火龙果组培苗体细胞无性系变异及其分子检测

Somaclonal variation of pitaya(Hylocereus undatus) in vitro plants and identification by molecular markers

【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-1 6-benzyladenine （6-BA）, successively in vitro shoots were subcuhured for four cycles. Sixty-nine plantlets subcuhured for 1 and 4 cycles as well as their stock plant which primarily derived from a seed were used to analyze the morphological 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 subcuhured 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 me3em3. A total of 405 bands were scored from the 70 samples by 24 ISSR prim- ers, 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 （me9em2 and nle3em3）; 12 plants and 9 polymorphie loci were detected by the IRAP primers 15f, 17r and 4r. The genetic similarity coeffi- cient among the 16 mutants and their stock plant varied from 0.77 to 0.97 based on statistical analysis of genetic diversity using NTSYSpc 2.10e software. Using unweighted pair group mathematics average （UPG- MA） 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 muhiplication, 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 me9em2. [ 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.