CN108260522B - Molecular breeding method for high-yield new soybean strain - Google Patents
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Abstract
The invention discloses a molecular breeding method of a new high-yield soybean strain. The method comprises the following steps: using a promoted soybean variety with the genotype of Ln and non-four-pod character as a receptor, and using a soybean variety with the genotype of Ln and a four-pod ratio obviously higher than that of the receptor as a donor to perform sexual hybridization; continuously backcrossing the receptor serving as a recurrent parent with a progeny soybean single plant with the genotype of ln to obtain a backcross progeny F1BCn(ii) a Selecting progeny soybean single plants with the genotype of ln and the characters that the four-grain pod ratio is obviously higher than the receptor and the hundred grain weight is not lower than the receptor, continuously selfing until the character inheritance is stable, and obtaining selfed progeny FnBCnThe new soybean line is the line with the genotype of ln and the character that the four-pod ratio is obviously higher than that of the receptor. Compared with the receptor for popularizing the soybean variety, the four-grain pod ratio and the average acre yield of the new high-yield soybean variety bred by the method are both obviously improved. The invention has important application value.
Description
Technical Field
The invention belongs to the field of plant breeding, and particularly relates to a molecular breeding method of a new high-yield soybean strain.
Background
High yield is a long-term major goal of soybean breeders. The soybean seed yield is mainly determined by the number of plants per unit area, the number of grains per plant and the weight of the grains per hundred, wherein the number of grains per plant is one of main improvement objects of high-yield breeding, and the number of grains per plant is obtained by multiplying the number of pods per plant by the average number of grains per pod. The different-grain pods of common cultivars of soybean include one-grain, two-grain, three-grain, and four-grain pods (five-grain pods are rarely seen), one-grain and two-grain pods being of the few-grain pod type, and three-grain and four-grain pods being of the multi-grain pod type.
The Soybean pod number trait is controlled by more than 10 quantitative trait sites (Chenyi, genetic analysis of Soybean per pod number; Soybean science, 1998 (2): 115-. In the case where soybean planting density (i.e., the number of plants per unit area) and the weight per hundred grains (i.e., the volume of grains) are relatively stable, generally, the greater the number of pods, the higher the yield of grains.
Currently, Ln genes controlling soybean pod number and leaf shape have been mapped and identified (Namhee Jeong, Plant Cell, 2012; Chao Fang et al, Journal of Genetics and Genomics, 2013). The Ln gene is finely positioned in a 57.6kb DNA fragment interval of the long arm of a No. 20 chromosome of soybean, 1 single base mutation of G → C is found in a 1 st exon sequence of the Ln gene, 1 amino acid variation (Asp → His) is caused, the phenotypic variation with the four-pod ratio obviously increased is caused, and the maximum contribution rate can reach more than 20%. The mutant genotype marker is genotype Ln (i.e., corresponding to the four pod phenotype), while the wild-type genotype marker is genotype Ln (i.e., corresponding to the non-four pod phenotype).
Disclosure of Invention
The invention aims to solve the technical problem of how to quickly obtain a new soybean strain with excellent properties.
In order to solve the technical problems, the invention firstly provides a molecular breeding method of a new soybean strain, which comprises the following steps:
(1) using soybean variety with Ln genotype and specific character I as receptor, using Ln genotype and specific character IDetermining soybean variety with character III as donor, and sexual hybridization to obtain hybrid F1Generation;
(2) in hybridization F1Selecting soybean single plant with the genotype of ln in the generation, and then backcrossing the soybean single plant with the receptor to obtain a backcross progeny F1BC1；
(3) Taking the receptor as a recurrent parent to backcross a descendant F1BC1Continuously backcrossing the soybean single plant with the middle genotype of ln to obtain a backcross progeny F1BCn；
(4) After backcross, the progeny F1BCnSelecting single soybean plant with ln genotype and specific character II, selfing continuously until obtaining selfing progeny F with stable character hereditynBCn；
Selfing progeny FnBCnIn the method, the strain with the genotype ln and the specific character II is a new soybean strain; the new soybean strain has excellent properties; the superior trait may be (a) and/or (b) and/or (c): (a) the four pod ratio was significantly higher than the receptor; (b) the yield per mu is obviously higher than that of a receptor; (c) the weight of the hundred grains is not less than that of the receptor.
In the molecular breeding method, the specific character I can be a character other than four pods. The specific character II can be a character that the four-pod ratio is obviously higher than that of a receptor and the weight of the hundred grains is not lower than that of the receptor. The specific trait iii may be a trait in which the four pod ratio is significantly higher than that of the recipient.
In the molecular breeding method, the soybean variety with the Ln genotype can be a soybean variety with the GG homozygous genotype based on the specific SNP locus. The soybean variety with the genotype ln can be a soybean variety with CC homozygote based on the genotype of the specific SNP locus. The specific SNP site can be the 74 th nucleotide from the 5' end of the sequence 1 in the sequence table in the soybean genome.
In the molecular breeding method, in the step (1), the soybean variety serving as the receptor can be a national examination and popularization soybean variety.
In the molecular breeding method, in the step (3), the continuous backcross may be specifically backcross for 2 generations.
In the molecular breeding method, the recipient is used as a recurrent parent to be backcrossed with a progeny F1BC1Continuously backcrossing the soybean single plant with the middle genotype of ln to obtain a backcross progeny F1BCn"specifically may be:
① progeny F after backcross1BC1F of the middle selection genotype ln1BC1The single plant is backcrossed with the receptor to obtain a backcross progeny F1BC2；
② progeny F after backcross1BC2F of the middle selection genotype ln1BC2The single plant is backcrossed with the receptor to obtain a backcross progeny F1BC3。
In the above molecular breeding method, in the step (4), the continuous selfing may be selfing for 3 generations.
In the above molecular breeding method, the "progeny F in backcross1BCnSelecting single soybean plant with ln genotype and specific character II, selfing continuously until obtaining selfing progeny F with stable character hereditynBCn"specifically may be:
① progeny F after backcross1BC3Selecting single soybean plant with ln genotype and specific character II, selfing to obtain selfed progeny F2BC3；
② in the self-bred progeny F2BC3Selecting single soybean plant with ln genotype and specific character II, selfing to obtain selfed progeny F3BC3；
③ in the self-bred progeny F3BC3Selecting single soybean plant with ln genotype and specific character II, selfing to obtain selfed progeny F4BC3。
In the molecular breeding method, the soybean variety with the genotype Ln and the specific character I is Zhonghuang 13 or Kedou No. 1; the soybean variety with the genotype of ln and the specific character III is Kefeng 17 or Heilong 40. Specifically, when Zhonghuang 13 is the recipient, Kefeng 17 is the donor; when Codou No. 1 is the acceptor, Heilong 40 serves as the donor.
The invention also protects the new soybean strain bred by adopting any one of the methods; the new soybean strain has excellent properties; the superior trait may be (a) and/or (b) and/or (c): (a) the four pod ratio was significantly higher than the receptor; (b) the yield per mu is obviously higher than that of a receptor; (c) the weight of the hundred grains is not less than that of the receptor.
The application of any of the methods described above in soybean breeding also belongs to the protection scope of the invention.
In the application, the soybean breeding is to use the new soybean strain bred by any one of the methods as a parent to be hybridized and/or backcrossed with other soybean varieties to obtain the new soybean strain with the genetic background of the other soybean varieties.
In the above application, the other soybean variety may be a soybean variety having the genotype Ln and the specific trait I.
Experiments prove that 4 new soybean lines (sequentially named as LZ904-1, KZ5-1, KZ5-5 and LZ904-7) are bred by using Zhonghuang 13 and Kefeng 17 by adopting the method provided by the invention, and 2 new soybean lines (sequentially named as KH5-2 and KK4-4) are bred by using Kedou No. 1 and Hei nong 40. The 6 new soybean strains are high-yield soybean strains, and particularly, KH5-2 and LZ904-7 have excellent comprehensive properties and large yield increase amplitude: the average yield per mu of KH5-2 is 202 kg, which is 6.88% higher than that of No. 1 bean of receptor control family; the average yield per mu of LZ904-7 was 201.6 kg, which is 10.16% greater than the yield of yellow 13 in the recipient control. The molecular breeding method for the new high-yield soybean strain provided by the invention has the advantages of short breeding period (only 3-4 years) and high selection efficiency. The invention has important application value.
Drawings
FIG. 1 is a technical route of a molecular breeding method for breeding a new high-yield soybean line by using Zhonghuang 13 and Kefeng 17.
FIG. 2 is a technical route of a molecular breeding method for breeding a new high-yield soybean strain by using Kodou No. 1 and Heilong 40.
FIG. 3 is the sequencing results for the genotype tests of 6 new soybean lines.
Fig. 4 is a graph of pod number distribution, average hundred weight and average yield per mu for 6 new soybean lines tested.
Detailed Description
The following examples are given to facilitate a better understanding of the invention, but do not limit the invention. The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified. The quantitative tests in the following examples, all set up three replicates and the results averaged.
The soybean variety of the yellow 13 is a product of limited liability company of the Yongmin breed of Anhui. Hereinafter, soybean variety Medium yellow 13 is abbreviated as Medium yellow 13. Zhonghuang 13 is the main summer soybean variety in the northern region of Huaihe river of Anhui province.
Soybean variety family No. 1 (described in the following documents: plum, new soybean variety "family No. 1" approved by farmer's consummation: general overview of the species, 2012 (2): 16-16) is a summer soybean variety developed by the municipalities of North Huaihe, Suzhou, Bozhou, etc., of Anhui province. Soybean variety heinong 40 is described in the following documents: xu shou Bian, high quality soybean variety Hei nong No. 40, China rural science and technology, 1999 (12). Hereinafter, soybean variety family bean No. 1 is abbreviated as family bean No. 1, and soybean variety heinong 40 is abbreviated as heinong 40.
The soybean variety kefeng No. 17 is obtained by sexual hybridization breeding of a soybean line 8511 and iron impurities by the institute of genetics and developmental biology of the Chinese academy of sciences, and is approved by the Committee of agricultural varieties in Beijing in 2004, and the approval number is 'Jingjudou 2004001'. Hereinafter, the soybean variety kefeng No. 17 is abbreviated as kefeng No. 17.
The following examples were used to determine whether the genotype of soybean was Ln or Ln as follows:
1. taking 0.5g of soybean leaves, and then extracting genome DNA by adopting a CTAB method to obtain the genome DNA of the soybean.
2. And (3) carrying out PCR amplification by taking the genome DNA of the soybean as a template and 5'-CCTGTGTTGTGATTTGCTGTTTA-3' and 5'-GCTTTTGTTGTTTCTTTGTTATTCA-3' as primers to obtain a PCR amplification product.
3. Taking the PCR amplification product, sequencing by adopting a DNA sequencer, analyzing a sequencing result and judging as follows: if the genotype based on the specific SNP locus is CC homozygous, the genotype of the soybean is ln; if the genotype based on the specific SNP locus is GG homozygous type, the genotype of the soybean is Ln; the specific SNP site is the 74 th nucleotide from the 5' end of the sequence 1 in the sequence table in the soybean genome.
Example 1 molecular Breeding method for breeding new high-yield soybean line
Specific character I: non-four pod trait.
Specific character II: the four-grain pod ratio is obviously higher than that of the receptor variety, and the hundred grain weight is not lower than that of the receptor variety.
Specific characteristics III: the four pod ratio was significantly higher than the trait of the recipient variety.
The four pod ratio is the percentage of the number of four pods to the total number of pods per plant.
First, 4 new soybean strains are bred by utilizing Zhonghuang 13 and Kefeng 17
The technical route of the molecular breeding method for breeding the new high-yield soybean line by utilizing the Zhonghuang 13 and the Kefeng 17 is shown in figure 1.
1. Medium yellow 13 was selected as the receptor. The Zhonghuang 13 is a national examination and popularization soybean variety with the genotype of Ln and specific character I.
2. Kefeng 17 was selected as donor. Kefeng 17 is a soybean variety with a genotype of ln and specific character III.
3. Preparing batch hybridization combinations (more than 30) from Kefeng 17 (donor) and Zhonghuang 13 (acceptor), wherein the number of emasculated and pollinated flowers of each combination is more than 40, and harvesting seeds of each hybridization combination, namely F1。
4. Taking F with the genotype ln obtained in the step 31The single plant and Chinese rhubarb 13 are prepared into batch backcross combinations (more than 30) for backcross, the number of flowers for emasculation and pollination of each combination is more than 40, and each backcross combination is used for harvesting seeds, namely F1BC1。
5. Taking F with the genotype ln obtained in the step 41BC1The single plant is backcrossed with Zhonghuang 13 in batch backcross combinations (more than 30 plants), and each groupThe number of flowers after emasculation and pollination is more than 40, and each backcross combination is used for harvesting seeds, namely F1BC2。
6. Taking F with the genotype ln obtained in the step 51BC2The single plant and Chinese rhubarb 13 are prepared into batch backcross combinations (more than 30) for backcross, the number of flowers for emasculation and pollination of each combination is more than 40, and each backcross combination is used for harvesting seeds, namely F1BC3。
7. Taking F with the genotype ln obtained in the step 6 and having specific character II1BC3Performing individual plant selfing to obtain seeds, namely F2BC3。
8. Taking F with the genotype ln obtained in the step 7 and having specific character II2BC3Respectively selfing the strains to obtain seeds, namely F3BC3。
9. Taking F with the genotype ln obtained in the step 8 and having specific character II3BC3Respectively selfing the strains to obtain seeds, namely the F with stable character inheritance4BC3。
In step 9, 4 new soybean lines with the gene type of ln and the specific character II are obtained and named as LZ904-1, KZ5-1, KZ5-5 and LZ904-7 in sequence.
Second, 2 new soybean strains are bred by utilizing Kedou No. 1 and Heilong 40
The technical route of the molecular breeding method for breeding the new high-yield soybean line by using the Kedou No. 1 and Heilong 40 is shown in figure 2.
1. Couma 1 was selected as the receptor. Codou No. 1 is a nationally examined high-quality soybean variety with the genotype of Ln and specific character I.
2. Melanong 40 was selected as the donor. Heilongnong 40 is a soybean variety with a genotype ln and a specific trait III.
3. Preparing batch hybridization combinations (more than 30) from Heilongnong 40 (donor) and Kedou No. 1 (acceptor), wherein the number of emasculated and pollinated flowers of each combination is more than 40, and harvesting seeds from each hybridization combination, namely F1。
4. Taking the radical obtained in step 3F with ln type1Backcrossing individual plant with batch backcross combination (more than 30) prepared from Kedou No. 1, emasculation and pollination of each combination having more than 40 flowers, and harvesting seeds from each backcross combination to obtain F1BC1。
5. Taking F with the genotype ln obtained in the step 41BC1Backcrossing individual plant with batch backcross combination (more than 30) prepared from Kedou No. 1, emasculation and pollination of each combination having more than 40 flowers, and harvesting seeds from each backcross combination to obtain F1BC2。
6. Taking F with the genotype ln obtained in the step 51BC2Backcrossing individual plant with batch backcross combination (more than 30) prepared from Kedou No. 1, emasculation and pollination of each combination having more than 40 flowers, and harvesting seeds from each backcross combination to obtain F1BC3。
7. Taking F with the genotype ln obtained in the step 6 and having specific character II1BC3Performing individual plant selfing to obtain seeds, namely F2BC3。
8. Taking F with the genotype ln obtained in the step 7 and having specific character II2BC3Respectively selfing the strains to obtain seeds, namely F3BC3。
9. Taking F with the genotype ln obtained in the step 8 and having specific character II3BC3Respectively selfing the strains to obtain seeds, namely the F with stable character inheritance4BC3。
In step 9, 2 new soybean strains with the genotypes being ln and the specific character II are obtained, and are sequentially named as KH5-2 and KK 4-4.
Thirdly, detecting the genotypes of the 4 new soybean strains obtained in the first step and the 2 new soybean strains obtained in the second step
The soybean to be detected is LZ904-1, KZ5-1, KZ5-5, LZ904-7, KH5-2, KK4-4, Zhonghuang 13, Kefeng 17, Kedou No. 1 or Heilong 40.
1. Taking 0.5g of soybean leaves to be detected, and then extracting genome DNA by adopting a CTAB method to obtain the genome DNA of the soybean to be detected.
2. And (3) performing PCR amplification by using the genomic DNA of the soybean to be detected as a template and 5'-CCTGTGTTGTGATTTGCTGTTTA-3' and 5'-GCTTTTGTTGTTTCTTTGTTATTCA-3' as primers to obtain a PCR amplification product.
3. Taking the PCR amplification product, sequencing by adopting a DNA sequencer, analyzing a sequencing result and judging as follows: if the genotype of the soybean to be detected based on the specific SNP locus is CC homozygoty, the genotype of the soybean to be detected is ln; if the genotype of the soybean to be detected based on the specific SNP locus is GG homozygous type, the genotype of the soybean to be detected is Ln; the specific SNP site is the 74 th nucleotide from the 5' end of the sequence 1 in the sequence table in the soybean genome.
The partial sequencing results are shown in FIG. 3(1 is Zhonghuang 13, 2 is KouDou No. 1, 3 is LZ904-1, 4 is KZ5-1, 5 is KZ5-5, 6 is LZ904-7, 7 is KH5-2, 8 is KK4-4, 9 is Kofeng 17, and 10 is Heilong 40). The sequencing results showed that the genotypes of the two recipient varieties (i.e., Zhonghuang 13 and Kedou No. 1) were Ln, while the genotypes of the two donor varieties (i.e., Kefeng 17 and Heiang 40), the 4 new soybean lines obtained in step one and the 2 new soybean lines obtained in step two were Ln.
Fourthly, detecting the pod grain number distribution, the average hundred grain weight and the average yield per mu of the 4 new soybean strains obtained in the first step and the 2 new soybean strains obtained in the second step
The soybean to be detected is LZ904-1, KZ5-1, KZ5-5, LZ904-7, KH5-2, KK4-4, Zhonghuang 13, Kefeng 17, Kedou No. 1 or Heilong 40.
The soybean seeds to be tested are taken and planted in 3 different test bases of Anhui and Henan, and then pod number distribution, average hundred-grain weight and average acre yield are counted. The pod number distribution is specifically a one-pod ratio (the number of one pod is a percentage of the total pod number per plant), a two-pod ratio (the number of two pods is a percentage of the total pod number per plant), a three-pod ratio (the number of three pods is a percentage of the total pod number per plant), and a four-pod ratio (the number of four pods is a percentage of the total pod number per plant).
The results are shown in FIG. 4(A is pod number distribution, B is average hundred weight, C is acre yield). The results show that compared with medium yellow 13, the four-pod ratio and the average yield per mu of the 4 new soybean lines are both remarkably improved (the four-pod ratio is increased by 16.8 to 23.8 percent compared with the medium yellow 13), and the average weight per hundred grains is not obviously reduced; both the four pod ratio and the average yield per mu were significantly improved for both new soybean lines compared to bean number 1 (four pod ratio increased by 15.5% and 18.8% over bean number 1) without significant reduction in average grain weight per hundred. Therefore, the 4 new soybean lines obtained in the step one and the 2 new soybean lines obtained in the step two are high-yield new soybean lines. In particular KH5-2 and LZ904-7 (although it is shown in FIGS. 4B and C that the average hundred grain weight and yield per mu are highest for LZ904-1, it is eliminated because the strain is infected by soybean mosaic virus), not only the comprehensive character is excellent, but also the yield is increased greatly: the average yield per mu of KH5-2 is 202 kg, which is 6.88% higher than that of No. 1 bean of receptor control family; the average yield per mu of LZ904-7 was 201.6 kg, which is 10.16% greater than the yield of yellow 13 in the recipient control.
<110> institute of genetics and developmental biology of Chinese academy of sciences
<120> molecular breeding method for new high-yield soybean strain
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<170>PatentIn version 3.5
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<213> Soybean (Glycine max (L.) Merr.)
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ctctaaaccc tttaactttc tctctcttat gactttgttg ttcctttaca ggagaccaga 60
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cctcgaagac catacctctt catccggtaa acttcatgat cataccaata tatatatata 180
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Claims (4)
1. A molecular breeding method of a new soybean strain comprises the following steps:
(1) using soybean variety whose genotype is Ln and possesses specific character I as receptor and using soybean variety whose genotype is Ln and possesses specific character III as donor, utilizing sexual hybridization to obtain hybrid F1Generation;
(2) in hybridization F1Selecting soybean single plant with the genotype of ln in the generation, and then backcrossing the soybean single plant with the receptor to obtain a backcross progeny F1BC1；
(3) After backcross, the progeny F1BC1F of the middle selection genotype ln1BC1The single plant is backcrossed with the receptor to obtain a backcross progeny F1BC2(ii) a After backcross, the progeny F1BC2F of the middle selection genotype ln1BC2The single plant is backcrossed with the receptor to obtain a backcross progeny F1BC3；
(4) After backcross, the progeny F1BC3Selecting single soybean plant with ln genotype and specific character II, selfing to obtain selfed progeny F2BC3(ii) a In the selfing progeny F2BC3Selecting single soybean plant with ln genotype and specific character II, selfing to obtain selfed progeny F3BC3(ii) a In the selfing progeny F3BC3Selecting single soybean plant with ln genotype and specific character II, selfing to obtain selfed progeny F4BC3；
Selfing progeny F4BC3In the method, the strain with the genotype ln and the specific character II is a new soybean strain; the new soybean strain has excellent properties; the excellent traits are (a) and/or (b) and/or (c): (a) the four pod ratio was significantly higher than the receptor; (b) the yield per mu is obviously higher than that of a receptor; (c) the weight of hundred particles is not less than that of the receptor;
the specific character I is a character of non-four-pod;
the specific character II is that the ratio of four pods is obviously higher than that of a receptor and the weight of one hundred grains is not lower than that of the receptor;
the specific character III is a character that the four-pod ratio is obviously higher than that of a receptor;
the soybean variety with the Ln genotype is a soybean variety which is homozygous for GG and based on the specific SNP locus;
the soybean variety with the genotype ln is a soybean variety with CC homozygote based on the genotype of the specific SNP locus;
the specific SNP site is the 74 th nucleotide from the 5' end of a sequence 1 in a sequence table in a soybean genome;
the acceptor is Zhonghuang 13, and the donor is Kefeng 17.
2. A molecular breeding method of a new soybean strain comprises the following steps:
(1) using soybean variety whose genotype is Ln and possesses specific character I as receptor and using soybean variety whose genotype is Ln and possesses specific character III as donor, utilizing sexual hybridization to obtain hybrid F1Generation;
(2) in hybridization F1Selecting soybean single plant with the genotype of ln in the generation, and then backcrossing the soybean single plant with the receptor to obtain a backcross progeny F1BC1；
(3) After backcross, the progeny F1BC1F of the middle selection genotype ln1BC1The single plant is backcrossed with the receptor to obtain a backcross progeny F1BC2(ii) a After backcross, the progeny F1BC2F of the middle selection genotype ln1BC2The single plant is backcrossed with the receptor to obtain a backcross progeny F1BC3；
(4) After backcross, the progeny F1BC3Selecting single soybean plant with ln genotype and specific character II, selfing to obtain selfed progeny F2BC3(ii) a In the selfing progeny F2BC3Selecting single soybean plant with ln genotype and specific character II, selfing to obtain selfed progeny F3BC3(ii) a In the selfing progeny F3BC3Selecting single soybean plant with ln genotype and specific character II, selfing to obtain selfed progeny F4BC3；
Selfing progeny F4BC3In the method, the strain with the genotype ln and the specific character II is a new soybean strain; the new soybean strain has excellent properties; the excellent traits are (a) and/or (b) and/or (c): (a) the four pod ratio was significantly higher than the receptor; (b) the yield per mu is obviously higher than that of a receptor; (c) the weight of hundred particles is not less than that of the receptor;
the specific character I is a character of non-four-pod;
the specific character II is that the ratio of four pods is obviously higher than that of a receptor and the weight of one hundred grains is not lower than that of the receptor;
the specific character III is a character that the four-pod ratio is obviously higher than that of a receptor;
the soybean variety with the Ln genotype is a soybean variety which is homozygous for GG and based on the specific SNP locus;
the soybean variety with the genotype ln is a soybean variety with CC homozygote based on the genotype of the specific SNP locus;
the specific SNP site is the 74 th nucleotide from the 5' end of a sequence 1 in a sequence table in a soybean genome;
the acceptor is Kedou No. 1, and the donor is Heilong 40.
3. Use of the method of claim 1 or 2 in soybean breeding.
4. Use according to claim 3, characterized in that: the soybean breeding is to use the new soybean strain bred by the method of claim 1 or 2 as a parent to be hybridized and/or backcrossed with other soybean varieties to obtain the new soybean strain with the genetic background of the other soybean varieties; the other soybean varieties are soybean varieties with the genotype of Ln and specific characters I.
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| CN108260522A (en) | 2018-07-10 |
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