Strategies to develop polymorphic markers for <Emphasis Type="Italic">Coffea arabica</Emphasis> L.

This study reports the implementation of three strategies for the development of genetic markers and their evaluation in both progenitors of an F2 population used for the construction of a genetic map of Coffea arabica. The strategies were Cleaved Amplified Polymorphic Sequences (CAPS), Single Strand Conformational Polymorphism (SSCP), and sequence analysis predicted Single Nucleotide Polymorphism (SNP). The methodologies were developed from different sequence sources: For CAPS, we used 25 COS sequences derived from Hedyotis spp. and 29 COSII sequences derived from Solanaceae and Rubiaceae species; for SSCP, we used 111 coffee EST sequences, 50 COSII sequences, and 10 C. arabica BAC end sequences. A low polymorphism was identified with the CAPS and SSCP methodologies. A total of 61 SNPs were identified in silico from 5,371 ESTs of coffee and from amplified, cloned, and sequenced COSII markers. Sixteen of these SNPs were validated with Luminex technology and 2 of them were polymorphic in C. arabica genotypes. This study highlights the difficulties of finding polymorphism in the species C. arabica where SNP identification seems to be the best strategy to search for polymorphic markers for this low diversity plant.     

AFLP analysis of introgression in coffee cultivars (Coffea arabica L.) derived from a natural interspecific hybrid

S.288 an offspring of a putative spontaneous interspecific hybrid between tetraploid Coffea Arabica (2n = 4x = 44) and diploid C. liberica(2n = 2x = 22) and 17 arabica coffee introgression lines (representing F₂ and F₄) derived from the cross S.288 x Kent arabica were evaluated for introgression of C. liberica genetic material. In all, 36 AFLP primer combinations were used in the analysis. The AFLP profiles of introgressed lines were compared to five accessions each of C. arabica and C. liberica. A total of 137 polymorphic bands were scored among the 29 accessions analysed. The introgressed genotypes exhibited 102 marker bands consisting of 65 additional bands and 37missing bands associated with introgression of C. liberica genetic material. C. liberica accessions of EA group (C. liberica var liberica of Guinean origin) seemed to be the likely progenitor in the origin of natural hybrid. Analysis of genetic relationships in the introgressed lines suggested that introgression was limited to few fragments. Segregation and wide variation in number of marker fragments in the F₂ and F₄progenies were attributed to chromosome recombinations. The differences in the level of introgression between introgressed parent, F₂ and F₄ groups was not pronounced. Therefore the alien genetic material appeared to be fixed and there was no elimination or counter-selection over generations, from introgressed parent to F₄.In C. arabica accessions, only 35 polymorphic bands were seen confirming the low genetic diversity. On the contrary, although representing a small amount of alien genome introgression, the Liberica-introgressed genotypes provided notable genetic diversity. Considering the fact that the diploid species of Coffea constitute a valuable source of genetic diversity, the potential implications of variability generated by Liberica-introgressed genotypes in C. arabica breeding are discussed. This revised version was published online in August 2006 with corrections to the Cover Date.     

Characterization of novel sugarcane expressed sequence tag microsatellites and their comparison with genomic SSRs

Microsatellites or simple sequence repeats (SSRs) are one of the most suitable markers for genome analysis as they have great potential to aid breeders to develop new improved sugarcane varieties. The development of SSR derived from expressed sequence tags (EST) opens new opportunities for genetic investigations at a functional level. In the present work, the polymorphism obtained with a subset of 51 EST–SSRs derived from sucest was compared with those generated by 50 genomic SSRs (gSSR) in terms of number of alleles, polymorphism information content, discrimination power and their ability to establish genetic relationships among 18 sugarcane clones including three Saccharum species (S. officinarum, S. barberi, S. sinense). The majority of EST–SSRs loci had four to six alleles in contrast to the seven to nine observed for the gSSRs loci. Approximately, 35% of the gSSRs had PIC values around 0.90 in contrast to 15% of the EST–SSRs. However, the mean discrimination power of the two types of SSR did not differ significantly as much as the average genetic similarity (GS) based on Dice coefficient. The correlation between GS of the two types of SSRs was high (r = 0.71/P = 0.99) and significant. Although differences were observed between dendrograms obtained with each SSR type, both were in good agreement with pedigree information. The S. officinarum clone IJ76‐314 was grouped apart from the other clones evaluated. The results here demonstrate that EST–SSRs can be successfully used for genetic relationship analysis, extending the knowledge of genetic diversity of sugarcane to a functional level.     

Anther Culture Response in Perennial Ryegrass (Lolium perenne L.)

20 diploid clones from 7 varieties, and 10 tetraploid clones from 3 varieties of Lolium perenne were tested in replicated anther culture experiments. Embryos or calluses, were obtained from all clones, and plants were regenerated from all clones except one. The total yield of plants (albino and green plants) ranged from 0 to 61 plants per 100 cultured anthers among genotypes, and there was a general tendency for tetraploic genotypes to be more responsive. Viable green plants were obtained from 5 diploid and 7 tetraploid clones representing 2 and 3 varieties, respectively. Their origin from reduced pollen was confirmed by a haploid chromosome number in some regenerants and by homozygosity for isozyme loci in spontaneously chromosome doubled plants produced from heterozygous diploid donor plants. A large number of the plants were successfully established in the soil. For most donor genotypes, green plants were rare exceptions, but two diploic clones consistently produced 2.3 and 3.8 green plants per 100 cultured anthers, respectively. Estimates of variance components from replicates with greenhouse and field-grown donor plants showed that genotypes accounted for about 73 per cent of the total variation in yield of embryos/calluses, while only 14—15 per cent of the total variation was due to replicates. Hence at present, emphasis should be placed on die selection of high-response genotypes in material of high agronomic potential.     

Breeding for low seed caffeine content of coffee (Coffea L.) by interspecific hybridization

Summary Seeds from open-pollinated flowers collected from hybrids of several Coffea species were analysed for caffeine content. The caffeine content was not always intermediary to that of the parents; higher and lower values were found. Diploid F₁ hybrids between accessions of C. eugenioides and C. salvatrix showed the lowest seed caffeine content. Seeds of the tetraploid hybrids C. arabica × C. salvatrix or C. arabica × C. eugenioides hybrids presented low caffeine content. The possibility of breeding coffee to reduce the caffeine content in the seeds by interspecific hybridization of C. arabica with other Coffea species is discussed.     

同工酶差异位点分析在蔬菜杂交种纯度检测中的应用

用10种同工酶和蛋白质分析体系，分析了7种重要蔬菜的71个杂交种与其亲本之间的差异位点，以及这些差异位点用于杂交种纯度检测的可能性和存在的问题。试验表明，作物的不同种类，杂交种与亲本之间的亲缘关系以及作物各类的遗传多态性，都会影响同工酶差异位点产生的多寡，从而影响到这一技术是否能用于此种作物的纯度检测。分析了不同同工酶在不同蔬菜中的多态性以及在蔬菜种子纯度检测中的表现。     

Genetic diversity of natural populations of Atriplex halimus L. in Morocco: An isoenzyme-based overview

Based on polyacrylamide gel electrophoresis, nine natural populations of Atriplex halimus L., a perennial shrub, collected in different regions of Morocco, were studied for their genetic variation using isoenzyme polymorphism of the highly active enzyme systems: esterases (EST), acid phosphatases (ACP) and glutamate oxaloacetate transaminase (GOT). Different allozyme frequencies from 7 different loci were obtained for all populations of this halophyte species. High levels of genetic diversity were revealed. The mean number of alleles per locus (A = 1.9–2.0), the percentage of polymorphic loci (p = 71.4–85.7) and the mean expected heterozygosity (H_e = 0.339–0.385) showed an important variability in all populations. Gene diversity was essentially explained by the within population component. The between populations differentiation accounted for 8% of the whole diversity (F_ST, averaged over all loci, is 0.08). The relationships among the 9 populations were inferred from the Nei’s genetic distances. Four major groups were formed. The northern population ‘Tanger’, forming a unique group, was highly divergent from the other groups. It appeared that the genetic distance between all groups was related to the geographic distance that separates them. This revised version was published online in August 2006 with corrections to the Cover Date.     

Detection by simple sequence repeat markers of introgression from Coffea canephora in Coffea arabica cultivars

Microsatellites or simple sequence repeats (SSR) were used to assess polymorphism among 16 Coffea arabica and four Coffea canephora accessions, and to identify DNA introgression fragments from C. canephora in four C. arabica lines. Thirty‐one primer pairs allowed for the identification of 92 polymorphic alleles distributed over 37 loci. The C. arabica accessions derived from the genetic bases ‘Typica’ and ‘Bourbon’ were grouped separately according to their genetic origin. Two genotypes derived from a spontaneous hybrid (C arabica×C. canephora) were classified with the C. canephora accessions from Central Africa. Coffea canephora from West Africa were separated from the other accessions studied. Four alleles related to introgression (i.e. present in C. canephora and introgressed lines, and absent in C. arabica) were identified. The SSR markers were used successfully for characterization of a particular cultivar (‘Veranero’) from Costa Rica, which is known for its late maturity.     

Cytological and genetical studies on hybrids between sorghum almum parodi (2n=40) and some diploid (2n=20) species of sorghum

Hybridization between S. almum (2n=40) and diploid (2n=20) species of Sorghum resulted in plants with 2n=40 and 2n=30 chromosomes. As segregation for many characters occurred in the offspring of those with 2n=40, hybrids of this type provide a means of transferring genes from the diploid to the tetraploid species of Eu-sorghum. Segregations for some genes in the progeny of these hybrids revealed heterozygosity in S. almum which may indicate that one of the ancestors of S. almum was a variety of S. vulgare very similar to the commercial grain sorghum.The triploids were only slightly fertile and the chromosome numbers of plants resulting from backcrossing to S. almum ranged from 30 to 46. Some of the plants with the higher chromosome numbers were self-fertile and segregation for genes which were present in the original diploid and tetraploid parents were observed in their off-spring. Backcrossing the triploid to the diploid parent produced fertile plants with 2n=20 and it is possible that the triploids could be used to transfer genes from the tetraploid to the diploid species of Eu-sorghum.The chromosome pairing in the triploids was similar to that expected in an autotriploid, but some non-homologous pairing was detected which may be the result of duplication of some chromosomes or chromosome segments within the genome (n=10) of S. vulgare.     

适于陆地棉品种身份鉴定的SNP核心位点筛选与评价

利用全基因组SNP信息, 筛选陆地棉品种特异的核心SNP位点组合, 可为陆地棉品种身份鉴定提供准确高效的检测手段。本研究利用棉花CottonSNP80K芯片对326份不同来源的陆地棉种质进行SNP分型。以南京农业大学陆地棉TM-1基因组Gossypium hirsutum (AD1) genome NBI v1.1版本为参考序列, 对SNP位点进行注释。结果表明, 93.85% (72 990/77 774)的位点检出率超过99%, 61 595 (79.20%)个SNP位点具有多态性, 其中76.32% (47 009)的位点最小等位基因频率(MAF)大于0.1。基于位点检出率大于0.99、位点具多态性、MAF大于0.2、杂合率小于0.05、每条染色体的SNP密度为400 kb/SNP左右等要求, 最终获得4857个覆盖全基因组的高质量核心SNP位点组合。这些核心SNP位点组合平均检出率接近100%; 平均MAF值为0.34; 平均杂合率为0.02; 99%以上的陆地棉材料均能够被准确鉴定。统计分析表明利用核心SNP位点组合与CottonSNP80K的鉴定结果呈极显著相关。本研究提供了包含4857个SNP位点, 适于陆地棉品种指纹图谱绘制的核心SNP位点组合, 可实现陆地棉品种身份鉴定和品种确权。     

AFLP fingerprinting in Medicago spp.: Its development and application in linkage mapping

Cultivated alfalfa (Medicago sativa L., 2n= 4x= 32) is one of the most important forage crops in temperate climates. The genus Medicago includes diploid species that are a valuable source of wild germplasm for studying the reproductive system of alfalfa and its abnormalities. A linkage map of an apomeiotic mutant of Medicago falcata (L.) Arcang. (2n= 2x= 16) that spanned 368.6 cM and included 29 amplified fragment length polymorphism (AFLP), 35 random amplified polymorphic DNA (RAPD) and three restriction fragment length polymorphism (RFLP) loci was constructed using a one-way pseudo-testcross mapping strategy. The success of such a strategy depends on the presence of sufficiently high levels of heterozygosity in the individual plant which is being mapped and on the informativeness of the marker system that is used. In general: (1) highly informative and reproducible RAPD and AFLP fingerprints were generated and several genome-specific primers selected; (2) of 67 marker loci mapped, 51 were arranged in 11 main linkage groups and eight additional couples of linked marker loci were detected; (3) mapping of an F₁ population theoretically allowed a better estimation of linkage distances since it avoided segregation distortion (x² analyses revealed segregation distortion in only 5.2% of marker loci); (4) the high frequency of unlinked marker loci obtained suggests that, in this alfalfa genotype, DNA markers are distributed throughout the genome. This type of genetic map should find application and prove useful in marker-assisted selection and map-based breeding programmes in meiotic mutants of alfalfa for which there is a lack of suitable genetic markers.     

Additional evidence for oligogenic inheritance of durable host resistance to coffee berry disease (<Emphasis Type="Italic">Colletotrichum kahawae</Emphasis>) in arabica coffee (<Emphasis Type="Italic">Coffea arabica</Emphasis> L.)

Breeding for host resistance to coffee berry disease (CBD) in arabica coffee (Coffea arabica) was initiated some 35–40 years ago in Kenya, Ethiopia and Tanzania in response to severe CBD epidemics. The release of CBD resistant cultivars to the coffee growers has been in progress since 1985. The resistance of cultivars like Ruiru 11 (Kenya) and Ababuna (and other cvs in Ethiopia) appears to be of a durable nature, since confirmed cases of a breakdown of host resistance under field conditions have not been reported over the past 20 years. Host resistance to the hemibiotrophic fungus Colletotrichum kahawae is of a quantitative nature, but nevertheless can be practically complete in some genotypes of arabica coffee. There is still no consensus on the genetics of CBD resistance, some claiming convincing evidence for oligogenes (1–3 major genes) and others for polygenes determining CBD resistance. Results from genetic studies with germplasm from the centre of genetic diversity for C. arabica in Ethiopia are presented here. These together with the recent identification of molecular markers associated with and the mapping of one major gene, provides additional evidence for oligogenic inheritance of CBD resistance. The development of cultivars combining yield and quality with durable host resistance to CBD has contributed greatly to increased sustainability of arabica coffee production in Africa. It has also considerable relevance to arabica coffee in Latin America and Asia, where CBD is still a quarantine disease but with a risk of becoming endemic one day, just as has happened earlier with coffee leaf rust (Hemileia vastatrix).     

Genetic strategies to determine the mode of 2n egg formation in diploid potatoes

Summary In this study, nineteen diploid potato clones (Solanum spp. 2n=2x=24) were identified as 2n egg producers on the basis of fruit set in 2x–4x crosses. The segregation of three genes mapped close to the centromere, Got-1 (1.1 cM), Pgm-2 (2.0 cM), and Sdh-1 (8.3 cM), were analyzed in the tetraploid offspring in these 2x–4x crosses to discriminate between First Division Restitution (FDR) and Second Division Restitution (SDR) modes of 2n egg formation. The co-dominant nature of these markers lead to more precise estimates of the recombinational frequencies as a result of completely classifying the segregating progenies. 2x–4x data revealed a predominance of SDR mechanisms occurring in 20 of the 21 families analyzed. With a SDR mode established, half-tetrad analysis (HTA) of four distal loci, 6-Pgdh-3, Mdh-1, Pgi-1, and Aps-1, revealed two SDR segregation patterns in some of the families. One pattern fit the expectations for the distal arm position. The gene-centromere map distances based upon SDR modes in the families following this pattern, were generally close to 4x–2x (FDR) estimates suggesting similar recombination rates between micro- and mega-sporogenesis. Heterozygosity transmission, on average, was 39.1%. In the other segregation pattern, in which the diploid parents were derived from S. chacoense PI 230580, higher than expected homozygosity levels were found in the female 2n gametophyte populations. A post-meiotic doubling of the reduced megaspore, which generates homozygous 2n eggs, is suggested to operate in three families. The common genetic background of the diploid clones suggested a heritable nature of this mechanism. Pooled data from these three deviant families calculated that 1.8% of the heterozygosity was transmitted to the tetraploid progeny.It is concluded that utilization of seven enzyme-coding loci, with previously established gene-centromere map distances, in 2x–4x crosses improved half-tetrad analysis (HTA) as a means to determine the mode of 2n gamete formation in megasporogenesis and megagametogenesis of diploid Solanum species.     

Waxy proteins in diploid, tetraploid and hexaploid wheats

Electrophoretic analyses of waxy proteins, encoded by genes present at the Wx‐1 loci, present in several cultivars and accessions of hexaploid wheat, Triticum aestivum, have permitted the detection of null alleles at the Wx‐B1 and Wx‐D1 loci. Polymorphism at the Wx‐A1 and Wx‐B1 loci was also investigated in several accessions of tetraploid wheats, Triticum durum, Triticum dicoccoides and Triticum timopheevi, and in diploid species, Triticum urartu, Triticum boeoticum and Triticum monococcum. One null allele at the Wx‐A1 locus and three polymorphic alleles at Wx‐B1 locus were detected in T. durum; a new allele at one of the two waxy loci was identified in the tetraploid wheat T. timopheevi; no polymorphism was detected in diploid species. Polymerase chain reaction techniques made possible the detection of further polymorphism existing at the Wx‐1 loci and the reason for the lack of expression of the null genotypes to be investigated. The null forms detected at each locus have been used to produce complete sets of partial and total waxy lines in durum and bread wheat.     

A method for testing drought tolerance in <Emphasis Type="Italic">Fragaria</Emphasis> based on fast screening for water deficit response and use of associated AFLP and EST candidate gene markers

Drought stress is one of the most important environmental factors that limit plant growth and development, thus reducing yield. The objective of the present research was to correlate the genetic structure of different Fragaria genotypes, as assessed by Expressed Sequence Tag (EST) and Amplified Fragment Length Polymorphism (AFLP) markers, and plant responses to drought stress. Firstly, physiological parameters related to the plant response to drought stress such as leaf relative water content (RWC) and water losing rate (WLR) were measured. WLR and RWC were compared for 20 cultivars of the octaploid Fragaria × ananassa, two ecotypes of the diploid species F. vesca and one octaploid species F. chiloensis. These parameters could discriminate genotypes showing a contrasting response to water stress. Secondly, AFLP and ESTs were compared in terms of their information content and efficiency in the study of genetic diversity and relationships among these 23 Fragaria genotypes. To evaluate the genetic basis for the observed variation in the measured physiological parameter, the effect of specific AFLP/EST loci on WLR and RWC for the different Fragaria genotypes was quantified by Kruskal–Wallis analysis. By Mantel testing, the hierarchical clustering of the Fragaria genotypes based on associated EST or AFLP markers was compared to the observed eco-physiological relevant grouping. A better discriminating capacity for associated markers was noted, enabling a functional marker selection approach to screen the strawberry gene pool for drought tolerance. Correlation of EST markers to leaf RWC and WLR enforces them as potential candidate genes in control of plant responses to drought stress in Fragaria sp.     

西瓜核心种质遗传多样性分析及指纹图谱构建

深入了解西瓜核心种质资源的群体结构及遗传多样性,可以准确指导亲本间杂交组合的有效配置,避免具有相似遗传背景材料的组合,以便提高育种效率。据此,本研究通过采用45对具有多态性的SSR标记对不同来源的78份西瓜核心种质进行了多元统计分析。结果表明,45个SSR标记共检测出175个位点,其中具有多态性位点数为165个,多态性比率达95.41%,且每对引物平均检测到等位基因位点3.67个,PIC大小范围在0.049~0.781之间,平均值为0.415,其有效等位基因数(Ne)、期望杂合度(He)、观测杂合度(Ho)、多样性指数(I)及Nei's基因多样性指数(H)在该群体水平上分别为2.258、0.479、0.521、0.850和0.476,同时筛选出了28对多态性高、带型稳定、易于统计的核心引物,构建了78份西瓜核心种质的SSR指纹图谱。群体结构分析表明,78份西瓜核心种质资源划分为2大类群和4个亚群,其中来自不同地域的美国材料和日本材料分别独立趋于2大类群中,而中国种质材料普遍交叉分布于2大类群中,且各亚群之间存在着明显的基因交流,分子方差分析表明种质间遗传多样性主要存在于品种间和居群间。     

用于区别不同棉花品种基因组特征的微卫星位点筛选

保守性强、重复性好、多态性高的微卫星位点可被有效用于构建作物DNA条形码。选取目前生产上主要推广种植、代表不同来源系统的12个棉花品种作为微卫星位点筛选材料,参考我室构建的四倍体栽培棉种种间高密度遗传图谱信息,从376对覆盖全基因组的SSR引物中,筛选出51对引物可扩增出带型清晰且多态性高的微卫星位点。这些引物在12个供试品种中共产生155个等位位点,每对引物揭示的等位基因位点在2~7之间,平均值为3.04。参照微卫星位点的染色体定位和多态信息,在每条染色体上选择一个多态性相对高的SSR位点,其相应的26对SSR引物被推荐为构建棉花品种DNA条形码的一套首选引物,并初步应用于12个品种的DNA条形码编制。其余25对引物作为候选引物。使用该套引物扩增出的微卫星位点可用于大量棉花品种DNA条形码构建,为棉花品种真实性和纯度的分子鉴定奠定基础。     

筛选普氏原羚粪便DNA中微卫星引物 并应用于个体识别

为了更好地保护极度濒危的普氏原羚物种,选择非损伤性样品-粪便作为研究材料,选用10对非洲糜羚微卫星引物和10对绵羊微卫星引物作为筛选普氏原羚基因组DNA微卫星位点的引物。通过非变性聚丙烯酰胺凝胶电泳检测微卫星PCR的扩增产物,结果发现20对引物中有8对引物在普氏原羚基因组DNA中扩增出了多态性位点。通过等位基因数目和等位基因频率对这8个位点的基因杂合度、多态性信息含量、有效等位基因数进行了计算,结果发现这8个位点在39个普氏原羚粪便样品中的基因杂合度介于0.71~0.84,平均杂合度为0.78;多态性信息含量介于0.79~0.66,平均多态信息含量为0.73;有效等位基因数介于3.40~6.08,平均有效等位基因为5.98,这表明所筛选到的8个微卫星基因座在研究普氏原羚粪便样品中均为中高度多态性基因座,具有比较明显的遗传变异,完全适合普氏原羚各种分子遗传分析。因此试验应用这8对多态性引物对39个粪便样品的个体进行识别,发现这39个粪便样品来自35个不同的个体。