10 in China
7hm
2, be equivalent to 1/3 of cultivated area.Needs along with exploitation and the afforestation in coastal saline-alkali area, the salt tolerance of turfgrass is proposed to more and more higher requirement, Zoysia plant is as a kind of important turfgrass, and the Zoysia new variety of plant of cultivating high-quality salt tolerant is a very important task.
Early-stage Study result shows, compares with other turfgrass, and Zoysia plant has stronger salt tolerance, but salt tolerance between the Different Provenances of not of the same race and same in Zoysia exists larger heritable variation.Therefore will select a kind of jielu grass of high-quality salt tolerant to carry out saline land greening, Salt-Tolerance Identification is a very important job.At present, evaluation about Zoysia plant salt endurance is all undertaken by water culture or earth culture method, although these two kinds of methods are before district examination, the most convictive Salt-Tolerance Identification method, but by above two kinds of methods, carry out Salt-Tolerance Identification and need very large workload, waste time and energy, and be easily subject to the impact of environment.When Selection of Salt-Tolerant material from material in enormous quantities, there is suitable difficulty.Jielu grass salt tolerance breeding process is greatly affected.
Genetic research result show the salt tolerance of jielu grass exist major gene loci (Guo Hailin etc. jielu grass (
zoysia japonica) genetic analysis of salt tolerance. plant genetic resources journal, 2012,13 (5): 733-738.).Therefore utilize molecular biological means, by building the genetic map of Zoysia plant, and to the major gene loci of its salt tolerance (Quantitative Trait Loci, QTL) analyze, find with the molecule marker of salt tolerance main effect QTL compact linkage the salt tolerance of kind is identified, to be cost-saving widely, improve breeding and efficiency of selection.
three, summary of the invention
technical problem:the present invention is directed to above-mentioned situation, a kind of molecule marking method of jielu grass salt tolerant major gene loci is provided, by detecting the chain molecule marker of these salt tolerance major gene locis, can predict that the salt tolerance of jielu grass is strong and weak, improve the efficiency of selection of salt tolerant jielu grass.
technical scheme:
Left end primer sequence 5 '-TGAGTCCAAACCGGTAG-3 ' and right-hand member primer sequence 5 '-GACTGCGTACGAATTGGT-3 ' with labeled primer Me12Em20
DNA to the separation of jielu grass blade increases, if can amplify respectively 220bp DNA fragmentation, indicates jielu grass salt tolerant major gene loci
qLF-2existence, this major gene loci is positioned at the 42.25cM place of the 5th linkage group, utilizing mapQTL5.0 software to record the contribution rate of jielu grass salt tolerance is 29.7%.
beneficial effect:
The molecule marking method of jielu grass salt tolerant major gene loci provided by the present invention, its beneficial effect is:
Two major gene loci jielu grass salt tolerant major gene locis of jielu grass salt tolerance are disclosed in the world first by the present invention
qLF-2, this major gene loci is positioned at the 42.25cM place of the 5th linkage group, and utilizing mapQTL5.0 software to record the contribution rate of jielu grass salt tolerance is 29.7%.。By the molecule marker Me12Em20-220 being associated with salt tolerant major gene loci, the salt tolerance of jielu grass is identified, can overcome water culture and earth culture method is easily affected by environment, the shortcomings such as workload is large, while particularly in enormous quantities kind of source material or filial generation being carried out to Salt-Tolerance Identification, will show obvious advantage, greatly reduce the waste of manpower and materials, improve breeding and efficiency of selection.
accompanying drawing explanation:
Fig. 1: jielu grass salt tolerance QTL linkage group positioning analysis schematic diagram, wherein: the chain section that comprises QTL is shown, and left side is the distance (cM) between mark, and right side is mark title.
four, embodiment
(1) structure and the Salt-Tolerance Identification of jielu grass F1 salt tolerant segregating population
To jielu grass salt-enduring species source material Z105(
z. japonica, salt tolerant coefficient is 0.72) and quick salt kind source material Z061 (
z. japonicasalt tolerant coefficient is 0.14) (be public kind, document sees reference: Li Ya, Geng Lei, Liu Jianxiu. Assessment on Salt-tolerance of Zoysia spp. in China. meadow journal, 2004,12 (1): 8-11,16.) by controlled pollination method, be cross-breeding, obtain F1 segregating population, comprise 120 F1 individualities.
By water culture (reference: Chen Jingbo, Yan Jun, Jiang Yanqin, Guo Hailin, Zhang Tingting, Chen Xuan, Liu Jianxiu. the preliminary assessment of warm season turf good choosing system and kind salt resistance. Acta Prataculture, 2009,18 (5): 107-114.) two parents and F1 colony thereof are carried out to Salt-Tolerance Identification, experiment is carried out at glasshouse, the method that specifically adopts salinity to increase gradually, with final salt concn, reach after 20g/L maintains 1 month and add up the withered and yellow rate of the blade of differing materials under salt stress, carry out Evaluation of Salt Tolerance.
Salt-Tolerance Identification result shows in salt treating processes, the processing of all material and synopsis reveal obvious difference, under salt is processed, Zoysia plant growth rate declines, there is withered and yellow phenomenon in blade, but the salt tolerance of differing materials also shows obvious difference (table 1), the statistics of the withered and yellow rate of blade shows, F
1the withered and yellow rate range of variation of blade of colony is 10% ~ 98.33%, average out to 50.39%, and the variation coefficient is 40.50%, and the withered and yellow rate of blade of maternal Z105 is 35%, and the withered and yellow rate of blade of male parent Z061 is 95%.
Table 1 filial generation Salt-Tolerance Identification result
| Offspring's numbering | The withered and yellow rate of blade (%) | Offspring's numbering | The withered and yellow rate of blade (%) | Offspring's numbering | The withered and yellow rate of blade (%) | Offspring's numbering | The withered and yellow rate of blade (%) |
| 129 | 10.00 | 67 | 33.33 | 47 | 50.00 | 121 | 66.67 |
| 89 | 11.67 | 161 | 33.33 | 58 | 50.00 | 132 | 66.67 |
| 74 | 15.00 | 32 | 35.00 | 59 | 50.00 | 163 | 66.67 |
| 73 | 16.67 | 37 | 35.00 | 116 | 50.00 | 84 | 68.33 |
| 153 | 16.67 | 90 | 36.67 | 93 | 51.67 | 63 | 70.00 |
| 19 | 17.50 | 100 | 36.67 | 97 | 51.67 | 105 | 70.00 |
| 130 | 18.33 | 115 | 36.67 | 65 | 53.33 | 111 | 70.00 |
| 71 | 20.00 | 81 | 38.33 | 15 | 55.00 | 123 | 70.00 |
| 135 | 20.00 | 35 | 40.00 | 79 | 55.00 | 8 | 71.67 |
| 34 | 21.67 | 104 | 40.00 | 149 | 55.00 | 80 | 71.67 |
| 76 | 23.33 | 109 | 40.00 | 43 | 56.67 | 88 | 71.67 |
| 120 | 23.33 | 112 | 40.00 | 61 | 56.67 | 138 | 71.67 |
| 125 | 23.33 | 141 | 40.00 | 70 | 56.67 | 142 | 72.50 |
| 45 | 25.00 | 150 | 40.00 | 110 | 56.67 | 82 | 73.33 |
| 86 | 25.00 | 7 | 43.33 | 3 | 57.50 | 137 | 73.33 |
| 143 | 25.00 | 33 | 43.33 | 87 | 60.00 | 44 | 75.00 |
| 10 | 26.67 | 57 | 43.33 | 103 | 60.00 | 6 | 76.67 |
| 75 | 26.67 | 68 | 43.33 | 119 | 60.00 | 56 | 76.67 |
| 106 | 26.67 | 118 | 43.33 | 31 | 61.67 | 156 | 77.50 |
| 96 | 28.33 | 133 | 43.33 | 134 | 61.67 | 145 | 78.33 |
| 122 | 28.33 | 155 | 46.67 | 159 | 61.67 | 36 | 80.00 |
| 4 | 30.00 | 158 | 46.67 | 162 | 61.67 | 154 | 80.00 |
| 25 | 30.00 | 85 | 48.33 | 29 | 63.33 | 83 | 81.67 |
| 72 | 30.00 | 131 | 48.33 | 38 | 63.33 | 22 | 83.33 |
| 98 | 30.00 | 136 | 48.33 | 50 | 63.33 | 11 | 85.00 |
| 124 | 30.00 | 140 | 48.33 | 53 | 63.33 | 113 | 85.00 |
| 127 | 32.50 | 1 | 50.00 | 62 | 63.33 | 17 | 86.67 |
| 128 | 32.50 | 13 | 50.00 | 126 | 63.33 | 114 | 88.33 |
| 51 | 33.33 | 24 | 50.00 | 2 | 65.00 | 46 | 95.00 |
| 60 | 33.33 | 41 | 50.00 | 146 | 65.00 | 23 | 98.33 |
(2) molecular marker analysis of F1 colony
(1) with New-type wide-spectrum plant genome DNA rapid extraction test kit (HF213), extract parent and each individual DNA of F1 colony thereof.
(2) first with the DNA of 400 couples of SRAP primer pair jielu grass two parent material Z105 and Z061, carry out polymorphism analysis, have 276 pairs of primers to show as polymorphism between two parents.SRAP-PCR adopts the reaction system of 10 μ L, comprising: 50ng template DNA, 10 * PCR damping fluid, Mg
2+1.5mmolL
-1, dNTPs 200 μ molL
-1, primer 0.2 μ molL
-1, Taq archaeal dna polymerase 0.5 U.SRAP-PCR amplification program is: 94 ℃ of denaturation 4min; 94 ℃ of sex change 1min, 37 ℃ of annealing 1min, 72 ℃ are extended 10sec, 5 circulations; 94 ℃ of sex change 1min, 50 ℃ of annealing 1min, 72 ℃ are extended 10sec, 35 circulations; After loop ends, 72 ℃ are extended 7min, 4 ℃ of preservations.Amplified reaction carries out on the TC-412 type PCR of Britain TECHNE company instrument.Amplified production carries out electrophoresis by 10% polyacrylamide gel and detects, and takes pictures on film illuminator, records result.Between parent, have in the primer F1 colony of polymorphism and analyze, obtain colony's genotype data.
(3) according to the labeling pattern of CP colony, mark to obtained F1 colony is added up, on the basis of labeling pattern statistics, application joinmap3.0 software, adopts Kosambi mapping function, carry out the structure of Zoysia plant F1 population genetic collection of illustrative plates, result is when LOD value equals 3.5, and 242 marks form 24 linkage groups, comprising 43 inclined to one side separation markings, 82 marks fail to locate in linkage group, and mapping site rate is 74.23%.In 24 linkage groups, the reference numerals of single linkage group is 2-48,10.08 of average out to; Collection of illustrative plates total length is 1210.99cM, and between mark, maximum map distance is 38.765cM, and minimal graph is apart from being 0cM, and mean chart is apart from being 5.00cM, and each linkage group length is between 3.34-205.17cM, and the shortest and the longest linkage group is respectively LG22 and LG3.
(4) molecular genetic linkage map based on having built, the Interval Mapping of application MAPQTL5.0 carries out linkage analysis to colony's genotype data of each molecule marker and each F1 strain corresponding to it in the withered and yellow rate of the blade under salt stress.Using the LOD value of linkage group 5% conspicuous level as the threshold value (every proterties is determined LOD threshold value through 1000 permutation tests) that detects QTL.When each LOD value is greater than threshold value, just think that this region exists a QTL, its position is in the peak value corresponding points of LOD value.The interval of the downward 1 GeLODZhi unit of the peak value of usining is as the fiducial interval of this QTL, and the QTL detecting navigates in linkage group by MAPChart software.Calculation result has been located 2 QTL(accompanying drawings 1 of the withered and yellow rate of blade), called after:
qLF-1with
qLF-2,
qLF-1be positioned at the 36.28cM place of the 4th linkage group, between mark Me5Em1-315 and Me12Em1-180, LOD value is 3.27, and contribution rate is 13.1%, with the map distance of mark Me5Em1-315 be 1.0 cM, with the map distance of mark Me12Em1-180 be 0.142cM;
qLF-2be positioned at the 42.25cM place of the 5th linkage group, LOD value is 2.88, and contribution rate is 29.7%, with mark Me12Em20-220 in linkage group in same position.The position of 2 QTL that detect, contiguous mark and effect are in Table 2 and accompanying drawing 1, and flag sequence is in Table 3.
The QTL location of table 2 jielu grass salt tolerance (the withered and yellow rate of blade and branches and leaves are pruned dry weight and reduced percentage)
The sequence of table 3 labeled primer and amplified fragments size
| Mark | Left end primer sequence | Right-hand member primer sequence | Clip size (bp) |
| Me12Em1 | 5’-TGAGTCCAAACCGGTAG-3’ | 5’-GACTGCGTACGAATTCAA-3’ | 180 |
| Me5Em1 | 5’-TGAGTCCAAACCGGTGC-3’ | 5’-GACTGCGTACGAATTCAA-3’ | 315 |
| Me12Em20 | 5’-TGAGTCCAAACCGGTAG-3’ | 5’-GACTGCGTACGAATTGGT-3’ | 220 |
SEQUENCE LISTING
<110> Institute of Botany
The molecule marking method of <120> jielu grass salt tolerance major gene loci qLF-2
<130> 0
<160> 2
<170> PatentIn version 3.1
<210> 1
<211> 17
<212> DNA
<213> is artificial
<220>
The left end primer sequence of <221> Me12Em20
<222> (1)..(17)
<223>
<400> 1
tgagtccaaa ccggtag 17
<210> 2
<211> 18
<212> DNA
<213> is artificial
<220>
The right-hand member primer sequence of <221> Me12Em20
<222> (1)..(18)
<223>
<400> 2
gactgcgtac gaattggt 18
Claims (1)
1.
detect jielu grass salt tolerance major gene loci qLF-2 molecule marking method, it is characterized in that:
Left end primer sequence 5 '-TGAGTCCAAACCGGTAG-3 ' and right-hand member primer sequence 5 '-GACTGCGTACGAATTGGT-3 ' with labeled primer Me12Em20
DNA to the separation of jielu grass blade increases, if can amplify respectively 220bp DNA fragmentation, indicates jielu grass salt tolerant major gene loci
qLF-2existence, this major gene loci is positioned at the 42.25cM place of the 5th linkage group, utilizing mapQTL5.0 software to record the contribution rate of jielu grass salt tolerance is 29.7%.
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| CN103602742B (en) * | 2013-11-21 | 2015-07-15 | 江苏省中国科学院植物研究所 | Molecular marking method for identifying variety of Suzhi No.1 hybrid zoysia |
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