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。     

Molecular Insights of Genetic Variation in Erianthus arundinaceus Populations Native to China

Background

E. arundinaceus (Retz.) Jeswiet is a warm-season, tall-growing perennial species native to much southern portion in China. The grass has been extensively used in sugarcane breeding and is recently targeted as a bioenergy feedstock crop. However, information on the genetic structure of the Chinese wild germplasm is limited. Knowledge of genetic variation within and among populations is essential for breeding new cultivars in the species. The major objective of this study was to quantify the magnitude of genetic variation among and within natural populations in China.

Methodology/Principal Findings

In this experiment, we analyzed genetic variation of 164 individuals of 18 populations collected from natural habitats in six Chinese provinces using 20 sequence-related amplified polymorphism (SRAP) primer pairs generating 277 polymorphic bands. Among and within the populations, the percentage of polymorphic bands (PPB) was 80.00% and 27.07%, genetic diversity (H_E) was 0.245 and 0.099, effective number of alleles (N_E) was 1.350 and 1.170, and Shannon''s information index (I) was 0.340 and 0.147, respectively. The populations were clustered into six groups exhibiting a high level of genetic differentiation, which was highly associated with geographic origins of respective germplasm populations, but was not significantly associated with geographic distances between the populations.

Conclusions/Significance

This is the first report indicating that large genetic variation exists in the Chinese E. arundinaceus germplasm based on the SRAP molecular marker analysis of native populations. The genetic structure of populations in the species has been substantially affected by geographic landforms and environments. The diverse collection will be highly valuable in genetic improvement in the species per se and likely in sugarcane.     

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。     

割手密野生资源抗逆性研究进展

割手密作为现代甘蔗遗传杂交育种史上最为成功的野生亲本,对多种不良环境都具有很强的抗逆性,被公认为是抗逆基因的主要来源。但目前真正被有效利用的割手密抗逆亲本和抗逆基因非常有限,我国自育和引进甘蔗主栽品种的抗逆性仍然比较单一且普遍偏弱,因此加强割手密优良抗逆亲本筛选和抗逆基因挖掘利用研究意义重大。本文综述了不同基因型割手密在非生物逆境(干旱、低温等理化因素)和生物逆境(病虫害侵染)下的抗逆性鉴定及其抗逆基因克隆和功能验证等国内外相关研究进展;并探讨了当前割手密资源抗逆材料筛选和抗逆基因挖掘利用中存在的问题和今后的研究方向,希望为高效利用割手密优异抗逆基因资源开展甘蔗多抗逆性聚合育种提供参考。     

80份甘蔗种质RAMP标记遗传多样性分析

采用RAMP分子标记技术对80份甘蔗种质(32份祖亲种、48份栽培品种或品系)的遗传基础进行了分析。从30对引物组合中筛选出4对多态性较强引物,构建了甘蔗80份种质的RAMP指纹图谱,这四对引物组合共扩增出84条带,其多态性为91.7%。80份种质的遗传相似系数变化范围在0.433～0.988,平均0.710。聚类分析表明,随着相似系数结合线的不同,可分别将参试的甘蔗种质从属间(甘蔗属与斑茅种)、野生种(割手密种、大茎野生种、印度种、中国种)与栽培种(热带种)间、栽培种与杂交栽培品种(或品系)间区别开来。各祖亲种与杂交栽培品种(或品系)的遗传相似性由大到小依次为热带种>印度种和中国种>大茎野生种＞云南割手密种＞其它割手密种＞斑茅。另外,本试验首次利用RAMP标记,获得了部分热带种、野生种及斑茅种特异片段,并发现这些特异片段能不同程度地在具有其血缘的栽培种中得到传递。     

Phylogenetic Analysis of Different Ploidy Saccharum spontaneum Based on rDNA-ITS Sequences

Saccharum spontaneum L. is a crucial wild parent of modern sugarcane cultivars whose ploidy clones have been utilized successfully in improving the stress resistance and yield related traits of sugarcane cultivars. To establish knowledge regarding the genetic variances and evolutional relationships of ploidy clones of Saccharum spontaneum collected in China, the rDNA-ITS sequences of 62 ploidy clones including octaploid clones (2n = 64), nonaploid clones (2n = 72), decaploid clones (2n = 80), and dodecaploid clones (2n = 96), were obtained and analyzed. The rDNA-ITS sequences of four species from Saccharum and Sorghum bicolor selected as controls. The results showed that decaploid clones (2n = 80) possess the most abundant variances with 58 variable sites and 20 parsim-informative sites in ITS sequences, which were then followed by octaploid clones with 43 variable sites and 17 parsim-informative sites. In haplotype diversity, all four population exhibited high diversity, especially nonaploid and decaploid populations. By comparing the genetic distances among four ploidy populations, the dodecaploid population exhibited the closest relationship with the nonaploid population, and then the relationship strength decreased successively for the decaploid population and then for the octaploid population. Population differentiation analysis showed that the phenomena of population differentiation were not found among different ploidy populations, and low coefficient of gene differentiation(Gst) and high gene flow(Nm) occur among these populations possessing close genetic relationship. These results mentioned above will contribute to the understanding of the evolution of different ploidy populations of Saccharum spontaneum and provide vital knowledge for their utilization in sugarcane breeding and innovation.     

甘蔗属不同种及优良甘蔗栽培品种的SSR标记遗传多样性分析

应用21对SSR引物与毛细管电泳技术,分析了52个甘蔗属品种的遗传多样性。共检测出327个SSR标记,平均每对引物检测15.6个。选择141个共显性标记构建SSR标记指纹图谱数据库,利用DNAMAN软件与UPGMA统计方法分析参试材料遗传多样性。DNAMAN软件同源分析显示,新台糖16号与台优1号之间的同源性最高(87%),品种之间最小的同源性为55%;利用UPGMA统计方法可把参试材料分成4个遗传相似性较高的类群。结果表明,SSR标记与毛细管技术的结合,可构建甘蔗种质资源SSR标记指纹图谱、分析甘蔗种质资源遗传多样性。聚类分析显示参试甘蔗材料的遗传基础相近,为了提高甘蔗选育种效率,应拓宽甘蔗选育种亲本的遗传基础,提高杂交栽培品种的抗虫、抗病等特性。     

Domestication to crop improvement: genetic resources for Sorghum and Saccharum (Andropogoneae)

BACKGROUND: Both sorghum (Sorghum bicolor) and sugarcane (Saccharum officinarum) are members of the Andropogoneae tribe in the Poaceae and are each other's closest relatives amongst cultivated plants. Both are relatively recent domesticates and comparatively little of the genetic potential of these taxa and their wild relatives has been captured by breeding programmes to date. This review assesses the genetic gains made by plant breeders since domestication and the progress in the characterization of genetic resources and their utilization in crop improvement for these two related species. GENETIC RESOURCES: The genome of sorghum has recently been sequenced providing a great boost to our knowledge of the evolution of grass genomes and the wealth of diversity within S. bicolor taxa. Molecular analysis of the Sorghum genus has identified close relatives of S. bicolor with novel traits, endosperm structure and composition that may be used to expand the cultivated gene pool. Mutant populations (including TILLING populations) provide a useful addition to genetic resources for this species. Sugarcane is a complex polyploid with a large and variable number of copies of each gene. The wild relatives of sugarcane represent a reservoir of genetic diversity for use in sugarcane improvement. Techniques for quantitative molecular analysis of gene or allele copy number in this genetically complex crop have been developed. SNP discovery and mapping in sugarcane has been advanced by the development of high-throughput techniques for ecoTILLING in sugarcane. Genetic linkage maps of the sugarcane genome are being improved for use in breeding selection. The improvement of both sorghum and sugarcane will be accelerated by the incorporation of more diverse germplasm into the domesticated gene pools using molecular tools and the improved knowledge of these genomes.     

割手密2C型蛋白磷酸酶ScPP2C基因的克隆与表达分析

割手密由于具有抗病、抗虫、抗逆等潜在的有利基因,历来被用于甘蔗的抗性遗传改良。在割手密中克隆编码2C型蛋白磷酸酶(protein phosphatase 2C, PP2C)的基因,并分析其在干旱胁迫下的差异表达。本研究利用RT-PCR方法从割手密叶片中克隆得到PP2C基因的CDS全长序列,利用生物信息学在线软件对PP2C蛋白的理化性质、蛋白结构进行预测分析,并采用实时荧光定量的方法分析该基因在不同干旱处理下的差异表达。结果显示从割手密中克隆到一个全长951 bp编码316个氨基酸的2C型蛋白磷酸酶基因,命名为ScPP2C,GenBank登录号为MG322120。荧光定量分析表明,随着干旱胁迫时间的延长,其表达量呈先上调后下调的表达模式,说明该基因是干旱胁迫诱导型基因。本研究通过挖掘甘蔗野生种割手密中的抗旱新基因ScPP2C,为甘蔗野生种质资源开发利用、转基因抗旱甘蔗新品种的选育提供了参考依据。     

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.     

Comparative analysis of QTLs affecting plant height and flowering among closely-related diploid and polyploid genomes.

Quantitative trait loci (QTLs) affecting plant height and flowering were studied in the two Saccharum species from which modern sugarcane cultivars are derived. Two segregating populations derived from interspecific crosses between Saccharum officinarum and Saccharum spontaneum were genotyped with 735 DNA markers. Among the 65 significant associations found between these two traits and DNA markers, 35 of the loci were linked to sugarcane genetic maps and 30 were unlinked DNA markers. Twenty-one of the 35 mapped QTLs were clustered in eight genomic regions of six sugarcane homologous groups. Some of these could be divergent alleles at homologous loci, making the actual number of genes implicated in these traits much less than 35. Four QTL clusters controlling plant height in sugarcane corresponded closely to four of the six plant-height QTLs previously mapped in sorghum. One QTL controlling flowering in sugarcane corresponded to one of three flowering QTLs mapped in sorghum. The correspondence in locations of QTLs affecting plant height and flowering in sugarcane and sorghum reinforce the notion that the simple sorghum genome is a valuable "template" for molecular dissection of the much more complex sugarcane genome.     

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。     

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。     

Genetic diversity assessment of bittersweet (Solanum dulcamara,Solanaceae) germplasm using conserved DNA‐derived polymorphism and intron‐targeting markers

Bittersweet (Solanum dulcamara), a European native weed, is widespread across a variety of habitats and often occurs as a coloniser of open, disturbed, ephemeral environments or wetlands, although it is also found in mountain habitats and on forest edges. As recent studies have shown the potential utility of the species in plant breeding programs, we assembled a collection of bittersweet germplasm from natural populations found in Europe. This collection was analysed with conserved DNA‐derived polymorphism (CDDP) and intron‐targeting (IT) markers to assess genetic diversity found within and among the populations. We found that there is limited genetic variability within the collected S. dulcamara accessions, with a greater proportion of allelic variation distributed among populations and considerably greater population structure at higher regional levels. Although bittersweet is an outcrossing species, its population structure might be affected by its perennial self‐compatible nature, reducing genetic diversity within regional populations and enhancing inbreeding leading to high interpopulation or spatial differentiation. We found that populations have been separated by local selection of alleles, resulting in regional differentiation. This has been accompanied by concurrent loss of genetic diversity within populations, although this process has not affected species‐level genetic diversity. Germplasm collecting strategies should be aimed at preserving overall genetic diversity in bittersweet nightshade by expanding sampling to southern Europe and to smaller regional geographic levels in northern and central Europe.     

基于SSR标记的紫花风铃木群体遗传多样性分析

为揭示紫花风铃木(Handroanthus impetiginosus)的群体遗传变异特征,对广东省6市12个群体72份种质材料进行遗传多样性和群体遗传结构分析。结果表明,9对引物共扩增出123个等位基因位点,引物的平均多态信息量为0.754,具有较高的多态性。12个群体均具有较高的遗传多样性,群体间平均有效等位基因数为3.272个,平均Shannon指数为1.159。AMOVA分析表明群体间遗传分化程度相对较低,群体内遗传分化程度较高。群体的总体遗传分化系数为0.077,处于中等程度。基于Structure分析、主坐标分析和NJ聚类分析均可将12个群体分为2大类群,分组结果具有一定相似性,表明供试紫花风铃木群体遗传结构较为简单。这为紫花风铃木优良种质资源的利用、遗传变异和科学育种提供了理论参考。     

Assessment of genetic diversity and population structure of swamp eel Monopterus albus in China

Swamp eel has become one of the most economically important fish in China. However, the wild swamp eel is facing the serious challenge of declining population and germplasm degeneration because most of farming swamp eel fingerlings was collected by fishing wild individuals. In this study, the genetic variation of Monopterus albus in six dominant farming regions was investigated based on the mitochondrial DNA D-Loop of 1008 bp in length. 180 individuals from 6 populations were examined and 74 haplotypes were observed. The overall genetic diversity was abundant and which its SD population was highest but CQ population was lowest. There was obvious genetic differentiation among investigated populations. Phylogenetic analysis revealed that these individuals were divided into four distinct genetic clades, clade A, B, C, and D. Clade A should be the most common ancestor clade. AH and CQ populations might originate from one single ancestor in maternal clade A. Clade C should be a native important clade in China. Though the genetic diversity did not suffered obvious decreasing, it is still imperative to take effective conservation measurements and establish an efficient selective breeding program.     

国内外割手密资源农艺性状表型遗传多样性分析

采用表型性状变异分析和聚类分析,对国内外不同地理来源的456份割手密资源13个性状进行评价,初步了解其遗传多样性特点,为解决种质创新与品种改良遗传基础狭窄问题提供思路。结果表明,456份割手密在株高、叶长、叶宽、锤度等多个性状都表现出了极高的遗传变异,多样性指数介于0.97～7.92之间;对嵌纹病、眼点病的抗病性及对金龟子的抗虫性变异范围广;国内外不同地理来源的割手密群间的遗传变异较大,以云南割手密群体多样性最丰富。聚类分析结果表明,国内外割手密资源聚成三大不同类群,云南独自为一大类,国外割手密被归为第二大类,其他为第三大类。各类群在植株生长特性及锤度方面具有极显著差异。     

基于遗传多样性构建金针菇的核心种质群体及分子身份证

金针菇Flammulina filiformis是我国产量最高的工厂化栽培食用菌。为提高优良工厂化栽培金针菇种质的育种效率,本研究以国内外收集的105份金针菇种质为材料,开展体细胞不亲和评价,并采用SSR分子标记的方法对所有种质进行遗传多样性分析和聚类分析。20对SSR引物在105份种质中共扩增得到209个等位基因位点,所有种质间的遗传相似系数为0.71-1.00,在遗传距离0.76处可分为5个大类群。105份金针菇种质共包含67种不同的遗传背景,野生金针菇种质比栽培种质具有更丰富的遗传多样性。基于SSR的聚类分析结果和体细胞不亲和评价结果既相互印证,又可互为借鉴。本研究构建了包含44份金针菇种质的核心种质群体,占所有供试材料的41.90%,保留了100%等位基因。核心种质群体覆盖区域广泛,最大限度地保留了原始群体的遗传多样性和表型变异,可为育种的亲本选择提供参考。进一步构建了能同时反映每份金针菇种质SSR分子标记指纹图谱、收集地区、子实体颜色和栽培性状的分子身份证编码,并转换成可视二维码,为金针菇种质的高效标识和快速溯源提供了科学依据。     

用AFLP技术分析四川核桃资源的遗传多样性

 利用AFLP分子标记技术, 运用EcoRⅠ/MseⅠ双酶切组合, 选用多态性高、分辨力强的4对选择性扩增引物组合E32/M48、E33/M61、E35/M61、E33/M62分别对四川省3个野生核桃(Juglans regia)种群和1个野生铁核桃(J. sigillata)种群共46个样品进行遗传多样性分析、居群遗传结构分析及种属关系探讨。结果表明: 1)共扩增出244个遗传位点, 其中146个多态位点, 多态率为59.84%; 核桃群体组和铁核桃群体的多态性百分率分别为55.33%和52.05%, 两个物种遗传多态性水平相当; 核桃群体组所检出的位点平均有效等位基因数Ae、Nei’s基因多样度H、平均Shannon信息指数I分别为1.322 9、0.190 8和0.286 3, 而铁核桃群体分别为1.339 9、0.196 1和0.289 8, 铁核桃群体遗传多样性水平略高于核桃群体。2)群体间特异带及群体间共有带占总扩增带数的15.16%, 其中铁核桃群体特异谱带最多, 群体特异谱带揭示了群体间的遗传差异及相似性。3) Shannon信息指数(I)、Nei’s基因多样性指数(H)和分子方差分析(AMOVA)表明核桃遗传多样性在群体间和群体内的分布分别为14.36%和85.64%、12.6%和87.4%、11.07%和88.93%, 表明群体内的遗传多样性大于群体间的遗传多样性; 核桃群体组与铁核桃群体的变异主要存在于群体组内, 组间的遗传变异仅占总变异的9.35%, 两者间的遗传分化系数Gst为0.093 5, 与AMOVA分析结果一致。4) 4个群体的Nei’s遗传距离在0.038 2～0.069 2之间, 遗传一致度在0.933 2～0.962 5之间, 表现出较高的遗传相似性; 运用Nei’s遗传一致度对供试种群进行了UPGMA聚类, 结果表明核桃的3个群体优先聚类, 大渡河流域群体与甘南地区群体聚类最近。AFLP所检测出的结果既是核桃与铁核桃生物学特性的反映, 又是其各自生态学特性的反映, 该研究结果对核桃种质资源的保护和育种提供一定的理论依据。