Characterisation of Erianthus sect. Ripidium and Saccharum germplasm (Andropogoneae - Saccharinae) using RFLP markers

A collection of 65 Erianthus Michx. sect. Ripidium Henrard accessions (representing seven accepted species) and 14 Saccharum L. representatives (S. officinarum L. and S. spontaneum L.) were studied by RFLP analysis using 14 dispersed nuclear single-copy probes from maize. An intergeneric distance (1–F) of 0.748 was revealed between Erianthus and Saccharum. Within the Erianthus collection, the greatest distances were found between E. elephantinus Hook f. or E. ravennae (L.) P. Beauv. (the two 2n=20 species), and the rest of the Erianthus collection. The smallest distances were found amongst the E. arundinaceus (Retz.) Jeswiet clones collected in Indonesia ((1–F)=0.005). In addition, a partition based on the geographical origin and consistent with the chromosome numbers, ie E. arundinaceus from Indonesia versus E. arundinaceus and E. procerus from India, was revealed. E. bengalense was intermediate. The study of the Saccharum individuals confirmed the greater variability of S. spontaneum compared to the so called noble cane, S. officinarum. The 2n=80 S. spontaneum genotypes were shown to be closely related to S. officinarum. The implication of these results on the involvement of S. spontaneum and Erianthus sect. Ripidium in the origin of S. officinarum is discussed. This revised version was published online in July 2006 with corrections to the Cover Date.     

用基因组原位杂交方法分析甘蔗-斑茅杂种及回交后代的染色体组成

斑茅(Erianthus arundinaceus Retz)是甘蔗的近缘属植物,具有多种优良特性,是甘蔗育种的重要种质资源之一。本实验使用基因组原位杂交(GISH)技术分析了甘蔗-斑茅杂种及回交后代F1、BC1和BC2的染色体构成与传递行为。结果表明:在F1代,来自斑茅HN92-77的染色体数目介于28～30条,来自热带种Badila的染色体数目介于38～40条,体细胞染色体数为2n=68～70,基本符合n+n的染色体传递方式;在BC1和BC2代,来自斑茅的染色体数分别为22～28条和13～15条,来自甘蔗的染色体数分别是87～94条和98～101条,其体细胞染色体数2n分别为110～121条和112～115条,基本上分别符合2n+n和n+n的染色体传递方式。在所观察的渐渗系中,均存在有染色体丢失的现象,但未观察到染色体发生交换与重组。     

斑茅cDNA中抗病基因同源序列的分离和表达特性分析

植物抗病基因具有一些特定的保守结构域。本研究根据已知植物同源抗病基因(RGAs)保守序列设计简并引物, 从甘蔗近缘植物斑茅的cDNA中扩增出6条抗病基因同源序列, 它们在NCBI上登录号分别为EU685835、EU685836、EU685837、EU685838、EU685839 和 EU685840。序列分析表明, 这些RGAs均含有典型的NBS-LRR类抗病基因保守结构域P-loop、Kinase-2a、Kinase-3a和疏水结构域(Hydrophobic domain, HD)。氨基酸序列的同源性比对表明,6条RGAs序列同11条参试的抗病基因之间的同源性为8.3%~93%,而6条RGAs之间的氨基酸序列同源为30.5%~45.6%。另外,本实验所克隆的6条斑茅抗病基因同源序列中, kinase-2 (LLVLDDVW/D)最后一个氨基酸皆为色氨酸,推测所克隆的NBS-LRR类抗病基因都属于non-TIR-NBS-LRR类。定量PCR分析表明, 6条斑茅抗病基因同源序列在根、茎和叶片中组成型表达,同时这些抗病基因同源序列的表达会受外源信号分子水杨酸和过氧化氢的上调作用,可能在斑茅的抗病性中具有一定的作用。     

Analysis of introgression of the Tulipa fosteriana genome into Tulipa gesneriana using GISH and FISH

Southern hybridization and genomic in situ hybridization (GISH) have demonstrated that ‘Purissima’ (2n = 2x = 24) is an interspecific hybrid comprised of one genome of Tulipa (T.) gesneriana and one genome of T. fosteriana. Backcrossing T. gesneriana with ‘Purissima’ was partially successful. Simultaneous GISH and fluorescence in situ hybridization (FISH) distinguished chromosomes from both parent genomes, as well as recombinant chromosomes, in interspecific hybrids and their progeny. Chromosome recombination was observed in all cultivars except ‘Purissima’ and ‘Kouki’ (2n = 3x = 36). ‘Kouki’ (2n = 3x = 36) had two genomes of the T. gesneriana and a single genome of the T. fosteriana. The number of nonrecombinant T. fosteriana chromosomes in ‘Judith Leyster’ (2n = 4x = 48) and ‘Purissima’ progeny varied from two in ‘Hatsuzakura’ to six in ‘Kikomachi’ and ‘Momotaro’. The number and type of recombinant chromosomes also differed among cultivars. The total number of translocations ranged from one in ‘Kikomachi’ to six in ‘Hatsuzakura’. Each was a combination of a single T. fosteriana fragment and a single T. gesneriana fragment, indicating that they resulted from a single crossover event. Sequential GISH and FISH analysis with rDNA probes yielded chromosome-specific markers that were used to identify most of the chromosomes in ‘Purissima’ progeny. This is the first report of introgression of T. fosteriana chromatin into the T. gesneriana genome.     

斑茅割手密杂种后代真实性鉴定及遗传分析

以斑茅GXA87-36(♀)与割手密GXS79-9(♂)杂交获得的经形态鉴别为真杂种的后代材料GXAS07-6-1 (斑茅割手密杂合体)及其双亲为材料,利用体细胞染色体计数以及SSR、SRAP分子标记对杂种进行真实性鉴定,并利用SRAP分子标记对斑茅GXA87-36、割手密GXS79-9及其杂种后代遗传位点的传递进行分析。结果表明, GXA87-36体细胞染色体数为2n=60,GXS79-9体细胞染色体数为2n=64,其杂种GXAS07-6-1染色体2n=62,其杂交的染色体遗传方式为n+n;筛选出3对SSR引物和5对SRAP引物可分辨后代的真伪;利用71对SRAP引物扩增后代和双亲,检测到1 095个遗传位点,多态性位点数为800;母本有279个位点,其中有 79 %传递给其后代;父本有439个位点,其中有73%传递给后代;用NTSYS-pc2.10e软件计算Jaccard遗传相似系数,双亲间为0.474,后代与母本、父本间分别为0.696、0.752,表明后代GXAS07-6-1偏向父本遗传。     

Development of molecular markers to assess the level of introgression of Populus tremula into P. alba natural populations

Morphological traits traditionally adopted to discriminate between Populus alba L. and P. tremula L. have frequently led to misclassifi‐cation of their spontaneous hybrid P. × canescens Sm. Moreover, they may not be of any help in cases of spontaneous backcross phenomena. These limitations can be overcome by molecular markers, which are not environmentally influenced nor subjectively assessed. In this study, the effectiveness of amplified fragment length polymorphism (AFLP) and simple sequence repeat (SSR) markers in species and hybrid discrimination was evaluated by analysing a set of reference samples of P. alba, P. tremula and P. × canescens. Species‐specific and species‐indicative AFLPs, as well as diagnostic SSR alleles, were recorded in both P. alba and P. tremula reference samples. The results allowed a clear distinction between the two poplar species and their hybrid. Using these diagnostic markers, a natural population of P. alba trees sampled along the Ticino river basin in northern Italy was analysed, and P. × canescens individuals, intermingled with P. alba trees, were detected.     

Development of EST-SSR markers of Ipomoea nil

Although Japanese morning glory (Ipomoea nil (L.) Roth.) has been used intensively for genetic studies, DNA markers have not been developed in Ipomoea nil sufficient to cover all chromosomes. Therefore, we conducted microsatellite (simple sequence repeats, SSR) marker development in I. nil for future genetic studies. From 92,662 expressed sequence tag (EST) sequences, 514 unique microsatellite-containing ESTs were identified. Primer pairs were designed automatically in 326 SSRs. Of 150 SSRs examined, 75 showed polymorphisms among strains. A phenogram based on the SSR genotypes revealed the genetic relation among seven Japanese morning glories from five different regions of the world and an ivyleaf morning glory (I. hederacea Jacq.). The developed SSR markers might be applicable for genetic studies of morning glories and their relatives.     

Evidence of gene introgression in apple using RAPD markers

Summary A genomic remnant of Malus floribunda clone 821 introgressed into the cultivated apple M. x domestica Borkh. was identified using randomly amplified polymorphic DNA (RAPD) markers obtained by the polymerase chain reaction (PCR). Using a set of 59 oligonucleotide decamer primers, polymorphic DNA markers were identified among three pooled DNA samples. Based on the presence or absence of bands among bulked apple scab-resistant selections and cultivars, bulked scab-susceptible cultivars, and a M. floribunda clone 821 sample, one primer, A 15, identified amplified fragments in the scab-resistant bulked sample that was also unique to the M. floribunda clone 821. The unique band from M. floribunda clone 821 was amplified in four out of 17 scab-resistant selections/cultivars. This RAPD, designated OA15₉₀₀, identifies an introgressed fragment that has as yet no known function.     

Estimation of between and within accession variation in selected Spanish melon germplasm using RAPD and SSR markers to assess strategies for large collection evaluation

The population structure of 15 Spanish melon (C. melo L.)accessions, mostly of Group Inodorus, was assessed by the analysis of 16individuals of each accession using 100 random amplified polymorphic DNA (RAPD) bands produced by 36 primers, and allelic variation at 12microsatellite (SSR) loci (23 alleles). A relatively high level of polymorphism (25.6%) was detected using RAPD markers, and eight SSR loci (66.7%) were useful in discriminating accessions. Cluster analysis using RAPD- and SSR-based genetic distance estimates resulted in similar and consistent groupings of most of the accessions studied. The mean genetic distance and standard error among accessions estimated by RAPD variation was 0.421 ± 0.099, and mean SSR-based genetic distance estimate was 0.285 ± 0.141. Albeit many dominant markers examined were fitted to a 3:1 test ratio, deviation from this ratio and from Hardy-Weinberg expectations for many SSR loci suggests that some populations were in genotypic disequilibrium. Moreover, a higher level of genetic variation was observed between Cassaba market classes than within accessions, suggesting that, depending upon the accession, allelic fixation has occurred in these populations. The relatively high level of heterogeneity observed (different band morphotypes and cluster grouping within a particular market class), however, indicates that the Spanish melons examined possess a relatively broad genetic background. An appraisal of accession population structure such as the one reported herein indicates that bulk sampling techniques coupled with molecular analysis techniques that employ a unique array of discriminating markers can provide information leading to effective strategies for diversity analyses of large collections. This revised version was published online in August 2006 with corrections to the Cover Date.     

Assessing genetic diversity in the cassava (Manihot esculenta Crantz) germplasm collection in Brazil using PCR-based markers

Knowledge of the origin, organization and nature of the cassava (Manihot esculenta Crantz) germplasm collection in Brazil is incomplete due to lack of critical information on several aspects of the collection. This study verifies the utility of SSR-primed PCR markers for germplasm assessment and then utilizes these markers as well as RAPD's to characterize the Brazilian collection. We specifically address the following questions: 1)what is the relationship of morphologically closely related species to cultivated cassava? 2) What is the genetic diversity of cultivars within and between different habitats in Brazil? 3) Do agronomic traits and molecular markers reveal the same relationship among cassava accessions? 4) How complete is the Brazilian cassava collection and how well is it represented in the Word Core Collection of cassava, maintained by CIAT? Results of the interspecific studies of cassava and its wild relatives confirms the close relationship of cassava, Manihot esculenta ssp. esculenta to Manihot esculenta ssp. flabellifolia as well identifying several other closely related wild species. Next, PCR-based markers indicate a strong grouping of varieties related to the region of cultivation in Brazil. Moreover, important regions such as Cerrados and Amazon are relatively poorly represented in germplasm collections. Interestingly, the relationships of accessions based on agronomic traits are not fully congruent with relationships revealed with RAPD markers. Finally, the genetic diversity of the Brazilian cassava collection is not fully represented in the Core of the Word Core Collection of CIAT.     

Assessment of genetic variation of bread wheat varieties using microsatellite markers

The DNA fingerprinting profiles of 11 bread wheat cultivars, grown in Turkey, was obtained by using 19 highly polymorphic wheat microsatellites. Up to six randomly selected individuals from each of these cultivars were subjected to analysis. Estimated polymorphism information content (PIC) values among 65 individual genotypes for 19 loci were between 0.36 and 0.87 with an average value of 0.68. The numbers of observed alleles were between 2 and 9, with an average value of 5.42. These relationships were assessed among cultivars and among their individual genotypes using the mean pairwise average square distance (D ₁) (or the delta mu measure of distance (Ddm)) matrix and neighbor-joining method based on `Nei-72' distance matrix, respectively. Two spring cultivars, `Kaklic-88' and `Orso', were closely related to each other; the other 9 winter and facultative cultivars were dispersed. Pedigrees of cultivars represented very little shared information among each other, therefore, they could not been utilized in comparisons. This revised version was published online in July 2006 with corrections to the Cover Date.     

黄麻应用核心种质的DNA分子身份证构建

构建并研究黄麻应用核心种质是促进黄麻遗传育种和挖掘优异基因的必要途径。在300份黄麻种质资源的农艺性状观察统计基础上,构建了黄麻应用核心种质,包含61份品种(系),可划分为高产、优质、抗病等16种应用类型。为准确鉴定这61份应用核心种质,以46对核心引物为基础,筛选出12对荧光核心引物,采用荧光标记毛细管电泳分析这12对引物的多态性,共检测出140个多态性位点。将毛细管电泳得到的分子量数据以数字+英文字母方式编码,选取了12对荧光核心引物的组合,构建出该应用核心种质的字符串DNA分子身份证,进而构建了相应的条形码和二维码DNA分子身份证,可迅速被电子设备识别。这些结果可促进黄麻种质资源的高效利用及快速分子鉴定。     

黄麻应用核心种质的DNA分子身份证构建

It is important to construct and study the applied core collection in order to promote jute genetic breeding and explore excellent genes. In this study, the applied core collection in jute, including 61 accessions divided into 16 application-oriented features, such as high yield, high quality, disease resistance and so on, was established based on the field performance from 300 jute germplasm. 12 fluorescent core primers were screened from 46 pairs of core primers. Fluorescence labeled capillary electrophoresis was used to analyze the polymorphism of these 12 pairs of primers, and a total of 140 polymorphism sites were detected. The precise DNA molecular ID cards were constructed by the combination of the 12 core primer pairs from the data coded in the form of numbers and English letters. Bar code and quick response code DNA molecular were established and can be quickly scanned and recognized by electric gadget. These results could be beneficial to increase the application efficiency and rapid molecular identification in jute germplasm resources.     

甘蓝型油菜中油杂8号种子纯度的SSR鉴定

（中国农业科学院油料作物研究所/国家油料作物改良中心/农业部油料作物遗传改良重点实验室,湖北武汉 430062）     

The taxonomic characterisation of annual Beta germplasm in a genetic resources collection using RAPD markers

Summary Annual beets in the genus Beta section Beta represent an important genetic resource. Representative accessions of annual beets from a beet germplasm collection were analysed using RAPD to assess the patterns of variation and relationships among them. Using arbitrary primers, markers showing variation across accessions were identified. A dendrogram of similarity was produced using these molecular markers. All the accessions analysed were classified into three major groups corresponding to species or subspecies macrocarpa, adanensis and maritima. Macrocarpa was shown to be the most divergent group in this section. Using RAPD molecular markers, it was possible to ascribe an accession to one of three taxonomic groups and overcome much of the confusion encountered when morphological traits are used for identification. The group of maritima was found to be more polymorphic than either the group of macrocarpa or adanensis at both accession and subspecies levels.     

Parentage analysis of tea cultivars in Japan based on simple sequence repeat markers

Tea cultivars have been bred by individual selection of landraces and by crossbreeding, but the validation of the parentage is limited. In this study, we performed parentage analysis of 79 tea cultivars in Japan based on SSR markers to confirm or identify the parent-offspring relationships among them. The effectiveness of nine SSR markers for parentage analysis was validated by comparing them to the existing cleaved amplified polymorphic sequence markers. The former markers were detectable more alleles than the latter. Simulation of parentage analysis of the tea cultivars predicted biparental origins for 12 cultivars (‘Houshun’, ‘Mie ryokuhou no. 1’, ‘Surugawase’, ‘Tenmyo’, ‘Yamanoibuki’, ‘Harumidori’, ‘Koushun’, ‘Minekaori’, ‘Okumusashi’, ‘Saemidori’, ‘Sofu’, and ‘Toyoka’), in the first five of which candidate parents of yet-to-be-defined pedigree were newly identified. Comparisons of a total of 41 SSR genotypes confirmed the newly-identified parentages of ‘Asahi’ for ‘Tenmyo’, ‘Rokurou’ for ‘Houshun’, ‘Surugawase’, and ‘Yamanoibuki’, and ‘Yamatomidori’ for ‘Mie ryokuhou no. 1’. The maternity of seven cultivars out of the 12 was also confirmed with chloroplast DNA sequences. Uniparental origins were confirmed for 25 cultivars. This parentage analysis has improved our knowledge of tea pedigrees and will aid in the development of new cultivars.     

Assessing the genetic diversity of Portuguese maize germplasm using microsatellite markers

A collection of Portuguese maize accessions representing a valuable source of genes for introduction into modern cultivars is stored at the Portuguese Plant Germplasm Bank (Banco Português de Germoplasma Vegetal—BPGV). To assess genetic diversity among inbreds, microsatellite analysis was carried out for 54 inbred lines representing the diversity of Portuguese dent and flint maize germplasm. Fifty American and other European elite inbreds were also analysed for comparison. Fifteen microsatellite loci distributed throughout the maize genome were chosen based on their repeat unit and base composition. A total of 80 alleles were detected with an average allele number of 5.33 per locus. Polymorphism information content (PIC) values and observed genetic distances showed the existence of large variability among inbreds. Cluster analysis indicated that almost all of the inbreds could be distinguished from each other and Portuguese inbreds were present in all clusters formed. These associations were consistent with the known pedigree records of the inbreds, confirming a mixed origin of Portuguese materials. Comparative analysis of microsatellite diversity among groups was established according to important traits for both breeding and line identification. This revealed that, although most of the genetic diversity (>95%) was attributable to differences among inbreds of different groups, the existence of phenotypic differentiation in endosperm colour, kernel type and cob colour could be suggested for grouping. These findings support the joint use of molecular and morphological traits in management of the germplasm collection. In this study, SSR markers proved to be effective to characterise and identify maize inbred lines, and demonstrate associations among them. This revised version was published online in July 2006 with corrections to the Cover Date.     

Screening global <Emphasis Type="Italic">Medicago</Emphasis> germplasm for weevil (<Emphasis Type="Italic">Hypera postica</Emphasis> Gyll.) tolerance and estimation of genetic variability using molecular markers

The genus Medicago encompasses many important forage species for both temperate and tropical regions. M. sativa L., commonly known as lucerne, is one of the most important forage species grown worldwide, but its production suffers seriously from weevil (Hypera postica Gyll.) infestation. The aim of this work was to identify species/accessions with tolerance to weevil and their molecular analysis using simple sequence repeat (SSR) markers. After screening 197 global germplasm encompassing 50 Medicago species for weevil tolerance, 22 lines representing 13 species were identified where leaf damage was ≤15% (P ≤ 0.05). In total, 37 accessions of the 22 lines, five Indian lucerne cultivars with leaf damage ≥75% and 10 accessions of the 13 Medicago species with low to high infestation (>25%) were molecularly assessed using 11 SSR markers (5 newly developed) to delineate closest to lucerne lines for breeding. In total, 33 bands were scored. The SAHN clustering using UPGM algorithm resulted into two main clusters supported with high boot strap values and with genetic similarity ranging from 0.33 to 0.96. Two accessions of M. tenoreana were observed closest to Indian lucerne cultivars. The rich variability revealed can be used as potential resource for transferring genes across species. Although the inter-specific hybridization is difficult preposition in genus Medicago largely due to post fertilization barrier, the identified species/accessions can be utilized on priority in breeding programs especially employing biotechnological tools like culturing of fertilized pods, ovule-embryo culture and electroporation.     

The development of specific SNP markers for chromosome 14 in cotton using next‐generation sequencing

Lacking of reference genome sequence for the development of stable molecular markers for specific chromosomes (intervals) remains to be a challenge in cotton, which was a necessary step in fine mapping of gene (QTL). In this study, the feasibility of development of single‐nucleotide polymorphism (SNP) markers between CS‐B14Sh (a substitution line for short arm of Chromosome 14) and TM‐1 (the recurrent parent) was explored using next‐generation sequencing (NGS) based on reduced representation libraries (RRLs). High‐quality genome sequences, representing about 7.1%, 8.8% and 10.4% of the tetraploid cotton genome, were generated for TM‐1, 3‐79 (the donor parent) and CS‐B14Sh, respectively. A total of 397 putative SNP markers were detected between CS‐B14Sh and TM‐1, and most (358) of them were also detected between TM‐1 and 3‐79. Allele‐specific PCR method was used for validation of 40 SNP markers, and 27 of them showed polymorphism between TM‐1 and CS‐B14Sh, and a linkage group comprising of 25 SNP markers and five SSR markers was constructed. The order of SNP markers agreed well with the position of them on Chr05 of D genome, which further approved the truth of SNPs detected. The results suggested that the development of SNP markers in specific genome region using NGS was efficient in substitution or near‐isogenic lines.     

叶绿体5S rDNA ITS和matK基因序列应用于刚竹属植物系统分类的可行性分析

本文应用CTAB法从37种竹类植物中提取基因组DNA,根据在GenBank中发表的5S rDNAITS和matK基因设计引物,通过PCR进行扩增.结果表明:37种竹子5S rDNA ITS序列PCR产物大小约为450 bp,在刚竹属内部无长度上的差异,但是舒竹(Phyllostachys shuchengensis)与其他刚竹属植物相比,有一个位点的差异(由A变为C).因此,5S rDNA ITS在属下水平上无法提供较大的信息量,变异性较低,不适于刚竹属属下水平的系统分类研究.同样,选取37种竹子中3个竹种的,matK基因的PCR扩增产物直接进行测序,结果显示marK基因在竹亚科的属间长度上无差异,产物的长度约为1 500 bp,将测序结果进行同源性比对,在变异位点附近寻找多态性酶切位点,碱基序列上有一个位点的差异(BstN Ⅰ).PCR-RFLP结果显示,共有3种竹子在此位点发生变异分别为:浙江淡竹(Phyllostachys meyeri)、安吉金竹(Phyllostachysparvifolia)和黄古竹(Phyllostachys angusta).刚竹属植物的mark基因序列相当保守,片段中刚竹属间的绝对核苷酸差异不到1个,所提供的信息量不够充分.因此,叶绿体5S rDNA ITS和marK基因序列不适用于刚竹属植物系统分类研究,但其可能适合在属或属以上分类等级竹类植物的系统分类中应用.