火龙果组培苗体细胞无性系变异及其分子检测
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摘要
【目的】了解火龙果离体快繁体细胞无性系的遗传稳定性,并揭示变异系间的遗传关系。【方法】以单粒种子萌发的丛芽扩繁第1代和第4代再生幼苗为材料,分析繁殖系数及棱的形态变化,并采用ISSR、SRAP和IRAP标记技术进行遗传变异分析。【结果】繁殖系数为14.9。快繁第4代试管苗中有3棱、4棱等6种形态学变异,其中5棱丛芽最多(50.54%),8棱丛芽最少(1%左右);3棱和4棱丛芽在RNA水平上(SRAP标记)表现出差异。24条ISSR引物、11对SRAP引物和17条IRAP引物在70个样品中共产生405条带,其中多态性带29条。扩繁第1代植株未发生DNA水平的变异,第4代植株的DNA条带出现了增加或缺失现象,植株变异频率为24.2%。16株变异株与原始植株(种子萌发植株)的遗传相似系数为0.77~0.97。在相似系数为0.90时,可将16个变异株分为4类,其中第Ⅰ类包括原始植株和12个变异株,第Ⅳ类与原始植株的差异最大,是GA3缺陷型矮化突变体。采用IRAP、SRAP、ISSR标记检测出的变异植株数量及多态性位点均存在差异。【结论】建立的火龙果快繁体系的繁殖系数为14.9,随快繁次数的增加,繁殖系数、生长势及遗传稳定性有下降的趋势。快繁第4代的体细胞无性系变异频率为24.2%,主要表现在DNA水平和棱茎形态上的变化。获得的16个变异株与原始植株的遗传相似系数为0.77~0.97,被分为4类,第Ⅳ类是GA3缺陷型矮化突变体。3棱和4棱丛芽差异可能是基因差异表达的结果。新开发的IRAP标记是检测火龙果无性系变异的有效手段。
【Objective】Pitaya production has become a new, special and excellent agricultural project.Usually, propagation of pitaya is conducted by using cuttings from field plants. However, multiplication rates are low and it is difficult to obtain enough true-to-type plants for both plantation and research use.Tissue culture is the main means of rapid propagation of plants, and also a good method to obtain somaclonal variation. Currently, the shoots derived from a clone were used as the explants to study the rapid propagation in vitro. After rapid propagation of several cycles, the plants growth and morphological traits were different among some regenerated plants. To better apply the rapid propagation system and make further use of the variants, it is necessary to evaluate the genetic fidelity of the in vitro plants derived from the current rapid propagation system as well as to reveal the genetic relationship among the variants.【Methods】Pitaya shoots from single seed germination as explants were multiplied on Murashige and Skoog(MS) medium with 0.1 mmol·L-1 naphthalene acetic acid(NAA) and 2 mmol·L-16-benzyladenine(6-BA), successively in vitro shoots were subcultured for four cycles. Sixty-nine plantlets subcultured for1 and 4 cycles as well as their stock plant which primarily derived from a seed were used to analyze themorphological variations as well as propagation coefficient, and the genetic fidelity was identified by ISSR, SRAP and IRAP markers. NTSYS 2.01 software was further used to illustrate the variation plants.【Results】The propagation coefficient of the rapid micropropagation system was 14.9. The 4 derived shoots regenerated from a seedling characterized in 4 arris. Six arris types, i.e. 3-, 4-, 5-, 6-, 7-and 8-arris, of cluster buds were investigated in the regenerated plants successively subcultured for 4 cycles, among which 5-arris buds were the most common, accounting for about 50.54% of the total, followed by the 4-arris buds which accounted for 27.2% of the total, and the 8-arris buds was the least, only about 1.0%. With the increase of arris number, the shoot diameter became larger, especially for 6-, 7-, and 8-arris shoot,and the opposite tend was observed in shoot length. No difference in DNA markers obtained from 11 pairs of SRAP primers was investigated between the 3 and 4 arris buds, however an aberrant band was detected in RNA level by primer me3 em3. A total of 405 bands were scored from the 70 samples by 24 ISSR primers, 11 pairs of SRAP primers and 17 IRAP primers, among which 28 were polymorphic, which included presence and absence of DNA bands in comparison with the stock plant. No polymorphic band was observed from the three plants which micropropagated in the first cycle, however 16 plants were detected as the variants among the 66 plants which subcultured 4 cycles, accounting for 24.2% of the total. Among the variants, 6 plants and 5 polymorphic loci were detected by the ISSR primers 811, 825 and 856; 9 plants and 14 polymorphic loci were detected by two pair of SRAP primers(me9 em2 and me3 em3); 12 plants and 9 polymorphie loci were detected by the IRAP primers 15 f, 17 r and 4 r. The genetic similarity coefficient among the 16 mutants and their stock plant varied from 0.77 to 0.97 based on statistical analysis of genetic diversity using NTSYSpc 2.10 e software. Using unweighted pair group mathematics average(UPGMA) analysis, 16 mutants might be divided into 4 groups taking the similarity coefficient 0.90 as the threshold. Group I included the stock plant and 12 mutants, the genetic similarity coefficient in the range of 0.91-0.97; mutants 7 and 10 were grouped into group II; mutants 41 and 33 were singly clustered into group III and IV, respectively. Mutant 33 showed the least genetic relatedness with the stock plant. After further micropropagation for 3 cycles, mutant 33 appeared no growth in height and no multiplication, and the regeneration plants were detected as the GA3-deficient dwarf mutant. The normal plant and 3 GA3-mutants could be distinguished by a pairs of SRAP primers me9 em2.【Conclusion】The propagation coefficient of the current micropropagation system in vitro was 14.9. However, the propagation coefficient,growth potential and genetic stability of somaclones demonstrated a decrease trend with increase of subculture cycles. Morphologically, the change of the shoot arris numbers, shoot length and diameter were also investigated herein. Further, the somaclonal variation rate after successively subculture for 4 cycles accounted for 24.2% of the total as revealed by molecular markers, and the variations mainly showed on present or absent amplified bands. The 16 mutants were divided into 4 groups, 12 of which and the stock plants were closely related, and the genetic similarity coefficient ranged from 0.91 to 0.97. Mutant 33 was a GA3-deficient dwarf mutant. The difference of 3-and 4-arris shoots might be ascribed to the differential expression of genes. The newly developed IRAP marker was more sensitive than ISSR and SRAP markers to detect the DNA mutation in different loci of the genome, therefore was an effective means for somaclonal variation detection in pitaya. The obtained results facilitated the rapid propagation, germplasm conservation and creation, as well as mutant detection in this fruit.
【Objective】Pitaya production has become a new, special and excellent agricultural project.Usually, propagation of pitaya is conducted by using cuttings from field plants. However, multiplication rates are low and it is difficult to obtain enough true-to-type plants for both plantation and research use.Tissue culture is the main means of rapid propagation of plants, and also a good method to obtain somaclonal variation. Currently, the shoots derived from a clone were used as the explants to study the rapid propagation in vitro. After rapid propagation of several cycles, the plants growth and morphological traits were different among some regenerated plants. To better apply the rapid propagation system and make further use of the variants, it is necessary to evaluate the genetic fidelity of the in vitro plants derived from the current rapid propagation system as well as to reveal the genetic relationship among the variants.【Methods】Pitaya shoots from single seed germination as explants were multiplied on Murashige and Skoog(MS) medium with 0.1 mmol·L-1 naphthalene acetic acid(NAA) and 2 mmol·L-16-benzyladenine(6-BA), successively in vitro shoots were subcultured for four cycles. Sixty-nine plantlets subcultured for1 and 4 cycles as well as their stock plant which primarily derived from a seed were used to analyze themorphological variations as well as propagation coefficient, and the genetic fidelity was identified by ISSR, SRAP and IRAP markers. NTSYS 2.01 software was further used to illustrate the variation plants.【Results】The propagation coefficient of the rapid micropropagation system was 14.9. The 4 derived shoots regenerated from a seedling characterized in 4 arris. Six arris types, i.e. 3-, 4-, 5-, 6-, 7-and 8-arris, of cluster buds were investigated in the regenerated plants successively subcultured for 4 cycles, among which 5-arris buds were the most common, accounting for about 50.54% of the total, followed by the 4-arris buds which accounted for 27.2% of the total, and the 8-arris buds was the least, only about 1.0%. With the increase of arris number, the shoot diameter became larger, especially for 6-, 7-, and 8-arris shoot,and the opposite tend was observed in shoot length. No difference in DNA markers obtained from 11 pairs of SRAP primers was investigated between the 3 and 4 arris buds, however an aberrant band was detected in RNA level by primer me3 em3. A total of 405 bands were scored from the 70 samples by 24 ISSR primers, 11 pairs of SRAP primers and 17 IRAP primers, among which 28 were polymorphic, which included presence and absence of DNA bands in comparison with the stock plant. No polymorphic band was observed from the three plants which micropropagated in the first cycle, however 16 plants were detected as the variants among the 66 plants which subcultured 4 cycles, accounting for 24.2% of the total. Among the variants, 6 plants and 5 polymorphic loci were detected by the ISSR primers 811, 825 and 856; 9 plants and 14 polymorphic loci were detected by two pair of SRAP primers(me9 em2 and me3 em3); 12 plants and 9 polymorphie loci were detected by the IRAP primers 15 f, 17 r and 4 r. The genetic similarity coefficient among the 16 mutants and their stock plant varied from 0.77 to 0.97 based on statistical analysis of genetic diversity using NTSYSpc 2.10 e software. Using unweighted pair group mathematics average(UPGMA) analysis, 16 mutants might be divided into 4 groups taking the similarity coefficient 0.90 as the threshold. Group I included the stock plant and 12 mutants, the genetic similarity coefficient in the range of 0.91-0.97; mutants 7 and 10 were grouped into group II; mutants 41 and 33 were singly clustered into group III and IV, respectively. Mutant 33 showed the least genetic relatedness with the stock plant. After further micropropagation for 3 cycles, mutant 33 appeared no growth in height and no multiplication, and the regeneration plants were detected as the GA3-deficient dwarf mutant. The normal plant and 3 GA3-mutants could be distinguished by a pairs of SRAP primers me9 em2.【Conclusion】The propagation coefficient of the current micropropagation system in vitro was 14.9. However, the propagation coefficient,growth potential and genetic stability of somaclones demonstrated a decrease trend with increase of subculture cycles. Morphologically, the change of the shoot arris numbers, shoot length and diameter were also investigated herein. Further, the somaclonal variation rate after successively subculture for 4 cycles accounted for 24.2% of the total as revealed by molecular markers, and the variations mainly showed on present or absent amplified bands. The 16 mutants were divided into 4 groups, 12 of which and the stock plants were closely related, and the genetic similarity coefficient ranged from 0.91 to 0.97. Mutant 33 was a GA3-deficient dwarf mutant. The difference of 3-and 4-arris shoots might be ascribed to the differential expression of genes. The newly developed IRAP marker was more sensitive than ISSR and SRAP markers to detect the DNA mutation in different loci of the genome, therefore was an effective means for somaclonal variation detection in pitaya. The obtained results facilitated the rapid propagation, germplasm conservation and creation, as well as mutant detection in this fruit.
引文
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[3]EVANS D A,SHARP W R,MEDINA-FILHO H P.Somaclonal and gametoclonal variation[J].American Journal of Botany,1984,71:759-774.
[4]SALES E K,BUTARDO N G.Molecular analysis of somaclonal variation in tissue culture derived bananas using MSAP and SSRmarkers[J].International Journal of Biological,2014,8(6):615-622.
[5]邹雪,肖乔露,文安东,胡芳,黄雪丽,王西瑶.通过体细胞无性系变异获得马铃薯优良新材料[J].园艺学报,2015,42(3):480-488.ZOU Xue,XIAO Qiaolu,WEN Andong,HU Fang,HUANG Xueli,WANG Xiyao.An excellent new potato material obtained by somaclonal variation[J].Acta Horticulturae Sinica,2015,42(3):480-488.
[6]胡盼盼,赵霞,李刚,李亮杰,秦豹,彭卓,周厚成.草莓组培苗体细胞无性系变异研究[J].中国农学通报,2016,32(16):77-82.HU Panpan,ZHAO Xia,LI Gang,LI Liangjie,QIN Bao,PENGZhuo,ZHOU Houcheng.Strawberry plantlets somaclonal variation[J].Chinese Agricultural Science Bulletin,2016,32(16):77-82.
[7]ZENG X H,WEN J,YI B,SHEN J,MA C,TU J,FU T.Effects of bleomycin on microspore embryogenesis in brassicanapus and detection of somaclonal variation using AFLP molecular markers[J].Plant Cell,Tissue and Organ Culture,2010,101(1):23-29.
[8]巩檑,杨亚琚,周坚.中国石蒜组培苗体细胞无性系变异的ISSR和SRAP检测[J].北方园艺,2011(22):108-112.GONG Lei,YANG Yaju,ZHOU Jian.Detection of somaclonal variation in micropropagated plants of lycoris chinensis by ISSRand SRAP markers[J].Northern Horticulture,2011(22):108-112.
[9]FAN Q J,ZHENG S C,YAN F X,ZHANG B X,QIAO G,WEN XP.Efficient regeneration of dragon fruit(Hylocereus undatus)and an assessment of the genetic fidelity of in vitro-derived plants using ISSR markers[J].Journal of Horticultural Science&Biotechnology,2013,88(5):631-637.
[10]CAMPBELL B C,LE M S,PIPERIDIS G,GODWIN I D.IRAP,a retrotransposon-based marker system for the detection of somaclonal variation in barley[J].Molecular Breeding,2011,27(2):193-206.
[11]KALENDAR R,GROB T,REGINA M,SUONIEMI A,SCHUL-MAN A H.IRAP and REMAP:two new retrotransposon-based DNA fingerprinting techniques[J].Theoretical and Applied Genetics,1999,98(5):704-711.
[12]刘洪章,刘艳军,苗博瑛.火龙果组培增殖快繁技术研究[J].天津农业科学,2012,18(5):32-34.LIU Hongzhang,LIU Yanjun,MIAO Boying.Tissue culture and rapid propagation of Hylocereus undatus[J].Tianjin Agricultural Sciences,2012,18(5):32-34.
[13]彭绿春,瞿素萍,苏艳,张艺萍,王丽花.火龙果两步成苗组培快繁技术研究[J].西南农业学报,2014,27(6):2529-2533.PENG Lüchun,QU Suping,SU Yan,ZHANG Yiping,WANG Lihua.Study on rapid propagation of Hylocereus undatus by two steps[J].Southwest China Journal of Agricultural Sciences,2014,27(6):2529-2533.
[14]张领,赵佐敏,陈红艳,张毅,李怀情.火龙果离体快繁与组培苗移栽驯化技术[J].贵州农业科学,2016,44(2):20-23.ZHANG Ling,ZHAO Zuomin,CHEN Hongyan,ZHANG Yi,LIHuaiqing.Rapid propagation in vitro and domestication technique of tisssue culture plantlets in pitaya[J].Guizhou Agricultural Sciences,2016,44(2):20-23.
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