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Abstract [Objectives] This study was conducted to better understand the variation law of sugarcane clones during tissue culture process， and to provide a reference for rapid propagation and detection of healthy sugarcane seedlings.
[Methods] The genetic stability of tissue culture clones of three sugarcane varieties was analyzed using the AFLP molecular marker technique.
[Results] The average number of polymorphic loci was 19.58 for each primer pair， and the percentage of polymorphic loci was 41.74%. Compared with the donor varieties， all tissue culture materials were mutated. There were 3-16 missing bands， with an average of 5.2bands， and there were 0-17 increased bands， with an average of 3.3 bands. The total number of missing and increased bands was 4-33， with an average of 8.5.The band difference rates were in the range of 0.009 4%-0.077 6%， with an average of 0.020 6%. The genetic similarity coefficients between materials ranged from 0.685 6 to 0.998 2， with an average of 0.818 4. The three sugarcane varieties and their tissue culture clones were clustered into three groups.
[Conclusions] Although variations occur in tissue culture， the variations are not too obvious， and the genetic stability is relatively high. It is recommended to minimize the number of subculture generations and cultivation time to reduce the occurrence of variation during tissue culture for rapid propagation of sugarcane seedlings.
Key words Sugarcane; Tissue culture; Clone; AFLP; Genetic stability
Sugarcane belongs to asexually propagated crops. Usually several years after planting， multiple pathogens repeatedly infect and accumulate in plants， which leads to the degradation of varietal characters， resulting in reduced yield and reduced quality， which significantly affect economic benefits. Experiments have shown that healthy sugarcane seedlings can restore the quality of sugarcane varieties， thereby increasing yield by 20%-40% and sucrose by 0.5%-1.0% （absolute value）[1]and significantly improving sugarcane quality. Therefore， healthy sugarcane seedlings are widely used internationally. It has also been gradually recognized by sugarcane farmers in our country， and the planting area has increased rapidly and has a bright future. Healthy sugarcane seedlings are produced by removing pathogenic bacteria through tissue culture techniques. There are two main ways， one of which is to produce through the induction of pathogenfree meristems， and the other is to propagate from axillary buds by physical and chemical methods in combination with tissue culture technology. The former is the method used in the early period of sugarcane micropropagation， and the latter is the method mainly used in the past 20 years. Both have in common the need to go through a tissue culture process. Studies have shown that somatic clone variation is a common phenomenon in plant tissue culture. During the cultivation of plant cells， tissues and organs， cells and regenerated plants can produce genetic or epigenetic mutations， which can be comprehensively detected and identified in many aspects such as morphology， cytology， biochemistry and molecular biology. Sugarcane is one of the earliest crops used in tissue culture research. Studies have confirmed that sugarcane clone variations obtained through meristematic tissue culture are very common[2-5]. Some varieties and materials have been screened through the characteristic of tissue culture process that it will cause genetic variation in cells[3，6]. However， for propagation of healthy sugarcane seedlings， variations should be avoided as much as possible. To this end， scientific and technological personnel have developed another sugarcane micropropagation approach， which directly separates axillary buds for rapid propagation while avoiding the callus culture process. Whether the healthy seedlings obtained through axillary buds will mutate has not been reported in any studies. In addition， the detection of sugarcane tissue culture clones has basically used the RAPD molecular marker technique[2-5]， and the application of AFLP has not been reported in any study. In this study， AFLP molecular marker technique with high sensitivity and large amount of information was used to detect sugarcane tissue culture plantlets obtained from different sources， and their genetic variations were analyzed， aiming to better understand the variation laws of sugarcane tissue culture clones in tissue culture and provide references for rapid propagation and detection of sugarcane healthy seedlings.
Materials and Methods
Experimental materials
The experimental materials were 13 sugarcane clones obtained through axillary bud tissue culture and onestep seedling formation using calli （Table 1）.
Experimental methods
Extraction of genomic DNA
The CTAB method was adopted referring to the method of Besse et al.[7]with some modifications. A small amount of PVP dry powder was added when grinding the sample， and isopropanol at a temperature ranging from -20 to -25 ℃was added when collecting the DNA precipitate. The DNA precipitate was washed with 70% ethanol for 2-3 times.
AFLP amplification of genomic DNA
Sugarcane genomic DNA was digested by Mse I and Eco R I at 37 ℃ for about 15 h. Then， the digested product was catalyzed by T4 Ligase at 22 ℃， and ligated with Mse Ⅰ adapter and Eco R I adapter for 2 h， respectively. After the ligation， the DNA sample was diluted 10 times， and preamplified with Mse IC and Eco R IA primer pairs. The PCR conditions were as follows： 95 ℃ for 2 min， 20 cycles of 94 ℃ for 30 s， 56 ℃ for 60 s and 72 ℃ for 60 s， and extension at 72 ℃ for 5 min. With the 20 times dilution of the preamplification product as template， PCR was then performed using selective primer pairs having 3 bases according to the following PCR program： 95 ℃ for 2 min， 13 cycles of 94 ℃ for 30 s， 65 ℃ for 30 s （0.7 ℃ reduction per cycle） and 72 ℃ for 60 s， 23 cycles of 94 ℃ for 30 s， 56 ℃for 30 s and 72 ℃ for 60 s， and extension at 72 ℃ for 5 min.All PCR reactions were performed on an Eppendorf gradient PCR instrument. After denaturing each amplification product at 95 ℃ for 10 min， it was electrophoresed on a 5% denaturing polyacrylamide gel （power 100 W） for 2.5-3.0 h. The gel was banded with silver staining method. After drying， the band was read and photographed. Data statistics and processing
The number of bands amplified by each pair of primers was counted， separately. The data was recorded with band as 1 and no band as 0. The genetic similarity coefficient was calculated with the obtained data using NTSYSpc 2.10esoftware， that is， Sij=2Nij/（Ni+Nj）， wherein Nij is the number of bands shared between sample i and sample j， and Ni and Nj are the numbers of individual bands in sample i and sample j， respectively. UPGMA cluster analysis was performed based on genetic similarity coefficients. The PowerMarker 3.25 software was used to calculate the genetic diversity index， that is， Ht=1-∑p2i， wherein pi is the frequency of the ithallele.
Results and Analysis
Analysis of AFLP amplification results
Amplification test was performed on the DNA template obtained by preamplification using AFLP primers， and 12 pairs of primers showing clear and stable bands were selected for formal amplification test. The sizes of the amplified product fragments were in the range of 30-600 bp. It can be seen from Table 2 that the numbers of amplified bands of the primer pairs were between 13 and 64， and a total of 563 bands were amplified by the 12 pairs of primers in the 13 materials， with an average of 46.92 bands per primer pair. ATC/CTA showed the highest polymorphism， which was 53.85%， while AAG/CTT showed the lowest value， only 32.43%. The percentages of polymorphic bands of other primers were in the range of 32.43%-53.85%， with an average of 41.74%. The values were much lower than the percentage of polymorphic bands between 61.5%-87.60%[7-11]in previous studies. Obviously， the polymorphism mainly came from the three donor varieties， and the genetic differences between the tissue culture clone materials and their donor varieties were not large， which was the main reason for the reduced polymorphism.
Analysis of genetic stability
It is a very common phenomenon that somatic clones appear in plant tissue culture. The degree of variation is related to various factors such as medium composition， explant type， explant genotype， subculture times and culture duration. It can be seen from Table 3 that all the tissue culture materials had mutated， that is， band missing or increase happened， or both of them happened. There were 3-16 missing bands， with an average of 5.2 and a maximum in YT60CH. There were 0-17 new bands， with an average of 3.3 bands and a maximum in YT60CH. The total number of missing and increased bands was 4-33， with an average of 8.5， and the maximum appeared in YT60CH， which was 33. The band difference rates were in the range of 0.009 4%-0.077 6%， with an average of 0.020 6% and a maximum in YT60CH. The root materials from the axillary bud clones， such as the root materials of CP65357AR and BadilaAR， had a higher difference rate than the leaf materials， such as the leaf materials of CP65357AL， BadilaAL and YT60AL. Whether it is associated with the fact the root system is immersed in the medium for long term needs further study. YT60CL was a material obtained by "onestep seedling formation"， which showed a band difference rate of 0.009 5% which was not high， because it was obtained from the differentiation of calli which were cultured for a short time and free of subculture. As to YT60CH， which was obtained from the tissue differentiation of calli which were subcultured for multiple generations， and was in a strange finehair tufted shape， its band difference rate was the highest （0.077 6%）， which might be caused by severer variation of DNA. CP65357AA was an albino plantlet， and CP65357ASM and CP65357ABF were plantlets with strange shapes， but their band difference rates were lower than normal plantlets， though all the three were from axillary buds. It seemed that the albino plantlet and these two oddshape plantlets were not necessarily caused by DNA mutations， they might be caused by problems in physiology and metabolism， or due to chromosomal aberrations， changes in methylation status， etc.， and AFLP technology failed to detect it. Fangye LAO et al. AFLP Analysis of Genetic Stability of Sugarcane Tissue Culture Clones
Analysis of genetic similarity
The degree of genetic similarity indicates the degree of intimacy of the samples. It can be seen from Table 4 that CP65357AR and CP65357AL had the highest genetic similarity coefficient （0.998 2）， and YT60CH and Badila had the lowest similarity coefficient （0.685 6）. In other words， the genetic similarity coefficient between the roots and leaves of CP65357s axillary bud micropropagation clone was the highest， while the oddshape tissue culture plantlet of YT60 （finehair tufted） had the lowest genetic similarity coefficient with Badila. The genetic similarity coefficient between the two sugarcane varieties CP65357 and YT60 was higher than 0.788 6， while those between the two and the sugarcane variety Badila were relatively lower， 0.703 4 and 0.701 6， respectively. The CP65357， YT60 and Badila varieties had very high genetic similarity coefficients with their respective clone materials， with average values of 0.989 0， 0.966 3 and 0.989 3， respectively， and among them， the average genetic similarity coefficients between YT60 and its clone materials were relatively low， which was mainly because of the relatively lower genetic similarity coefficients between YT60CH （finehair tufted oddshaped plantlet）and other three materials in the group， which might be caused by NDA mutations in YT60CH. The genetic similarity coefficients between any two of the 13 materials ranged from 0.685 6 to 0.998 2， with an average of 0.818 4.
From the clustering chart （Fig. 2）， it can be seen that when the genetic similarity coefficient threshold was about 0.705 3， the 13 samples could be divided into 2 groups. A total of 10 samples from the CP65357 series and the YT60 series fell into the same group， and other three materials of the Badila series were clustered into a large group， which clearly separated sugar cane and fruit cane， indicating that the former two sucrose varieties were closer to each other and relatively far away from Badila. When the genetic similarity coefficient threshold was about 0.795 4， the 13 samples can be divided into 3 groups， i.e.， the CP65357 series， the YT60 series and the Badila series， that is， the 3 donor varieties were clustered with their tissue culture clones， respectively， suggesting that although variations occur in tissue culture， the variations will not be too obvious， and the genetic stability is still relatively high. When the threshold of genetic similarity coefficient was about 0.941 1， the 13 samples can be divided into 4 groups， with the CP65357 series and the Badila series unchanged the YT60 series divided into 2 groups and the YT60CH independently clustered into 1 group. YT60， YT60AL YT60CL and YT60CL fell into the same group， which indicated that DNA variation had occurred to some extent in YT60CH， and there were also obvious differences in morphology in that the plantlets could not grow up and were finehair tufted. Discussion
During the process of dedifferentiation， differentiation and regeneration of plantderived tissues or cell cultures， due to the induction of abiotic factors， somatic cell clones can produce a wide range of mutations， and the mutation frequency is much higher than the natural mutation frequency. There are mainly the following types： chromosome aberrations （such as chromosome number and structural variations such as polyploidy， aneuploidy， translocation and breakage）， point mutations， replication and deletion， activation of transposable factors， methylation status changes， changes in cytoplasmic DNA， epigenetic variation， etc. Somatic clones have unique value in the research of breeding theory， selection of highquality crops， new resistant strains， and creation of new germplasms. Therefore， they have been widely used in crop breeding and have achieved good results[6]. However， when applying tissue culture techniques to breed good seeds and produce healthy seedlings， the abovementioned mutations should be avoided as much as possible to ensure the purity and stability of the original species. Studies have shown that the occurrence of variations is related to the genetic composition of explants， the type of culture， the type of explants （i.e.， tissue source）， medium composition， subculture times， selection pressure， and temperature treatment. About 20 years ago， tissue culture for fine varieties was mainly achieved through callus induction and differentiation. The process was relatively complicated （the use of many types of growth regulators and long subculture time）， and the incidence of variation was high. This study shows that YT60CH， which was obtained from calli through multiple times of subculture and differentiation， had the highest mutation rate （Table 3）， confirming once again that this method is not suitable for fine variety reproduction. However， YT60CL， which was obtained by "onestep seedling formation"， had also passed the callus stage， but had not been subjected to subculture， resulting in less growth regulators and a short culture time. Therefore， the mutation rate was much lower than that of YT60CH， and even lower than those of CP65357AL， YT60AL and BadilaAL which were propagated from axillary buds （Table 3）. It can be seen that "onestep seedling formation" can be used for the breeding of good varieties， and even if the explants are axillary buds， the number of subculture generations should not be too high， and should not exceed 8 generations， otherwise the incidence of mutation will increase significantly. Because somatic clone mutations may occur at different levels （such as individual， organ， tissue， cell， and molecular levels）， their detection methods are also diverse， mainly including morphological observation， cytogenetics （or karyotype） analysis， the content of secondary metabolites （such as anthocyanins or flavonoids）， isoenzymes and molecular marker. The AFLP used in this study is only one of the molecular marker techniques， and therefore， certain types of somatic mutations such as chromosomal aberrations and changes in methylation status could not be detected. For example， the albino plantlets （CP65357AA） and oddshape plantlets （CP65357ASM and CP65357ABF） in this study were significantly different from normal plantlets in appearance， but the mutation rates were not high， even smaller than normal plantlets （Table 3）， which could not be interpreted by AFLP molecular markers alone. For what kind of mutation it is， or is it only a transient phenomenon and can it return to normal， further research is still needed. In production， if these situations are encountered， it is best to eliminate seedlings with abnormal shapes as soon as possible. The above analysis shows that to assess the genetic stability of sugarcane tissue culture clones， using only one molecular marker method is not comprehensiveand accurate. If possible， it is best to use "morphology + cytology + molecular biology" to perform comprehensive evaluation.
The molecular marker evaluation techniques developed in the past 20 years include RFLP， RAPD， SSR， AFLP， MSAP， etc. Early applications of RFLP and RAPD were more[2-5]， and they are fast， convenient， and economical. The AFLP molecular marker technique that was developed subsequently combines the advantages of RFLP and RAPD. It is convenient and fast， and also has the advantages of large amount of information， strong stability and more polymorphic information. It has been applied to the analysis and identification of many plant somatic clones. Although only the AFLP was used to analyze the genetic stability of sugarcane tissue culture clones in this study and variations at the cell level and morphological level could not be detected to a certain extent， but this study shows that AFLP is a very effective technique. Clones， that could not be discerned by naked eyes， could be classified one by one. In the case of morphological variations， it could also be known from which original plant the mutant plant had mutated. Under the condition of tissue culture， there were certain limitations in the clone variation， because there was no introduction of foreign genes， and even if the morphological variation was very obvious， like YT60CH， the genetic similarity coefficient with the original donor variety was still high， reaching 0.941 4. Studies have shown that the genetic similarity coefficients of different sugarcane varieties will not be so high[8-12]， and even from the same cross combination， such as "CP721210×Yacheng 82108"， the highest genetic similarity coefficient was only 0.839 8[8]. Therefore， when using AFLP molecular marker technique for sugarcane variety identification and analysis， if there are sufficient numbers of amplified bands， for example， 400 or more， it can be considered that the two are the same variety in the condition that the genetic similarity coefficient is above 0.950 0. References
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[6] PING WEN， YANG TIEZHAO. Somaclonal variation and its application in crop breeding[J]. Acta Agriculturae Borealioccidentalis Sinica， 2005， 14（5）： 23-31. （in Chinese）
[7] BESSE P， MCINTYRE CL， BERDING N. Ribosomal DNA variations in Erianthus， a wild sugarcane relative （AndropogoneaeSaccharinae）[J]. Theor. Appl. Genet.， 1996， 92： 733-743.
[8] LAO FANGYE， LIU RUI， HE HUIYI， et al. AFLP analysis of genetic diversity in series sugarcane parents developed at HSBS[J]. Molecular Plant Breeding， 2008， 6（3）： 517-522. （in Chinese）
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[Methods] The genetic stability of tissue culture clones of three sugarcane varieties was analyzed using the AFLP molecular marker technique.
[Results] The average number of polymorphic loci was 19.58 for each primer pair， and the percentage of polymorphic loci was 41.74%. Compared with the donor varieties， all tissue culture materials were mutated. There were 3-16 missing bands， with an average of 5.2bands， and there were 0-17 increased bands， with an average of 3.3 bands. The total number of missing and increased bands was 4-33， with an average of 8.5.The band difference rates were in the range of 0.009 4%-0.077 6%， with an average of 0.020 6%. The genetic similarity coefficients between materials ranged from 0.685 6 to 0.998 2， with an average of 0.818 4. The three sugarcane varieties and their tissue culture clones were clustered into three groups.
[Conclusions] Although variations occur in tissue culture， the variations are not too obvious， and the genetic stability is relatively high. It is recommended to minimize the number of subculture generations and cultivation time to reduce the occurrence of variation during tissue culture for rapid propagation of sugarcane seedlings.
Key words Sugarcane; Tissue culture; Clone; AFLP; Genetic stability
Sugarcane belongs to asexually propagated crops. Usually several years after planting， multiple pathogens repeatedly infect and accumulate in plants， which leads to the degradation of varietal characters， resulting in reduced yield and reduced quality， which significantly affect economic benefits. Experiments have shown that healthy sugarcane seedlings can restore the quality of sugarcane varieties， thereby increasing yield by 20%-40% and sucrose by 0.5%-1.0% （absolute value）[1]and significantly improving sugarcane quality. Therefore， healthy sugarcane seedlings are widely used internationally. It has also been gradually recognized by sugarcane farmers in our country， and the planting area has increased rapidly and has a bright future. Healthy sugarcane seedlings are produced by removing pathogenic bacteria through tissue culture techniques. There are two main ways， one of which is to produce through the induction of pathogenfree meristems， and the other is to propagate from axillary buds by physical and chemical methods in combination with tissue culture technology. The former is the method used in the early period of sugarcane micropropagation， and the latter is the method mainly used in the past 20 years. Both have in common the need to go through a tissue culture process. Studies have shown that somatic clone variation is a common phenomenon in plant tissue culture. During the cultivation of plant cells， tissues and organs， cells and regenerated plants can produce genetic or epigenetic mutations， which can be comprehensively detected and identified in many aspects such as morphology， cytology， biochemistry and molecular biology. Sugarcane is one of the earliest crops used in tissue culture research. Studies have confirmed that sugarcane clone variations obtained through meristematic tissue culture are very common[2-5]. Some varieties and materials have been screened through the characteristic of tissue culture process that it will cause genetic variation in cells[3，6]. However， for propagation of healthy sugarcane seedlings， variations should be avoided as much as possible. To this end， scientific and technological personnel have developed another sugarcane micropropagation approach， which directly separates axillary buds for rapid propagation while avoiding the callus culture process. Whether the healthy seedlings obtained through axillary buds will mutate has not been reported in any studies. In addition， the detection of sugarcane tissue culture clones has basically used the RAPD molecular marker technique[2-5]， and the application of AFLP has not been reported in any study. In this study， AFLP molecular marker technique with high sensitivity and large amount of information was used to detect sugarcane tissue culture plantlets obtained from different sources， and their genetic variations were analyzed， aiming to better understand the variation laws of sugarcane tissue culture clones in tissue culture and provide references for rapid propagation and detection of sugarcane healthy seedlings.
Materials and Methods
Experimental materials
The experimental materials were 13 sugarcane clones obtained through axillary bud tissue culture and onestep seedling formation using calli （Table 1）.
Experimental methods
Extraction of genomic DNA
The CTAB method was adopted referring to the method of Besse et al.[7]with some modifications. A small amount of PVP dry powder was added when grinding the sample， and isopropanol at a temperature ranging from -20 to -25 ℃was added when collecting the DNA precipitate. The DNA precipitate was washed with 70% ethanol for 2-3 times.
AFLP amplification of genomic DNA
Sugarcane genomic DNA was digested by Mse I and Eco R I at 37 ℃ for about 15 h. Then， the digested product was catalyzed by T4 Ligase at 22 ℃， and ligated with Mse Ⅰ adapter and Eco R I adapter for 2 h， respectively. After the ligation， the DNA sample was diluted 10 times， and preamplified with Mse IC and Eco R IA primer pairs. The PCR conditions were as follows： 95 ℃ for 2 min， 20 cycles of 94 ℃ for 30 s， 56 ℃ for 60 s and 72 ℃ for 60 s， and extension at 72 ℃ for 5 min. With the 20 times dilution of the preamplification product as template， PCR was then performed using selective primer pairs having 3 bases according to the following PCR program： 95 ℃ for 2 min， 13 cycles of 94 ℃ for 30 s， 65 ℃ for 30 s （0.7 ℃ reduction per cycle） and 72 ℃ for 60 s， 23 cycles of 94 ℃ for 30 s， 56 ℃for 30 s and 72 ℃ for 60 s， and extension at 72 ℃ for 5 min.All PCR reactions were performed on an Eppendorf gradient PCR instrument. After denaturing each amplification product at 95 ℃ for 10 min， it was electrophoresed on a 5% denaturing polyacrylamide gel （power 100 W） for 2.5-3.0 h. The gel was banded with silver staining method. After drying， the band was read and photographed. Data statistics and processing
The number of bands amplified by each pair of primers was counted， separately. The data was recorded with band as 1 and no band as 0. The genetic similarity coefficient was calculated with the obtained data using NTSYSpc 2.10esoftware， that is， Sij=2Nij/（Ni+Nj）， wherein Nij is the number of bands shared between sample i and sample j， and Ni and Nj are the numbers of individual bands in sample i and sample j， respectively. UPGMA cluster analysis was performed based on genetic similarity coefficients. The PowerMarker 3.25 software was used to calculate the genetic diversity index， that is， Ht=1-∑p2i， wherein pi is the frequency of the ithallele.
Results and Analysis
Analysis of AFLP amplification results
Amplification test was performed on the DNA template obtained by preamplification using AFLP primers， and 12 pairs of primers showing clear and stable bands were selected for formal amplification test. The sizes of the amplified product fragments were in the range of 30-600 bp. It can be seen from Table 2 that the numbers of amplified bands of the primer pairs were between 13 and 64， and a total of 563 bands were amplified by the 12 pairs of primers in the 13 materials， with an average of 46.92 bands per primer pair. ATC/CTA showed the highest polymorphism， which was 53.85%， while AAG/CTT showed the lowest value， only 32.43%. The percentages of polymorphic bands of other primers were in the range of 32.43%-53.85%， with an average of 41.74%. The values were much lower than the percentage of polymorphic bands between 61.5%-87.60%[7-11]in previous studies. Obviously， the polymorphism mainly came from the three donor varieties， and the genetic differences between the tissue culture clone materials and their donor varieties were not large， which was the main reason for the reduced polymorphism.
Analysis of genetic stability
It is a very common phenomenon that somatic clones appear in plant tissue culture. The degree of variation is related to various factors such as medium composition， explant type， explant genotype， subculture times and culture duration. It can be seen from Table 3 that all the tissue culture materials had mutated， that is， band missing or increase happened， or both of them happened. There were 3-16 missing bands， with an average of 5.2 and a maximum in YT60CH. There were 0-17 new bands， with an average of 3.3 bands and a maximum in YT60CH. The total number of missing and increased bands was 4-33， with an average of 8.5， and the maximum appeared in YT60CH， which was 33. The band difference rates were in the range of 0.009 4%-0.077 6%， with an average of 0.020 6% and a maximum in YT60CH. The root materials from the axillary bud clones， such as the root materials of CP65357AR and BadilaAR， had a higher difference rate than the leaf materials， such as the leaf materials of CP65357AL， BadilaAL and YT60AL. Whether it is associated with the fact the root system is immersed in the medium for long term needs further study. YT60CL was a material obtained by "onestep seedling formation"， which showed a band difference rate of 0.009 5% which was not high， because it was obtained from the differentiation of calli which were cultured for a short time and free of subculture. As to YT60CH， which was obtained from the tissue differentiation of calli which were subcultured for multiple generations， and was in a strange finehair tufted shape， its band difference rate was the highest （0.077 6%）， which might be caused by severer variation of DNA. CP65357AA was an albino plantlet， and CP65357ASM and CP65357ABF were plantlets with strange shapes， but their band difference rates were lower than normal plantlets， though all the three were from axillary buds. It seemed that the albino plantlet and these two oddshape plantlets were not necessarily caused by DNA mutations， they might be caused by problems in physiology and metabolism， or due to chromosomal aberrations， changes in methylation status， etc.， and AFLP technology failed to detect it. Fangye LAO et al. AFLP Analysis of Genetic Stability of Sugarcane Tissue Culture Clones
Analysis of genetic similarity
The degree of genetic similarity indicates the degree of intimacy of the samples. It can be seen from Table 4 that CP65357AR and CP65357AL had the highest genetic similarity coefficient （0.998 2）， and YT60CH and Badila had the lowest similarity coefficient （0.685 6）. In other words， the genetic similarity coefficient between the roots and leaves of CP65357s axillary bud micropropagation clone was the highest， while the oddshape tissue culture plantlet of YT60 （finehair tufted） had the lowest genetic similarity coefficient with Badila. The genetic similarity coefficient between the two sugarcane varieties CP65357 and YT60 was higher than 0.788 6， while those between the two and the sugarcane variety Badila were relatively lower， 0.703 4 and 0.701 6， respectively. The CP65357， YT60 and Badila varieties had very high genetic similarity coefficients with their respective clone materials， with average values of 0.989 0， 0.966 3 and 0.989 3， respectively， and among them， the average genetic similarity coefficients between YT60 and its clone materials were relatively low， which was mainly because of the relatively lower genetic similarity coefficients between YT60CH （finehair tufted oddshaped plantlet）and other three materials in the group， which might be caused by NDA mutations in YT60CH. The genetic similarity coefficients between any two of the 13 materials ranged from 0.685 6 to 0.998 2， with an average of 0.818 4.
From the clustering chart （Fig. 2）， it can be seen that when the genetic similarity coefficient threshold was about 0.705 3， the 13 samples could be divided into 2 groups. A total of 10 samples from the CP65357 series and the YT60 series fell into the same group， and other three materials of the Badila series were clustered into a large group， which clearly separated sugar cane and fruit cane， indicating that the former two sucrose varieties were closer to each other and relatively far away from Badila. When the genetic similarity coefficient threshold was about 0.795 4， the 13 samples can be divided into 3 groups， i.e.， the CP65357 series， the YT60 series and the Badila series， that is， the 3 donor varieties were clustered with their tissue culture clones， respectively， suggesting that although variations occur in tissue culture， the variations will not be too obvious， and the genetic stability is still relatively high. When the threshold of genetic similarity coefficient was about 0.941 1， the 13 samples can be divided into 4 groups， with the CP65357 series and the Badila series unchanged the YT60 series divided into 2 groups and the YT60CH independently clustered into 1 group. YT60， YT60AL YT60CL and YT60CL fell into the same group， which indicated that DNA variation had occurred to some extent in YT60CH， and there were also obvious differences in morphology in that the plantlets could not grow up and were finehair tufted. Discussion
During the process of dedifferentiation， differentiation and regeneration of plantderived tissues or cell cultures， due to the induction of abiotic factors， somatic cell clones can produce a wide range of mutations， and the mutation frequency is much higher than the natural mutation frequency. There are mainly the following types： chromosome aberrations （such as chromosome number and structural variations such as polyploidy， aneuploidy， translocation and breakage）， point mutations， replication and deletion， activation of transposable factors， methylation status changes， changes in cytoplasmic DNA， epigenetic variation， etc. Somatic clones have unique value in the research of breeding theory， selection of highquality crops， new resistant strains， and creation of new germplasms. Therefore， they have been widely used in crop breeding and have achieved good results[6]. However， when applying tissue culture techniques to breed good seeds and produce healthy seedlings， the abovementioned mutations should be avoided as much as possible to ensure the purity and stability of the original species. Studies have shown that the occurrence of variations is related to the genetic composition of explants， the type of culture， the type of explants （i.e.， tissue source）， medium composition， subculture times， selection pressure， and temperature treatment. About 20 years ago， tissue culture for fine varieties was mainly achieved through callus induction and differentiation. The process was relatively complicated （the use of many types of growth regulators and long subculture time）， and the incidence of variation was high. This study shows that YT60CH， which was obtained from calli through multiple times of subculture and differentiation， had the highest mutation rate （Table 3）， confirming once again that this method is not suitable for fine variety reproduction. However， YT60CL， which was obtained by "onestep seedling formation"， had also passed the callus stage， but had not been subjected to subculture， resulting in less growth regulators and a short culture time. Therefore， the mutation rate was much lower than that of YT60CH， and even lower than those of CP65357AL， YT60AL and BadilaAL which were propagated from axillary buds （Table 3）. It can be seen that "onestep seedling formation" can be used for the breeding of good varieties， and even if the explants are axillary buds， the number of subculture generations should not be too high， and should not exceed 8 generations， otherwise the incidence of mutation will increase significantly. Because somatic clone mutations may occur at different levels （such as individual， organ， tissue， cell， and molecular levels）， their detection methods are also diverse， mainly including morphological observation， cytogenetics （or karyotype） analysis， the content of secondary metabolites （such as anthocyanins or flavonoids）， isoenzymes and molecular marker. The AFLP used in this study is only one of the molecular marker techniques， and therefore， certain types of somatic mutations such as chromosomal aberrations and changes in methylation status could not be detected. For example， the albino plantlets （CP65357AA） and oddshape plantlets （CP65357ASM and CP65357ABF） in this study were significantly different from normal plantlets in appearance， but the mutation rates were not high， even smaller than normal plantlets （Table 3）， which could not be interpreted by AFLP molecular markers alone. For what kind of mutation it is， or is it only a transient phenomenon and can it return to normal， further research is still needed. In production， if these situations are encountered， it is best to eliminate seedlings with abnormal shapes as soon as possible. The above analysis shows that to assess the genetic stability of sugarcane tissue culture clones， using only one molecular marker method is not comprehensiveand accurate. If possible， it is best to use "morphology + cytology + molecular biology" to perform comprehensive evaluation.
The molecular marker evaluation techniques developed in the past 20 years include RFLP， RAPD， SSR， AFLP， MSAP， etc. Early applications of RFLP and RAPD were more[2-5]， and they are fast， convenient， and economical. The AFLP molecular marker technique that was developed subsequently combines the advantages of RFLP and RAPD. It is convenient and fast， and also has the advantages of large amount of information， strong stability and more polymorphic information. It has been applied to the analysis and identification of many plant somatic clones. Although only the AFLP was used to analyze the genetic stability of sugarcane tissue culture clones in this study and variations at the cell level and morphological level could not be detected to a certain extent， but this study shows that AFLP is a very effective technique. Clones， that could not be discerned by naked eyes， could be classified one by one. In the case of morphological variations， it could also be known from which original plant the mutant plant had mutated. Under the condition of tissue culture， there were certain limitations in the clone variation， because there was no introduction of foreign genes， and even if the morphological variation was very obvious， like YT60CH， the genetic similarity coefficient with the original donor variety was still high， reaching 0.941 4. Studies have shown that the genetic similarity coefficients of different sugarcane varieties will not be so high[8-12]， and even from the same cross combination， such as "CP721210×Yacheng 82108"， the highest genetic similarity coefficient was only 0.839 8[8]. Therefore， when using AFLP molecular marker technique for sugarcane variety identification and analysis， if there are sufficient numbers of amplified bands， for example， 400 or more， it can be considered that the two are the same variety in the condition that the genetic similarity coefficient is above 0.950 0. References
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