甘蔗野生种滇蔗茅利用研究 Ⅲ.滇蔗茅杂种F1群体的表型变异与遗传多样性分析

选用8个数量性状和18个质量性状对滇蔗茅杂种F1群体的表型变异、多样性及聚类关系进行分析。结果表明：数量性状的遗传变异主要来自于有效茎、单茎重及糖分;质量性状的叶鞘背毛群与内叶耳形状多样性丰富,根点排列、芽位、茎形、节间性状、生长裂缝等5个性状均表现一致;聚类分析表明62份后代材料可分为4大类群和10个亚类群。遗传多样性和聚类分析为杂交后代的筛选及其杂交或回交利用提供了科学依据。     

滇蔗茅杂交F_1双抗SCSMV和SrMV鉴定与评价

以我国蔗区甘蔗花叶病的2种主要病原甘蔗条纹花叶病毒分离物(SCSMV-JP1,Gen Bank登录号JF488064)和高粱花叶病毒分离物(Sr MV-HH,Gen Bank登录号DQ530434)为接种毒源,采用人工切茎接种和RT-PCR检测相结合的方法,于2015-2016年2次对由热带种路打士与滇蔗茅云滇95-19杂交获得的41份滇蔗茅杂交F_1及亲本进行了双抗SCSMV和Sr MV鉴定与评价。结果表明,41份滇蔗茅杂交F_1及亲本中,对SCSMV表现1级高抗到3级中抗的有23份,占53.49%,4级感病到5级高感的有20份,占46.51%;对Sr MV表现1级高抗到3级中抗的有31份,占72.09%,4级感病到5级高感的有12份,占27.91%。综合分析结果显示,10份滇蔗茅杂交F_1对SCSMV和Sr MV均表现1~2级抗病,占23.26%,其中云09-604、云09-607、云09-619、云09-633、云09-656、云滇95-19等6份滇蔗茅杂交F_1对2种病毒均表现为1级高抗,占13.95%。研究结果明确了41份滇蔗茅杂交F_1及亲本对甘蔗花叶病2种主要致病病原的抗性,筛选出10份双抗SCSMV和Sr MV的滇蔗茅杂交F_1,为深入开展抗甘蔗花叶病育种提供了优良抗源种质和参考依据。     

甘蔗近缘种滇蔗茅(Erianthus rockii)表型性状遗传多样性研究

为有效评价和利用蔗茅种质资源,挖掘其优良性状,以滇、黔、川考察收集的29份蔗茅为材料,选取5个数量性状为指标,对其多样性指数、变异系数、数量性状之间的相关性、数量性状与经纬度、海拔的相关性进行研究。分析结果表明：(1)共采集到25份高海拔种质资源,其中海拔超过2800米有4份,进一步丰富了甘蔗野生种质资源库;(2)蔗茅种质资源数量性状的Shannon-Wiener多样性指数均较高,株高多样性指数最高(1.441),锤度多样性指数最低(1.291);数量性状遗传变异较丰富,各性状变异系数范围为21%～38%,变异系数最大的为株高(38%),最小的为叶长(21%);(3)蔗茅种质资源性状差异显著,叶长、叶宽、株高、茎径存在较大相关性,而与锤度不相关;叶宽与经度呈正相关,与纬度、海拔呈负相关。(4)聚类分析结果表明,蔗茅种质资源可分为四大类群,其中Ⅱ类群(EF-27)具有良好的数量性状和锤度品质潜力,可推荐作为甘蔗育种杂交利用首选材料。     

甘蔗-滇蔗茅杂交F_1花粉母细胞减数分裂过程GISH分析

研究甘蔗属与滇蔗茅属间远缘杂交F1染色体遗传行为具有重要科学意义,可为发掘利用滇蔗茅野生优异基因资源提供细胞学依据。本研究采用常规压片技术对可育父本及不育杂交F1花粉母细胞减数分裂过程进行比较观察,结果显示可育父本云南95-19减数分裂正常,而不育杂交F1分裂异常;进一步对F1花粉母细胞减数分裂过程进行基因组荧光原位杂交(GISH,genome in situ hybridization)分析,结果表明:滇蔗茅与甘蔗属热带种的亲缘关系较远,双亲染色体在F1细胞中不能进行同源配对,终变期,15条滇蔗茅染色体以单价体形式存在,花粉母细胞减数分裂细胞中存在滞后染色体、染色体丢失和不均衡分离现象。甘蔗-滇蔗茅属间远缘杂交F1花粉母细胞减数分裂异常因而杂交F1花粉完全败育。     

甘蔗近缘种滇蔗茅(Erianthus rockii)表型性状遗传多样性研究

选用6个数量性状和23个质量性状对滇蔗茅野生资源的表型变异、多样性及聚类关系进行分析。结果表明,数量性状变异系数在12.72%~22.38%之间,最大的是田间锤度,为22.38%,其次是茎径,为22.27%,最小的是叶长,为12.72%;质量性状的曝光后节间颜色多样性丰富,茎形、芽形、生长裂缝、节间形状、花序形状等5个性状均表现一致;聚类分析表明51份滇蔗茅无性系材料可分为4大类群和5个亚类群。遗传多样性和聚类分析为资源杂交利用和优异基因挖掘提供参考。     

甘蔗与斑茅割手密复合体杂交后代的分子标记鉴定

为有效利用甘蔗野生种质拓宽甘蔗遗传基础,本研究利用甘蔗与斑茅割手密复合体进行杂交,同时应用序列相关扩增多态性(SRAP)和微卫星(SSR)分子标记技术鉴定其后代。两种分子标记鉴定结果相互印证表明:3个杂交组合的后代中,经表型鉴定含斑茅血缘的34个后代为真杂种。该真杂种后代为进一步综合利用斑茅、割手密的优异基因改良甘蔗品种提供了优良的创新种质。     

甘蔗近缘种蔗茅Erianthus fulvus考察收集与表型性状初步研究

为有效评价和利用蔗茅种质资源,挖掘其优良性状,以滇、黔、川考察收集的29份蔗茅为材料,选取5个数量性状为指标,对其多样性指数、变异系数、数量性状之间的相关性,数量性状与经纬度、海拔的相关性进行研究。分析结果表明:(1)共采集到25份高海拔种质资源,其中海拔超过2800m的有4份,进一步丰富了甘蔗野生种质资源库;(2)蔗茅种质资源数量性状的Shannon-Wiener多样性指数均较高,其中株高的最高为1.441,锤度的最低为1.291;数量性状遗传变异较丰富,各性状变异系数范围为21%~38%,变异系数最大的为株高(38%),最小的为叶长(21%);(3)蔗茅种质资源性状差异显著,叶长、叶宽、株高、茎径存在较大相关性,而与锤度不相关;叶宽与经度呈正相关,与纬度、海拔呈负相关。(4)聚类分析结果表明,蔗茅种质资源可分为4大类群,其中第Ⅱ类群(EF-27)具有良好的数量性状和锤度品质潜力,可推荐作为甘蔗育种杂交利用首选材料。     

SSR分子标记鉴定山葡萄和河岸葡萄种间杂种

利用SSR分子标记技术,对山葡萄和河岸葡萄种间杂交后代的真伪性进行鉴别。从12对多态性SSR引物中筛选出能扩增出父本特异性条带的7对引物,作为杂种鉴定的标记。用这7对引物对239株山葡萄和河岸葡萄的杂交后代进行鉴定。结果表明,有161株后代具有父本的特异性条带,结合田间形态学分析,确认为真杂种。另外,后代中还出现了新的条带,表明杂交后代产生了丰富的变异。因此,SSR标记可以有效地对葡萄属种间杂交后代进行真实性鉴定,可作为葡萄种质创新的有效辅助手段。     

甘蔗与斑茅BC1分子鉴定、抗黑穗病和花叶病初步评价

为了解甘蔗(Saccharum)与斑茅(Erianthus arundinaceus)杂交后代作为抗病亲本的利用价值,通过特异引物PCR鉴定出78份甘蔗与斑茅杂交BC_1真实杂种;通过人工接种花叶病毒和黑穗病菌,初步评价了甘蔗和斑茅杂交BC_1的抗病表现。结果表明,甘蔗和斑茅杂交BC_1的抗花叶病具有普遍性,而黑穗病抗性则出现分离。初步筛选出BC_1无性系YCE01-48、YCE01-71、YCE01-105、YCE01-125、YCE02-184和YCE01-118可同时抗花叶病和黑穗病,有望成为甘蔗杂交利用的高抗病源亲本。     

斑茅割手密复合体(GXAS07-6-1)及其与甘蔗F_1的GISH分析

斑茅割手密复合体(GXAS07-6-1)是广西蔗茅属斑茅和广西甘蔗属割手密的属间杂种,聚集了双亲的优点。本研究利用基于Alu-like的PCR鉴定方法对GXAS07-6-1及甘蔗与GXAS07-6-1的3份F_1材料进行真实性鉴定,基于基因组原位杂交技术(GISH)对父本GXAS07-6-1及其3份F_1染色体组成及核型进行分析。研究结果表明:3份F_1材料为GXAS07-6-1的真杂种;父本GXAS07-6-1的染色体众数为62条,其中30条来自蔗茅属斑茅,32条来自甘蔗属割手密,核型分类属于1B,其染色体按"n+n"方式传递;GXASF_108-2-17、GXASF_108-2-22、GXASF_108-2-32的染色体数目为78~80条,其中69~71条来自甘蔗属,9~11条来自蔗茅属斑茅,3份F_1的核型分类分别属于2B、1B、1B,染色体传递方式均为"n+n"。父本GXAS07-6-1及3份F_1材料中均未发现有染色体的交换或易位现象。甘蔗与斑割复合体杂交,蔗茅属斑茅染色体在亲子间传递过程存在丢失现象。     

Molecular contribution to selection of intergeneric hybrids between sugarcane and the wild species Erianthus arundinaceus.

Erianthus arundinaceus has great potential as a germplasm source for better ratoonability, vigour, tolerance to environmental stresses, and disease resistance in sugarcane. Many unsuccessful attempts have been made to introduce these characters into modern sugarcane cultivars. We report on significant progress made since molecular tools were implemented. Sequence-tagged PCR, revealing size variation in the 5S rDNA cluster, was performed on intact leaf tissue to identify genuine hybrids six weeks after germination. This early screening of seedlings avoids the loss of genuine hybrids due to competition with selfed progeny. Of 96 crosses made involving female Saccharum officinarum or sugarcane cultivars (Saccharum spp.) and male E. arundinaceus, 26 were fertile producing 1328 seedlings. Thirty-seven genuine hybrids were unequivocally identified but only 19 have survived. Genuine hybrids were produced from only three crosses, all involving S. officinarum as the female parent. Chromosome elimination was observed in all seven hybrids analyzed using genomic in situ hybridization (GISH). Very little cross-hybridization was observed between the genomes of the two species after GISH, confirming recent molecular studies which showed that E. arundinaceus is quite distant from the genus Saccharum. The major limitation in the introgression of E. arundinaceus resides now in the apparent sterility of the hybrids.     

Evaluation of maize microsatellite markers for genetic diversity analysis and fingerprinting in sugarcane.

The use of maize microsatellite markers as a potential cost-effective method for molecular analysis of sugarcane was evaluated. Of the 34 primer pairs obtained from maize genomic libraries, 14 showed repeatable amplifications in Saccharum species clones, commercial hybrids, and the related genera Erianthus, accounting for 41.17% cross transferability. Complex banding patterns were encountered in sugarcane with the number of amplified fragments ranging from 7 to 14 with an average of 10 per primer, indicating the high polyploidy and heterozygosity existing in sugarcane. Phenetic analysis of the SSR polymorphisms produced by nine primers could clearly differentiate the different species of Saccharum and Erianthus and revealed the relationships that existed between them. Genetic similarity co-efficient indicated low diversity existing among the S. officinarum clones (82%) and a relatively higher level of diversity in the S. spontaneum clones (69.7%). Higher level of divergence of Erianthus from Saccharum was also clearly estabilished. Five primers produced genus- and species-specific fragments for Erianthus, S. spontaneum, S. officinarum, and S. barberi. The polymorphic primers, when tested on a panel of 30 commercial sugarcane cultivars, revealed a broad range (32.4-83.3%) of pair-wise similarity values, indicating their ability to detect high levels of polymorphism. A combination of two primers could differentiate all the varieties, further emphasizing their potential in fingerprinting and varietal identification.     

Preliminary Report of RAPD Analysis in Paeonia suffruticosa Subsp. spontanea and Paeonia rockii

Ten different oligonucleotide primers of arbitrary sequence were used in Capillary and Air Thermocycler to amplify genomic total DNA of Paeonia suffruticosa subsp. spontanea and P. rockii isolated from several local populations in Shanxi, Shaanxi and Gansu provinces. Under the strictly standardized amplification condition for all the primers, these primers yielded clear and reproducible bands corresponding to amplified products and separable by agarose gel electrophoresis. Among a total of 71 bands amplified, 16(22. 5%) were polymorphic in a single individual of P. suffruticosa subsp. spontanea, while among a total of 76 bands 21(27.6%)were polymophic in a single individual of P. rockii. On an average,the pairwise marker difference between band profiles of conspecific individuals (different populations) was 7.9 for P. suffruticosa subsp. spontanea and 8.7 for P. rockii respectively. The average marker difference between P. suffruticosa subsp. spontanea and P. rockii was 10.3. Obviously, greater number of plants and primers will be required to detect satisfactorily level of genetic diversity. These preliminary results showed that intraspecific genetic diversity was low for the two endangered species. RAPD as a molecular marker was useful and feasible for detecting the genetic variation within species of wild moutans. And it was also potential for studying evolution and relationships between species.