甘蔗、斑茅及其杂种的过氧化物酶同工酶

甘蔗属(Saccharum L.)主要包括热带种(S.officinarum L.)、中国种(S.sinensisRoxb)、印度种(S.barberi Jeswiet)、“割手密”种(S.spontaneum L.)和大茎野生种(S.robustum Brandes)。甘蔗属的近缘植物——斑茅(Arundinaceum Retz)应该划归甘蔗属、蔗茅属(Erianthus Michx)或另立一属,意见不一。Dutt 等将斑茅列入蔗茅属,耿以礼等则将其划归甘蔗属。甘蔗糖业栽培的甘蔗(糖蔗)是热带种与割手密种、大     

29份云南甘蔗创新种质的SSR遗传多样性分析和指纹图谱构建

该研究以16份甘蔗骨干亲本为参照,对29份云南甘蔗创新种质进行SSR指纹图谱构建和遗传多样性分析,以明确创新种质与16份亲本间的遗传基础和多样性水平。结果表明:6对引物共扩增出104条带,其中101条为多态性条带,多态性条带比例为97.25%;45份材料的遗传相似性系数为0.235 3~0.891 3,平均值为0.563 3;其中16份甘蔗骨干亲本的遗传相似性系数为0.301 6~0.755 6,甘蔗创新种质与甘蔗骨干亲本的特异条带比例为14∶1,涵盖了割手密、大茎野生种、斑茅和滇蔗茅等基因源。根据骨干亲本间的相似性系数范围,在相似性系数为0.43处,可将种质分为6大类群,亲缘关系相对较远,适宜作为种质间的杂交利用。通过引物区分效率分析,6对引物扩增的多态信息量为0.967 9~0.975 8,其中MSSCIR21引物区分效率最高,利用MSSCIR21和SMC1047HA引物组合构建了云南甘蔗创新种质标准指纹图谱,在相似性系数为0.85处即可区分所有种质,图谱的鉴别准确率为100%,每份资源都有唯一的指纹图谱,可将29份创新种质和16份骨干亲本区分鉴别出来。该研究能够为后续杂交利用、种质鉴定和知识产权保护提供依据。     

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。     

利用SSR标记与毛细管电泳对甘蔗属进行的遗传分析

为探讨甘蔗属内不同种之间的遗传多样性,利用SSR标记与毛细管电泳技术,对来自甘蔗属3个不同种的12个材料19对引物进行检测,共检测到229个DNA多态性条带,19对引物扩增的DNA条带范围集中在100～260bp之间。12个甘蔗材料的Jaccard遗传相似度,最小0.09,最大0.65,平均为0.26。通过遗传相似性系数分析,UPGMA聚类图内12个甘蔗材料可分为两个群,三个割手密种材料分为一个亚群,甘蔗栽培品种与甘蔗热带种合为一个亚群。结果表明:热带种比割手密种具有和甘蔗栽培品种更亲近的遗传关系;SSR分子标记与毛细管技术结合,相比别的分子标记技术或电泳技术,具有更准确、简便、自动化等优点。     

中国十倍体割手密资源抗逆功能标记的遗传多样性分析

整倍体割手密是现代甘蔗品种重要的野生亲本和抗逆性状基因源,为了明确其抗逆相关基因的遗传背景,该研究选用来自脱水绑定因子DREB、水通道蛋白AQP、热激蛋白HSP70、WRKY1转录因子和随机引物组成的5对抗旱和耐高温相关功能标记,以63份甘蔗常用亲本材料和8份甘蔗原种作为对照,对中国保育的50份十倍体割手密(Saccharum spontaneum L.)进行遗传多样性分析。结果显示:(1)5对引物共获得119个扩增片段,其中110个为多态性条带,平均多态性条带比例为92.44%,平均多态信息量为92.53%,十倍体割手密抗旱功能标记多态性最高,甘蔗常用亲本材料耐高温功能标记多态性最高。(2)十倍体割手密在抗旱功能标记上具有较低的平均相似性系数值(0.541)和最大的分布范围(0.347~0.800),且主要集中在0.400~0.600之间,而在耐高温功能标记上甘蔗常用亲本材料虽拥有最大的分布范围(0.238~1.000),但甘蔗原种具有最低的平均相似性系数值(0.481),且相似性系数范围主要集中在0.400~0.600之间。(3)UPGMA聚类可将参试材料划分为较明显的两个大组,十倍体割手密与甘蔗常用亲本和甘蔗原种在聚类关系上表现出明显的不同。研究表明,十倍体割手密资源无论是在抗旱功能标记DBF、Aqua方面,还是在耐高温功能标记SCB174、SCB190方面都与甘蔗常用亲本和甘蔗原种具有较大的遗传差异,而且在抗旱功能标记方面表现出更为丰富的遗传变异,因此在后续甘蔗品种抗逆性状改良和亲本创新上应进一步加大十倍体割手密资源的利用。     

斑茅割手密复合体杂交利用过程野生特异基因遗传分析

揭示斑茅割手密复合体在杂交利用过程中的斑茅、割手密野生特异基因在各世代的遗传规律,为利用斑割复合体创制甘蔗育种新亲本提供理论依据。利用AFLP-PCR分子标记结合毛细管电泳技术对斑割复合体在杂交利用过程中的斑茅、割手密野生特异基因在各世代的传递动态进行分析,并研究它们之间的遗传关系。29对AFLP引物组合共扩增出3695个位点,多态性比例为97.89%。斑茅和割手密对斑割复合体的遗传贡献率分别为43.96%和56.04%。斑茅特异位点在F1、BC1和BC2 3个世代的平均遗传率分别为8.25%、1.90%和0.63%,割手密特异位点在F1、BC1和BC2 3个世代的平均遗传率分别为16.98%、2.40%和0.21%,特异遗传物质均呈逐代减少趋势。比较不同世代甘蔗栽培种亲本遗传到后代的特异位点比率,F1的GT02-761特异位点比率最高,BC1的GT05-2743特异位点比例平均高达92.75%,BC2的ROC23遗传率最低,为49.09%,FN39遗传率最高,达94.32%。聚类分析结果表明斑割复合体偏向父本遗传,斑割复合体杂交后代偏向甘蔗栽培种遗传,与分子遗传关系分析结果一致。研究表明,经过3代的遗传重组,斑割复合体后代的遗传物质与斑割复合体相比已发生了很大的改变;研究明确了斑茅、割手密2个亲本在3个世代的遗传贡献规律,为进一步的杂交选育提供理论支持。     

11份割手密遗传多样性的SSR分析

利用10对多态性丰富的SSR引物,以国家甘蔗种质资源圃中保存的14份具有代表性的割手密为对照,对未收集过的云南地区11份割手密（Saccharum spontaneum L.）野生资源进行多样性分析。结果共扩增出233条DNA谱带,与对照相比,新采集材料的多态性条带为207条,其中14条为特有条带,多态性条带比率为0.89。遗传相似性系数和UPGMA聚类分析表明,新采集的材料并没有单独聚为一类,而是比较分散,在相似性系数为0.64处做切割线,参试材料可分为三个类群：第一类群主要由龙门割手密、河边村割手密和福建仙游1号组成;第二类群中包含19份材料,其中新采集的样品有上岗割手密、他拉割手密、安乐割手密、勐根割手密、芒美割手密、贺海割手密、回落割手密、里拉割手密和曼亨割手密,对照材料主要包含了云南、四川、越南、老挝、泰国地区的割手密,其共同特点是均分布在内陆地区;第三类群包括3个材料,分别是海南1号、海南92-2和广东化州割手密,其中不包含新采集的材料。而在相似性系数为0.654处作切割线又能将上述第二类群分为较细的三个亚群。由此可见,新采集的11份割手密资源具有丰富的遗传多样性,与已收集的资源相比,具有一定的差异性。说明依靠云南高山峡谷等立体气候特点,分布着遗传差异显著的割手密无性系。     

甘蔗种质间亲缘关系及特异标记的RAPD分析

采用RAPD技术分析甘蔗种质问的亲缘关系及特异标记。筛选出25个扩增多态性较强的随机引物,构建了41份甘蔗种质的RAPD指纹图谱,并对RAPD数据进行UPGMA聚类分析。甘蔗栽培品种之间、栽培品种与近缘种之间以及近缘种相互间的遗传相似系数变异范围为0．56～0．92,表明所研究的甘蔗种质之间的亲缘关系较近。此外,发现某些甘蔗亲本种质具有特异RAPD标记带。     

甘蔗细茎野生种云南不同生态类型的RAPD分析

利用25个随机引物对来自云南不同生态类型的82份甘蔗细茎野生种（Saccharnm spontanenm L．）和4份国外种材料进行RAPD标记,结果表明：云南甘蔗细茎野生种不同生态类型的遗传变异较大,具有丰富的遗传多样性,低续度类型的遗传多样性明显高于高纬度类型,在相同的纬度范围内,随着海拔的升高,其多态性逐渐减少,基于分子聚类分析,86份材料被划分为8个不同群体,表现出明显的地理分布的特点,结果初步证明了云南甘蔗细茎野生种可能起源于云南南部低海拔,低纬度地区,而后逐渐向高海拔,高纬度的西北和东北部演化,扩散,提出了云南南部可能是野生甘蔗起源中心之一的观点。     

烟草种质不同群体量遗传完整性的SSR研究

本研究以普通烟草种质红花大金元、豌口红土烟、白花黑烟、云烟87以及野生种N.alata为试验材料,利用SSR分子标记技术结合构建DNA混合基因池的方法对种质不同群体量的遗传完整性性进行研究。结果表明,960对引物对红花大金元、豌口红土烟、白花黑烟以及云烟87进行全基因组扫描,在前3份种质中未筛选到多态性引物,而在云烟87中筛选出3对多态性引物,3对多态性在云烟87的80个单株中扩增出6条特异性条带,将群体量降为10株时仍能检测到6条特异性条带,因此普通烟草种质繁殖更新群体等于或大于10株便能代表群体的遗传完整性。野生种N.alata从608对引物中筛选出11对多态性引物,扩增出44条DNA 条带, 其中多态性DNA 片段有19条,并对不同的群体量进行遗传多样性参数的比较,得出大于20株的群体能代表野生种质的遗传完整性。     

Species-specific accumulation of interspersed sequences in genus Saccharum

The genus Saccharum consists of two wild and four cultivated species. Novel interspersed sequences were isolated from cultivated sugar cane S. officinarum. These sequences were accumulated in all four cultivated species and their wild ancestral species S. robustum, but were not detected in the other wild species S. spontaneum and the relative Erianthus arundinaceus. The species-specific accumulation of interspersed sequences would correlate to the domestication of sugar canes.     

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.     

Detailed alignment of saccharum and sorghum chromosomes: comparative organization of closely related diploid and polyploid genomes.

The complex polyploid genomes of three Saccharum species have been aligned with the compact diploid genome of Sorghum (2n = 2x = 20). A set of 428 DNA probes from different Poaceae (grasses) detected 2460 loci in F1 progeny of the crosses Saccharum officinarum Green German x S. spontaneum IND 81-146, and S. spontaneum PIN 84-1 x S. officinarum Muntok Java. Thirty-one DNA probes detected 226 loci in S. officinarum LA Purple x S. robustum Molokai 5829. Genetic maps of the six Saccharum genotypes, including up to 72 linkage groups, were assembled into "homologous groups" based on parallel arrangements of duplicated loci. About 84% of the loci mapped by 242 common probes were homologous between Saccharum and Sorghum. Only one interchromosomal and two intrachromosomal rearrangements differentiated both S. officinarum and S. spontaneum from Sorghum, but 11 additional cases of chromosome structural polymorphism were found within Saccharum. Diploidization was advanced in S. robustum, incipient in S. officinarum, and absent in S. spontaneum, consistent with biogeographic data suggesting that S. robustum is the ancestor of S. officinarum, but raising new questions about the antiquity of S. spontaneum. The densely mapped Sorghum genome will be a valuable tool in ongoing molecular analysis of the complex Saccharum genome.     

Unraveling the genome structure of polyploids using FISH and GISH; examples of sugarcane and banana

We review here the progress that has been achieved using molecular cytogenetics to analyze the genome structure of sugarcane (Saccharum spp) and banana (Musa spp), two crops that are polyploid, of interspecific origin and with chromosomes not distinguishable by their gross morphology. In Saccharum, molecular cytogenetics enabled us to determine the basic chromosome number of two species, Saccharum officinarum and S. spontaneum, involved in the origin of modern cultivars, to quantify the proportion of chromosomes of these species in the genome of modern cultivars, to assess the extent of interspecific chromosome recombination and to clarify the origin of the related species S. barberi. These techniques are also used to monitor introgression with related genera. In Musa, GISH enabled us to differentiate the four genomes involved in banana cultivars and allowed us to determine the genome constitution of several cultivars. FISH was used to analyze the distribution of repeated sequences along the genome.     

Species-specific abundant retrotransposons elucidate the genomic composition of modern sugarcane cultivars

Chromosoma - Modern sugarcane cultivars are highly polyploid and derived from the hybridization of Saccharum officinarum and S. spontaneum, thus leading to singularly complex genomes. The complex...     

Inference of subgenomic origin of BACs in an interspecific hybrid sugarcane cultivar by overlapping oligonucleotide hybridizations

Sugarcane (Saccharum spp.) breeders in the early 20th century made remarkable progress in increasing yield and disease resistance by crossing Saccharum spontaneum L., a wild relative, to Saccharum officinarum L., a traditional cultivar. Modern sugarcane cultivars have approximately 71%-83% of their chromosomes originating from S. officinarum, approximately 10%-21% from S. spontaneum, and approximately 2%-13% recombinant or translocated chromosomes. In the present work, C(0)t-based cloning and sequencing (CBCS) was implemented to further explore highly repetitive DNA and to seek species-specific repeated DNA in both S. officinarum and S. spontaneum. For putatively species-specific sequences, overlappping oligonucleotide probes (overgos) were designed and hybridized to BAC filters from the interspecific hybrid sugarcane cultivar 'R570' to try to deduce parental origins of BAC clones. We inferred that 12?967 BACs putatively originated from S. officinarum and 5117 BACs from S. spontaneum. Another 1103 BACs were hybridized by both species-specific overgos, too many to account for by conventional recombination, thus suggesting ectopic recombination and (or) translocation of DNA elements. Constructing a low C(0)t library is useful to collect highly repeated DNA sequences and to search for potentially species-specific molecular markers, especially among recently diverged species. Even in the absence of repeat families that are species-specific in their entirety, the identification of localized variations within consensus sequences, coupled with the site specificity of short synthetic overgos, permits researchers to monitor species-specific or species-enriched variants.     

RFLP Mapping in Cultivated Sugarcane (Saccharum Spp.): Genome Organization in a Highly Polyploid and Aneuploid Interspecific Hybrid

Sugarcane cultivars are polyploid, aneuploid, interspecific hybrids between the domesticated species Saccharum officinarum and the wild relative S. spontaneum. Cultivar chromosome numbers range from 100 to 130 with ~10% contributed by S. spontaneum. We have undertaken a mapping study on the progeny of a selfed cultivar, R570, to analyze this complex genome structure. A set of 128 restriction fragment length polymorphism probes and one isozyme was used. Four hundred and eight markers were placed onto 96 cosegregation groups, based on linkages in coupling only. These groups could tentatively be assembled into 10 basic linkage groups on the basis of common probes. Origin of markers was investigated for 61 probes and the isozyme, leading to the identification of 80 S. officinarum and 66 S. spontaneum derived markers, respectively. Their distribution in cosegregation groups showed better map coverage for the S. spontaneum than for the S. officinarum genome fraction and occasional recombination between the two genomes. The study of repulsions between markers suggested the prevalence of random pairing between chromosomes, typical of autopolyploids. However, cases of preferential pairing between S. spontaneum chromosomes were also detected. A tentative Saccharum map was constructed by pooling linkage information for each linkage group.