CN112877468B - CMS molecular tag based on kenaf mitochondrial gene non-coding region, primer pair and application thereof - Google Patents

CMS molecular tag based on kenaf mitochondrial gene non-coding region, primer pair and application thereof Download PDF

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CN112877468B
CN112877468B CN202110429317.7A CN202110429317A CN112877468B CN 112877468 B CN112877468 B CN 112877468B CN 202110429317 A CN202110429317 A CN 202110429317A CN 112877468 B CN112877468 B CN 112877468B
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廖小芳
侯文焕
唐兴富
赵艳红
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Guangxi Zhuang Nationality Autonomous Region Academy of Agricultural Sciences
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Abstract

The invention belongs to the technical field of biology, relates to kenaf breeding, and particularly relates to a CMS molecular tag based on a kenaf mitochondrial gene non-coding region, a primer pair and application thereof. The CMS molecular signature is based on mitochondrial genescobThe upstream non-coding region 535bp and 995 bp have deletions of 1bp and 101 bp respectively and are named as HMBQ 102. The invention develops a CMS molecular tag based on the noncoding region of kenaf mitochondrial gene, a primer pair and application thereof based on the difference of kenaf cytoplasmic male sterile line and maintainer line mitochondrial genome, the tag has simple operation, can quickly and accurately identify kenaf male sterile cytoplasm, and has important effects on the breeding of kenaf cytoplasmic male sterile line and heterosis utilizationThe application value is required.

Description

CMS molecular tag based on kenaf mitochondrial gene non-coding region, primer pair and application thereof
Technical Field
The invention belongs to the technical field of biology, relates to kenaf breeding, and particularly relates to a CMS molecular tag based on a kenaf mitochondrial gene non-coding region, a primer pair and application thereof.
Background
Cytoplasmic Male Sterility (CMS) is one of the major means of hybrid breeding of kenaf. However, the single cytoplasmic type of kenaf is easy to cause the potential risk of large-area occurrence of physiological races of a certain disease, and on the other hand, the dominance and resistance expression of hybrids have obvious cytoplasmic effect, so creating multiple sterile cytoplasmic germplasms is a key problem to realize continuous utilization of kenaf hybrid dominance and needs to be solved urgently. Molecular marker assisted breeding is an important means for accelerating the breeding of cytoplasmic male sterile line of kenaf.
An innovation team of Zhou Reyang professor of Guangxi university discovers 1 male sterile plant in winter-bred kenaf wild species UG93 in Hainan in 3 months in 2001, takes the male sterile plant as a cytoplasm donor, has bred 7 kenaf CMS lines, combines 4 red excellent line series three-line hybrid species, and the utilization of hybrid vigor of kenaf in China is at the international leading level. Research shows that most fertile strains in the kenaf UG93 population can maintain the CMS characteristics of the UG93 male sterile strain, and UG93A and a fertile strain UG93B (the fertile strain with the CMS characteristics maintained in UG 93) are bred by taking the fertile strain as a nucleus donor. UG93A is presumed to be a CMS mutant of UG93B and UG93B is its wild type, since both are derived from the same cultivar population. Because the cytoplasmic near isogenic line is adopted, the method provides a valuable research material for researching the molecular mechanism of the kenaf CMS. The inventor continuously researches the mitochondrial genomes of kenaf cytoplasmic male sterile line UG93A and maintenance line UG93B for the first timecobThe sequence difference between the sterile line and the maintainer line is found in the non-coding region at the upstream of the gene.
Disclosure of Invention
The invention develops the CMS molecular tag based on the non-coding region of the kenaf mitochondrial gene and the primer pair and the application thereof based on the difference of kenaf cytoplasmic male sterile line and maintainer line mitochondrial genome.
The technical scheme of the invention is realized as follows:
CMS molecular signature based on non-coding region of kenaf mitochondrial gene, wherein the CMS molecular signature is based on mitochondrial genecobThe upstream non-coding region 535bp and 995 bp have deletions of 1bp and 101 bp respectively and are named as HMBQ 102.
The primer pair is HMBQ102F with the sequence of 5'aggggtcatcctgggttactg3' and HMBQ102R with the sequence of 5'tcgtcgcttacatggattaccac 3'.
The CMS molecular tag is applied to the breeding of cytoplasmic male sterile line of kenaf.
According to the mitochondrial gene of the sample to be testedcobWhether deletion of 1bp and 101 bp exists at 535bp and 995 bp positions of the upstream non-coding region respectively is judged, and whether the cytoplasm is kenaf male sterile cytoplasm is judged.
If the mitochondrial gene of the sample to be testedcobThe upstream non-coding region 535bp and 995 bp respectively have deletion of 1bp and 101 bp, and are kenaf male sterile cytoplasm; if the mitochondrial gene of the sample to be testedcobThe upstream non-coding region is 535bp and 995 bp without deletion of 1bp and 101 bp, and is kenaf fertile cytoplasm.
The CMS molecular tag primer pair is applied to breeding of cytoplasmic male sterile lines of kenaf.
The size of the band amplified by the CMS molecular tag primer in the kenaf cytoplasmic male sterile line is 980bp, the size of the band amplified in the kenaf maintainer line is 1082bp, and the kenaf cytoplasmic male sterile line is bred through the difference of the amplified bands.
The invention has the following beneficial effects:
1. plant mitochondrial genomes are complex and diverse, ranging in genome size from 208 kb for oilseed rape (Kubo et al, 2000) to 11.3 Mb for turfgrass (Sloan et al, 2012), but the coding region sequences of mitochondrial genes are well conserved, while the non-coding region sequences are complex and diverse (Liao et al, 2018), and thus the mitochondriaRearrangement of non-coding sequences in the mitochondrial genome is the main cause of complex and variable mitochondrial genomes in plants (Alverson et al, 2011). In the present invention, mitochondrial genes are present in cytoplasmic male sterile line UG93A of kenafcobThe upstream non-coding regions 535bp and 995 bp respectively have deletions of 1bp and 101 bp, while the maintainer line UG93B does not have the sequence deletion characteristic, and PCR detection is further carried out on sterile lines and maintainer lines of different types, so that the molecular characteristic can be stably inherited in sterile lines and maintainer lines of different types of kenaf, and the molecular tag is proved to have the characteristic of stably identifying male sterile cytoplasm and fertile cytoplasm of kenaf. And the characteristic can not be changed along with the structural change of the mitochondrial genomes of different cytoplasmic types of the kenaf, so that the molecular tag is further explained to be closely related to the male sterility of the kenaf. The invention has important guiding significance for breeding of different cytoplasmic types of male sterile lines of the kenaf and utilization of heterosis.
2. The existing kenaf CMS molecular tags are developed based on the difference of kenaf mitochondrial gene coding regions and 3' flanking sequences thereof in kenaf sterile lines and maintainer lines, and the report of the molecular tags related to kenaf mitochondrial genome non-coding regions is not found; and since the sequences of plant mitochondrial gene regions are well conserved during evolution, the differences in these gene regions are preserved as the mitochondria evolve. The invention is based on mitochondrial gene in kenaf cytoplasmic male sterile line UG93AcobThe upstream non-coding region 535bp and 995 bp respectively have deletion of 1bp and 101 bp, while the maintainer line UG93B does not have the sequence deletion characteristic, and a molecular tag HMBQ102 for identifying kenaf male sterile cytoplasm is developed based on the sequence deletion characteristic. The main advantages of the present invention are, firstly: the invention reports the gene based on the kenaf for the first timecobThe upstream non-coding molecular tag HMBQ102 is developed by maintaining sequence difference between the kenaf sterile line and the kenaf male sterile cytoplasm and the fertile cytoplasm and is used for specifically identifying the kenaf male sterile cytoplasm and the fertile cytoplasm; secondly, the method comprises the following steps: in the practical process of kenaf field breeding, individual fertile mutant individuals (such as UG93AR1 and UG93AR 2) often appear in sterile line population, while sterile mutant individuals (such as UG93AR 2) appear in maintainer line populationThe materials used UG93BS1 and UG93BS 2), how the cytoplasm of such mutant individuals changes, and molecular marker detection developed based on differences in the mitochondrial genomes of the sterile line and the maintainer line, made this problem open. The invention detects four mutant individuals UG93AR1, UG93AR2, UG93BS1 and UG93BS2 in sterile line and maintainer line populations respectively based on kenaf molecular tag HMBQ102, and the result shows that UG93AR1 and UG93AR2 have sterile cytoplasm characteristics (figure 3a, lanes 11 and 12), which shows that the nuclear genomes of the two individuals contain fertility restorer genes, and UG93BS1 and UG93BS2 also have sterile cytoplasm characteristics (figure 3a, lanes 5 and 6), which shows that the cytoplasmic mitochondrial genes are recombined. UG93A, UG93AR, UG93B and UG93BS are all derived from the same population, are near isogenic lines of cytoplasmic nuclear nucleus level, and provide ideal materials for researching kenaf male sterile nuclear-cytoplasmic interaction. The application of the HMBQ102 molecular tag provides a molecular basis for cytoplasmic identification of the kenaf AR and BS mutant strains. Thirdly, the method comprises the following steps: the non-coding region is a recombination active region of a plant mitochondrial genome, the molecular tag can be stably inherited in different types of kenaf sterile lines and maintainer lines, and cannot be changed along with the structural change of different cytoplasmic types of the mitochondrial genomes of kenaf, so that the molecular tag is closely related to kenaf male sterility.
3. The main improvements of the application are as follows: (1) the operation is simple and convenient: the method can identify the kenaf male sterile cytoplasm fertile cytoplasm through simple PCR reaction, and is simpler and easier to operate compared with a method that some molecular labels can identify the sterile cytoplasm and the fertile cytoplasm through a gel method after enzyme digestion; in the preparation process of the PCR reaction system, the invention adopts 2 xTaq Mix reagent (containing PCR buffer, Mg) 2+ Enzyme kit loading buffer and other reagents) to replace the traditional PCR buffer and Mg 2+ Adding the reagent such as enzyme into the mixed reaction system independently, thereby saving Mg 2+ The optimization step and the loading buffer and other steps must be added in the gel electrophoresis loading process, so that the operation is simpler and more convenient. (2) The process is simplified: the method identifies the male sterile cytoplasm and the fertile cytoplasm of the kenaf in an agarose gel electrophoresis mode, and the result is clearClearly, the identification result is stable and reliable, and the operation process is more simplified and convenient compared with other molecular identification methods using SDS-PAGE gel electrophoresis; (3) the results show that: based on the difference that the size of the PCR product of the HMBQ102 molecular label in the sterile cytoplasm and the fertile cytoplasm of the kenaf is only 102 bp, the invention specifically uses the 100bp DNA ladder Marker as an indicating band, can clearly and intuitively indicate the size of a difference strip, and compared with the 2000 bp DNA Marker used for other molecular labels, the electrophoresis result is more intuitive and clear.
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In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the drawings without creative efforts.
FIG. 1 shows the alignment of differences between the mitochondrial genome sequences of kenaf UG93A and UG 93B. Note: the red box indicates the primer sequence of kenaf molecular marker HMBQ 102.
FIG. 2 shows the identification of different cytoplasmic types by the molecular tag of kenaf HMBQ 101. Note: m,100bp DNA ladder, 1, F3A, 2, P3A, 3, P7A, 4, P8A, 5, 722A, 6, L23A, 7, P4A, 8, 917A, 9, Furanou 13A, 10, UG93A, 11, UG93B, 12, F3B, 13, P3B, 14, P7B, 15, P8B, 16, 722B, 17, L23B, 18, 917B, 19, P4B.
FIG. 3 shows the screening of cytoplasmic male sterile germplasm resources of kenaf by utilizing kenaf HMBQ101 molecular tags. (a) M,100bp DNA ladder, 1, H4, 2, H6, 3, H7, 4, H12, 5, UG93BS1, 6, UG93BS2, 7, S1, 8, S2, 9, S3, 10, S4, 11, UG93AR1, 12, UG93AR2, 13, UG93A, 14, UG93B, 15, H1, 16, H2, 17, H3, 18, H5, 19, H8, and B) M,100bp DNA ladder, 1, L23A/PA258, 2, B4/B19, 3, KN142, 4, KN250, 5, UG93A, 6, UG 64793 5, 7, F302, PA258, 9, PA299, 10B, 299, and 19. Wherein H1-H8 and H12 are individual plants of kenaf field germplasm resources.
Detailed Description
The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments of the present invention, and it should be understood that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be obtained by a person skilled in the art based on the embodiments of the present invention without inventive step, are within the scope of the present invention.
The sources of kenaf materials in this application are:
(1) kenaf cytoplasmic male sterile line (with kenaf male sterile cytoplasm)
UG 93A: the male sterile plant in the kenaf wild species UG93 is obtained by saturation backcross breeding by taking a fertile plant line in a UG93 population as a nucleus donor. Green stem, lobe, yellowish crown.
F3A: the Guangxi discriminator [2007] No. 177, cytoplasmic male sterile line bred by Zhou Rui Yang teacher of Guangxi university, is bred by saturation backcross with UG93 as cytoplasmic donor and F3 as nuclear donor. Green stem, lobe, yellowish crown.
P3A: also Yuefeng No. 1A, see patent ZL 200510019454.4 (publication No. CN 100415083C).
P7A: the cytoplasmic male sterile line bred by Zhouyiyang teacher of Guangxi university, Guideng (Ma) 2015027, is bred by saturation backcross by using male sterile plants found in UG93, ZB90, KN142 and KN250 as cytoplasmic donors and P7 as nuclear donors. Green stem, lobe, yellowish crown.
P8A: guideng (Ma) 2015028, a cytoplasmic male sterile line bred by Zhou Rui Yang teacher of Guangxi university, is bred by saturation backcross with male sterile strains S found in UG93, ZB90, KN142 and KN250 as cytoplasmic donors and P8 as nuclear donors, and has green stems and cracked leaves.
722A: the cytoplasmic male sterile line bred by Guangxi university Zhou Rui Yang teacher, Gui Ke's No. 2004 and 152, is bred by saturation backcross with UG93 as cytoplasmic donor and 722 as nuclear donor, and has complete leaves and green stem.
L23A: guike, No. [2007]177, cytoplasmic male sterile line bred by Zhou Rui Yang teacher of Guangxi university.
P4A: guideng (Ma) 2015033 is prepared from the wild-abortive cytoplasmic male sterile line P3A of kenaf, which is bred at Guangxi university, as a cytoplasmic donor, and P4B as a nuclear donor through saturation backcross breeding, and the leaves and stems are light red.
917A: the cytoplasmic male sterile line bred by Guike's identifier [2004] No. 152, Zhou Rui Yang teacher of Guangxi university, etc. is bred by saturation backcross with UG93 as cytoplasmic donor and 917 as nuclear donor, and has light red stem and cracked leaf.
FYH 13A: introduced by Fujian agriculture and forestry university, takes 722B as a nucleus donor, and is formed by saturated backcross breeding, and the cracked leaves and the stalks are light red.
(2) Kenaf cytoplasmic male sterile line (with kenaf fertile cytoplasm)
UG 93B: introduced from the research institute of hemp of Chinese academy of agricultural sciences, can maintain the male sterility characteristics of UG93A, and is a recurrent parent (nucleus donor parent) of UG93A and a corresponding maintainer line of UG 93A.
F3B: fuhong No. 3 was introduced by Fujian university of agriculture and forestry as a recurrent parent (nuclear donor parent) of F3A, and was a corresponding maintainer line of F3A.
P3B: also named Yuefeng No. 1B, the male sterility maintainer line of the cytoplasmic male sterile line P3A bred by Zhouyiyang teacher of Guangxi university, is disclosed in patent ZL 200510019454.4 (publication No. CN 100415083C).
P7B: the hybrid is bred by F6 generation through excellent single strain/NA 126 hybrid selected from K03B, is a recurrent parent (nucleus donor parent) of P7A, and is a corresponding maintainer line of P7A. Wherein K03B is a maintainer line of 'Jinguang non-thorny' (introduced by Fujian agriculture and forestry university) breeding, and NA126 (native Nigeria, introduced from the hemp research institute of Chinese academy of agricultural sciences).
P8B: the excellent single strain selected from K03B/NA 126 hybrid F6 generation bred green stem and leaf, and the recurrent parent (nucleus donor parent) of P8A are corresponding maintainer line of P8A.
722B: also named 722, kenaf variety bred by the institute of hemp research of the Chinese academy of agricultural sciences is a recurrent parent (nucleus donor parent) of 722A, and a corresponding maintainer line of 722A.
L23B: the recurrent parent (nuclear donor parent) of L23A, the corresponding maintainer line of L23A.
917B: 917, kenaf variety bred by the institute for hemp, academy of agricultural sciences, china, is the recurrent parent of 917A (the nucleus donor parent), the corresponding maintainer line of 917A.
P4B: the K03A/Cuba 6F 7 generation strain, which is the recurrent parent (nucleus donor parent) of P4A, the corresponding maintainer line of P4A.
L23A/PA 258: and (3) carrying out hybridization by taking L23A as a female parent and PA258 as a male parent to obtain a progeny.
B4/B19: and B4 is taken as a female parent, and B19 is taken as a male parent to obtain offspring through hybridization.
KN 142: native kenya was introduced from the institute for hemp, national academy of agricultural sciences.
KN 250: native kenya was introduced from the institute for hemp, national academy of agricultural sciences.
F302: the general kenaf variety is described in "discovery of cytoplasmic SNP molecular tag of CMS (cytoplasmic SNP) of kenaf Patrinia (CMS) (King Kong, Zhou Rui Yang, on-line scientific and technological paper of China).
PA 258: common kenaf varieties are native to pakistan, which is introduced from the institute of pockmarks of the Chinese academy of agricultural sciences.
PA 299: the general kenaf variety is described in "discovery of cytoplasmic SNP molecular tag of CMS (cytoplasmic SNP) of kenaf Patrinia (CMS) (King Kong, Zhou Rui Yang, on-line scientific and technological paper of China).
B19: the general kenaf variety is described in "discovery of cytoplasmic SNP molecular tag of CMS (cytoplasmic SNP) of kenaf Patrinia (CMS) (King Kong, Zhou Rui Yang, on-line scientific and technological paper of China).
(3) Sources of germ plasm resource materials in kenaf field
H1: the K03A/Cuba 6 selfing progeny selected line is full-leaf, yellow flower, and fertile in field.
H2: the K03A/Cuba 6 selfing progeny selected line is full-leaf, purple-flower and fertile in field.
H3: FHY 13/722B/compound filial generation of the round leaf of the sweet polder, and the field shows fertility.
H4: the filial generation of the P4A/polder round leaf is fertile in field.
H5: round leaves of the sweet polder, a common kenaf variety, are introduced from the town of the sweet polder in Nanning, Guangxi Zhuang nationality, and conventional varieties are planted locally.
H7: the yellow flower and cracked leaf strains in the P4B are sterile in field.
H6: the dehiscent leaves and yellow flowers of the P4A/P4B filial generation are bred, and the field shows sterility.
H8: also known as Taihong 763. The common kenaf variety is originated from the research institute of hemp of Chinese academy of agricultural sciences and native to Thailand.
H12: the P4A/P4B filial generation has dehiscent leaf and purple flower line and is sterile in field.
UG93BS 1: sterile individual 1 in kenaf maintainer line UG 93B.
UG93BS 2: sterile individual 2 in kenaf maintainer line UG 93B.
S1: the hybrid of kenaf UG93A/UG93R and the sterile single plant 1 in F1 is disclosed in the invention patent ZL 201310710995.6 (publication No. CN 103740817B) wherein UG93R is derived from a restorer line bred by a kenaf wild species UG93 group.
S2: the hybrid of kenaf UG93A/UG93R and sterile single plant 2 in F1 is disclosed in the invention patent ZL 201310710995.6 (publication number CN 103740817B), wherein UG93R is derived from a restorer line bred by a kenaf wild species UG93 population.
S3: the hybrid F1 of kenaf UG93A/UG93R is a sterile single plant 3, wherein UG93R is derived from a restorer line bred by kenaf wild species UG93 in a group, and the invention is disclosed in patent ZL 201310710995.6 (publication No. CN 103740817B).
S4: the hybrid of kenaf UG93A/UG93R and the sterile single plant 4 in F1, wherein UG93R is derived from a restorer line bred by kenaf wild species UG93 in a group, and is disclosed in the invention patent ZL 201310710995.6 (publication No. CN 103740817B).
UG93AR 1: fertile individual plant 1 in kenaf sterile line UG 93A.
UG93AR 2: fertile individual plant 2 in kenaf sterile line UG 93A.
The CMS molecular tag is based on a non-coding region of a kenaf mitochondrial gene, is named as HMBQ102 on the basis that 535bp and 995 bp of the upstream non-coding region of a mitochondrial gene cob have deletions of 1bp and 101 bp respectively.
The primer pair is HMBQ102F with a sequence of 5'aggggtcatcctgggttactg3' and HMBQ102R with a sequence of 5'tcgtcgcttacatggattaccac 3'.
The CMS molecular tag is applied to breeding of cytoplasmic male sterile line of kenaf.
And identifying whether the cytoplasmic male sterile line of the kenaf exists according to whether the 535bp and 995 bp non-coding regions of the mitochondrial cob upstream of the sample to be detected have deletions of 1bp and 101 bp respectively.
If the 535bp and 995 bp of the mitochondrial cob upstream non-coding region of the sample to be detected have deletions of 1bp and 101 bp respectively, the cytoplasmic male sterile cytoplasm is determined; if the 535bp and 995 bp of the mitochondrial cob upstream non-coding region of the sample to be detected do not have deletion of 1bp and 101 bp, the sample is kenaf fertile cytoplasm.
The CMS molecular tag primer pair is applied to breeding of cytoplasmic male sterile lines of kenaf.
The size of the band amplified by the CMS molecular tag primer in the kenaf cytoplasmic male sterile line is 980bp, the size of the band amplified in the kenaf maintainer line is 1082bp, and the kenaf cytoplasmic male sterile line is bred through the difference of the amplified bands.
Example 1
Extraction of total DNA of kenaf
The suction head, the mortar and the extraction buffer solution used in the extraction process of the general genome DNA of the kenaf are firstly autoclaved for 20 min, and are taken out for standby application, and the DNA is extracted according to the following steps:
(1) preheating CTAB (0.1 mol/L Tris-HCl pH8.0, 0.025 mol/L EDTA-Na2 pH8.0, 1.4 mol/L NaCl, 2% CTAB (w/v)) extract in a 65 ℃ water bath;
(2) grinding 2-3 g of fresh or-80 ℃ frozen leaves in liquid nitrogen;
(3) adding CTAB extractive solution rapidly, mixing, pouring into centrifuge tube, and water bath at 65 deg.C for 30 min. Continuously and lightly shaking the mixture;
(4) adding chloroform/isoamyl alcohol with the same volume, uniformly mixing, centrifuging at 12000 rpm for 10 min at normal temperature;
(5) taking the supernatant, and mixing the extract: 5M NaCl: absolute ethanol = 1: 1: 1, adding 5M NaCl and absolute ethyl alcohol to prepare a mixed solution, and reversing and uniformly mixing;
(5) sucking the supernatant, and transferring the mixed solution to a DNA purification column;
(6) 12000 rpm, centrifuging for 30s at normal temperature, discarding the liquid in the collecting tube, and repeating the steps until the mixed solution is completely transferred;
(7) adding 700 μ L75% ethanol, centrifuging at 12000 rpm at normal temperature for 30s, discarding the liquid in the collection tube, and repeating the step for 1 time;
(8) 12000 rpm, empty column for 2 min;
(9) placing the column into a new 1.5 ml EP tube, adding 50 μ L ddH20 water into the center of the column, and standing for 2 min;
(10) centrifuging at 12000 rpm for 1min, discarding the centrifugal column, and collecting the liquid as purified DNA;
(11) DNA purity and integrity were checked by 1% agarose gel electrophoresis and stored at-20 ℃ until use.
Molecular label primer for cytoplasmic male sterility of kenaf line and PCR detection
Based on the fact that the mitochondrial genome of the kenaf cytoplasmic male sterile line UG93A has deletions of 1bp and 101 bp respectively at 535bp and 995 bp of the mitochondrial gene cob upstream compared with the maintenance line UG93B (figure 1), a molecular tag for specifically marking kenaf cytoplasmic sterility is developed, and is named that the amplification product size of HMBQ102 in the maintenance line UG93B is 1082bp, and the amplification product size in the sterile line UG93A is 980 bp. The sequence information of the molecular tag primers is as follows:
HMBQ102F: aggggtcatcctgggttactg；
HMBQ102R: tcgtcgcttacatggattaccac。
amplification and gel electrophoresis detection
The molecular marker primer is used for PCR detection, wherein a 20 mul reaction system comprises 50ng kenaf DNA template, 10 mul of 2 xTaq Mix (Nanjing Novoweb), 0.6 mul upstream and downstream primers, and ddH is used 2 O make up to 20. mu.L. The reactionThe amplification procedure for the mixture was as follows: pre-changing at 94 ℃ for 3min, pre-changing at 94 ℃ for 30s, pre-changing at 56 ℃ for 20s, pre-changing at 72 ℃ for 20s, amplifying at 35 cycles, finally extending at 72 ℃ for 3min, detecting PCR reaction products by using 1.5% agarose gel electrophoresis, and loading 5 mu L.
Example 2
Application of HMBQ102 marker in breeding of cytoplasmic male sterile line of kenaf
The molecular marker is used for detecting the existing known kenaf cytoplasmic male sterile line and maintainer line materials in the subject group, and the result shows that the size of a band amplified by the molecular marker in the known kenaf cytoplasmic male sterile lines with different cytoplasmic types is 980bp (lanes 1-10) and the size of a band amplified in the maintenance is 1082bp (lanes 12-19) as shown in figure 2, which indicates that the molecular marker can accurately identify the cytoplasmic male sterile line resources of the kenaf with different cytoplasmic types.
Example 3
Stability of HMBQ102 labeling repeat experiment
According to the stable characteristic that the ambary HMBQ102 molecular tag can amplify a 980bp band in the sterile cytoplasm and amplify a 1082bp band in the maintainer line, the ambary field planting resources are screened and identified, and the result is shown in figure 3: in the embodiment, 16 germplasm resources with consistent cytoplasmic characteristics with UG93A and 10 germplasm resources with consistent cytoplasmic characteristics with UG93B are identified. The identification of the materials provides a molecular basis for the breeding of cytoplasmic male sterile lines of the kenaf. On the other hand, the two germplasm resources of UG93BS1 and UG93BS2 (fig. 3a, lane 5 and lane 6) are two sterile individuals found in the maintainer line UG93B population, and the detection of HMBQ102 molecular tag shows that the two individuals have cytoplasmic type consistent with that of the sterile line UG93A, so that the rearrangement of mitochondrial genomes of the two sterile individuals is presumed, and further suggests that the molecular tag region may be an active recombination region of kenaf mitochondrial genome. And the two single plants of UG93AR1 and UG93AR2 are two fertile plants found in the sterile line UG93A, but the HMBQ101 molecular tag shows that the two single plants also have the cytoplasm type consistent with the sterile line UG93A, which indicates that the nuclear genomes of the two single plants carry the restoring gene, and based on the cytoplasm characteristics and the field fertility performance of the four materials, the molecular tag provides a theoretical basis for constructing near-isogenic line groups of the cytoplasmic male sterile line, the maintainer line and the restorer line of the kenaf, and also provides an ideal material for researching the molecular mechanism and the fertility restoring mechanism of the cytoplasmic male sterile of the kenaf.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.
SEQUENCE LISTING
<110> Guangxi Zhuang nationality college of autonomous region agro-sciences
<120> CMS molecular tag based on non-coding region of kenaf mitochondrial gene, primer pair and application thereof
<130> 202104-0524
<160> 2
<170> PatentIn version 3.5
<210> 1
<211> 21
<212> DNA
<213> Artificial sequence
<400> 1
aggggtcatc ctgggttact g 21
<210> 2
<211> 23
<212> DNA
<213> Artificial sequence
<400> 2
tcgtcgctta catggattac cac 23

Claims (2)

1. The application of CMS molecular label primer pair based on kenaf mitochondrial gene non-coding region in breeding kenaf cytoplasmic male sterile line is characterized in that: the CMS molecular tag primer pairs are HMBQ102F and HMBQ102R, wherein the HMBQ102F sequence is 5'aggggtcatcctgggttactg3', and the HMBQ102R sequence is 5'tcgtcgcttacatggattaccac 3'.
2. Use according to claim 1, characterized in that: the CMS molecular tag primer pair has the amplified band size of 980bp in the kenaf cytoplasmic male sterile line and the amplified band size of 1082bp in the kenaf maintainer line, and the kenaf cytoplasmic male sterile line is bred through the amplification of different band sizes.
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