摘要
麻疯树(Jatropha curcas L.)为大戟科麻疯树属小乔木或大灌木,起源于墨西哥和中美洲,现广泛分布于热带和亚热带地区；麻疯树易扦插繁殖,种子籽油可以制备生物柴油,为一种高效可再生的生物能源树种。
本试验首次对不同国家种子特性、种苗生长；家系、无性系田间生长指标；AFLP分子标记3个方面阐述了麻疯树遗传变异及多样性情况,并对麻疯树EST-SSR引物开发进行了初步研究。研究结果阐明了不同种源麻疯树的亲缘关系及引种可能性,为培育稳产、高产、含油量高的优良品种提供了理论依据。主要结果如下：
1.本试验对来自8个国家40个地区的种子特性及苗期生长状况进行了检测,种子种仁千粒重在种源间差异显著；印度种源的种子、种仁千粒重较轻,中国种子与平均值相当,马里种源种子、种仁千粒重值较大；印度种源和中国种源的种子与其他国家相比均比较小；种子、种仁平均含油量为29.65%,48.45%；种子、种仁含油量变幅为14.85—40.17%,34.02—61.39%；2009年8月在越南林科院苗圃育苗试验结果调查统计发现,种子出苗率变幅为1.7—90.80%,2个月后平均株高、地径分别为22.69cm、0.80 cm；3个月后为25.73 cm、1.09 cm；种子长、宽、重及含油量、种苗发芽率、生长量方面不同种源间均存在变异,经综合考虑得到10个种源可应用在以后的育种中。
2.从形态学上调查了海南麻疯树试验基地2年生195个家系及1年37个无性系的田间生长指标。家系平均成活率为93.65%、树高1.97 m、地径6.85 cm、每棵树结果枝数1.38个,结果数量为20个；家系成活率在家系间差异显著而在省级水平上差异不显著,树高、地径、结果枝数及结果等级在省及县(市)水平差异极显著而在家系间差异不显著。无性系成活率为84%,平均株高为101.48 cm,平均一级枝量2.77,二级枝量1.67,平均结果量16.5个；方差结果显示树高在无性系及县级水平上差异不显著,存活率及第二分枝数在无性系和县级水平差异均显著,第一分枝数及每棵树的结果总量在各无性系间差异显著并均达到极显著水平,但在县级水平差异不显著。
由于麻疯树分布区自然条件差异较大,来自不同省份、县(市)的麻疯树家系、无性系在生长指标特征上存在显著差异,不同区域分布的麻疯树遗传基础存在差异,具有丰富的遗传变异。
3.利用9对AFLP引物组合中国6省37个种源及印尼1个种源进行了分子标记分析,9对引物扩增出246条条带,其中多态性条带为72条,多态性比率为26.99%。评价引物的PIC,MI和RP三个属性均能很好的代表引物的区分能力,且均与遗传多样百分率呈很强的正相关(r2>0.9)。
Jaccard相似系数介于0.866和0.977之间,表明这38个种源的遗传多样性很低。非加权聚类平均法UPGMA及主成份分析聚类结果与地理种源的地理位置没有很大的相关性。中国麻疯树种源相对较低的遗传多样性暗示在以后的育种工作中应当引进原产地种源以扩大遗传基础。
4.采用4对荧光AFLP的方法对10个国家不同地区的64个种源310个个体进行遗传多样性分析,4对引物共扩增出89条清晰的多态性条带,平均多态性百分率(Pp)为18.31%,平均每位点等位基因数(Na)为1.183,平均有效等位基因数(Ne)为1.117,平均Nei's基因多样性指数(H)为0.070,平均香农指数(I)为0.103,群体间的遗传分化系数Gst为0.5057,麻疯树种源间的遗传变异占总变异的50.57%,稍大于种源内变异,基因流Nm为0.473。
10个国家中Pp,Na,Ne,H和I变化趋势相同,墨西哥国家麻疯树的遗传多样性最丰富,其次为越南、印度、泰国、巴布亚新几内亚、中国、菲律宾、马里、印度尼西亚,遗传多样性最低的为老挝。
麻疯树种源间的遗传距离介于0.002—0.308之间；以Nei's(1973)遗传距离D为参数对麻疯树64个种源进行UPGMA聚类,64个种源可划分为4个组,第一组为混合亚洲组,第二组为印度组,第三组为中国种源组,第四组为墨西哥组；另外海南的一个种源HN7单独据为一类；由此可以推断聚为同一类的种源可能由同一原产地引种而来,聚类结果与地理区划有一定的相关性,但是在每一个国家内聚类与地理位置没有相关性。
5.从NCBI上已经公布的13,201条麻疯树EST中得到了37个SSR-ESTs；在这37个序列中,二核苷酸重复单元占59.46%,GA、GT各占28.205%；三核苷酸重复单元占40.54%,AAG重复单元最多,占17.949%；SSR长度在18—45bp之间,平均长度27.69 bp；设计的37对EST-SSR引物,28对引物可在麻疯树材料中能够扩增出目标片段,其中7对引物具有多态性,扩出的等位位点数从2—14个不等。
该研究结果揭示了不同种源在种子特性、田间生长指标方面存在差异,通过选择可以获得高产、稳产、含油量高的品种；同时试验结果进一步确定了中国麻疯树种源的遗传多样性低,在以后的育种中应引进更多的不同基因型的材料,扩大麻疯树的遗传基础。
Jatropha curcas L. belonging to the family Euphorbiaceae is a deciduous, multipurpose shrub or small tree up to 6 m tall. Its natural distribution range is centre in Mexico and Central America. It has been extensively cultivated outside its natural range, and become naturalized throughout the tropical and subtropical regions due to the ease of propagation either by seed or cuttings. The species is a highly effective, renewable bio-energy tree species because the seed can be extracted for bio-diesel oil.
The main objective of this study was to determine the genetic diversity of J. curcas using genetic material deriverd from sources within China and many other countries. Field growth indices:seed morphology, branching habit, fruit yield, seed oil content and molecular diversity were determined. The results showed the relationship between J. curcas populations and suggested the probable sources of original introductions into China. The results also provided a basis for genetic improvement by selection of superior genotypes in terms of growth, high seed yield and high oil content. Main results obtained from this study are described as follows.
1. J. curcas seeds from 8 countries 40 sources were included in the study. The 1000-seed weightand 1000-seed kernel weight were quite different between populations. The 1000-seed weight,1000-kernel weight, seed length, seed width, seed oil content, kernel oil content and seed germination and seedling growth were determined. The results showed that seeds from Mali were the heaviest and those from India were generally the lightest; Seed weight of the Chinese sources was around the overall mean. In term of seed size, Indian and Chinese seeds were smaller than other countries. The range of seed oil content among the 40 sources was 14.85-40.17%, while the kernel oil content was 34.02-61.39%. Seed germination varied form 1.7% to 90.80%. Mean diameter and height of three-month-old seedlings were 25.73 cm and 1.09 cm respectively. The highest oil content was recorded for seed from Mali. Overall, there were marked variations in seed length, seed width, seed weight and oil content. At last 10 populations with high oil content could be used for further research.
2. The field growth indices at 2 years age of 195 families of China and at 1 year old of 37 clones were assessed. Analysis results revealed that survival differed significantly at family level but not at province level, while height, diameter at ground level, number of fruiting branches and fruit yield varied at province and county levels but not at family level. The family trial results showed that overall mean survival, height, diameter at ground level, number of fruiting branches and fruit yield were 93.65%,1.97 m,6.85 cm,1.38 per tree and 20 per tree respectively. Clone trial results showed that mean survival, height, first order branch (FOB), second order branch (SOB) and fruit yield were 84%,101.48 cm, 2.77,1.67 and 16.5 respectively. Analysis of variance indicated that there were no differences in height at county and clone levels. Survival and SOB differed at county and clone levels. FOB and fruit yield varied at clone level but did not differ at county level.
The results described above show that families and clones of J. curcas from different counties and provinces performed differently in many growth characteristics. Thus, genetic variations in J. curcas exist and it is possible to make selection for superior genotypes.
3. Amplified fragment length polymorphism (AFLP) was used to determine the genetic diversity among 37 Chinese populations and 1 Indonesia of J. curcas grown in a seed source trial in Hainan. Nine AFLP primer combinations were used to generate a total of 246 fragments, of which 72 (26.99%) were polymorphic. Three marker attributes: polymorphism information content (PIC), marker index (MI) and Resolving power (RP), were all found to be reliable to determine the discriminatory power of the nine primer combinations.
The Jaccard's similarity coefficient showed a high similarity range from 0.866 to 0.977, suggesting a low genetic diversity among the 38 populations. The UPGMA-based dendrogram and biplot of a principal component analysis did not reveal a clear pattern of groupings by geographic locations of the seed sources; populations from across different provinces were mixing in the same groups. The low genetic diversity and a lack of variation pattern among the populations in China suggest that it is necessary to import new germplasm preferably from the species'natural distribution range to broaden the genetic base for improvement program.
4. Four selected AFLP fluorescence primer combinations were employed to test the genetic diversity of 310 individuals from 64 populations from ten countries. Eighty nine AFLP markers were observed. The genetic diversity parameters at species level was PP= 18.31%, Na= 1.183, Ne= 1.117, H= 0.070, I= 0.103. The genetic differentiation coefficient was 0.5057 which indicated that 50.57% variability occurred between populations. The gene flow was 0.473.
All the genetic diversity parameters Pp, Na, Ne, H and I had similar tendency in these ten countries. Mexico had the highest genetic diversity followed by Vietnam, India, Thailand, Papua New Guinea, China, Philippines, Mali and Indonesia.
The genetic distance of these 64 populations varied form 0.002 to 0.308. The UPGMA dendrogram was built according to Nei's genetic distance. The 64 populations were clustered into 4 main groups, i.e. mixed Asian group, India group, China group and Mexico group. The populations clustered in the same group are likely to be introduced from the same original population. The cluster result had some relationship with geographic distribution, but within each group there was no relationship with geographic distribution.
5. Thirty seven SSR-ESTs were obtained from 13,201 EST sequences. Among these 37 SSR-ESTs, dinucleotide repeat was more dominant motif than trinucleotide repeat. For dinucleotide repeat, GA and GT were the most abundant motif. As for trinucleotide, AAG was dominant. The length of SSR repeat was between 18 bp and 45 bp, the mean length was 27.69 bp. At last 37 EST-SSR primers were gained, of which 28 worked for J. curcas, and 7 primers were polymorphic. The amplified loci ranged from 2 to 14.
This study showed variations in many growth characteristics of J. curcas from a wide range of seed sources through seed and field growth assessment. These results indicate that it is feasible to select genotypes which possess fast growth and high seed oil contents. Molecular analysis confirmed low genetic diversity among Chinese populations, suggesting the need to introduce more genetic material to broaden the genetic base in China.
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