Genetic variation among Southern African cultivated peanut (Arachis hypogaea L.) genotypes as revealed by AFLP analysis

The amplified fragment length polymorphism (AFLP) technique, employing two different rare cutters, EcoRI and MluI in combination with the frequent cutter MseI, was used to assess genetic diversity and relationships among 21 closely related cultivated Southern African peanut genotypes. A dendrogram was constructed using Jaccard's coefficient and the UPGMA clustering method. Low levels of polymorphism (on average 2.78%) were detected. Results indicated that both EcoRI/MseI and MluI/MseIAFLP enzyme combinations efficiently detected polymorphism within closely related cultivated peanut, although the EcoRI/MseI enzyme combination detected more fragments per primer combination (on average 67.8) as opposed to29.7 by the MluI/MseI enzyme combination. All 21 genotypes could be uniquely distinguished from each other with a minimum of three MluI/MseI primer combinations. Genetic data correlated well with known species and pedigree data, dividing the 21 genotypes into two main groups corresponding to the two subspecies of Arachis hypogaea namely fastigiata and hypogaea. Divisions within the two main groups correlated with botanical types and pedigree data. This is the first report where MluI/MseI primer combinations were used on cultivated peanut and also the first successful detection of polymorphisms between closely related cultivated peanut genotypes worldwide. This revised version was published online in July 2006 with corrections to the Cover Date.     

Random amplified polymorphic DNA analysis of Australian rice (Oryza sativa L.) varieties

Summary The genetic relationships between rice varieties were analysed by using the polymerase chain reaction (PCR), with arbitrary oligonucleotide primers in the random amplified polymorphic DNA (RAPD) method. PCR with 22 arbitrary primers applied to 37 varieties produced 144 useful markers, of which 67% were polymorphic. Thus, with selected primers sufficient polymorphism could be detected to allow identification of individual varieties. Visual examination of electrophoresis gels and analysis of banding patterns confirmed that commercial Australian and USA lines and their relatives were very closely related, with similarity indices of 88–97%. Three varieties originating from more distant geographical centres were easily distinguished, producing variety-specific amplification profiles and expressing a lower similarity index of 80% to all other varieties tested. PCR offers a potentially simple, rapid and reliable method for rice genotype identification and recognition of lines that could contribute genetic diversity to new commercial varieties.Abbreviations PCR Polymerase Chain Reaction - RAPD Random Amplified Polymorphic DNA     

Simple sequence repeat markers for botanical varieties of cultivated peanut (Arachis hypogaea L.)

Cultivated peanut (Arachis hypogaea L.) consists of six botanical varieties. Identification of DNA markers associated with botanical varieties would be useful in plant genotyping, germplasm management, and evolutionary studies. We have developed 130 simple sequence repeat (SSR) markers in peanut, 38 of which were used in this study because of their ability in detecting genetic polymorphism among 24 peanut accessions. Eight SSR markers were found useful to classify botanical varieties. Among them, six SSR markers were specific to botanical varieties fastigiata and vulgaris, one to botanical varieties hypogaea and hirsuta, and one to botanical varieties peruviana, and aequatoriana. Also, three of them derived from peanut expressed sequence tags (ESTs) were associated with putative genes. As botanical varieties have different morphological traits and belong to different subspecies in A. hypogaea, these markers might be associated with genes involved in the expression of morphological traits. The results also suggested that SSRs (also called microsatellites) might play a role in shaping evolution of cultivated peanut. Multiplex PCR of botanical variety-specific markers could be applied to facilitate efficient genotyping of the peanut lines.     

Integration of AFLP markers into a linkage map of sugar beet (Beta vulgaris L.)

The AFLP (amplified fragment length polymorphism) technique has been applied in establishing an extended linkage map of sugar beet. A total of 120 AFLPs were integrated into an existing linkage map based on RFLP markers. Four primer combinations yielded between 19 and 40 polymorphic bands in an F₂ population consisting of 94 plants. The AFLP loci were evenly distributed over the nine linkage groups, with the exception of linkage group V where the number of AFLPs was significantly low. The AFLPs were found to be reproducible even against the background of different combinations of Taq DNA polymerases and buffers. However, the quantity of higher molecular weight fragments (>400 bp) was reduced when using plant DNA of poor quality as a template. The results of these experiments are discussed, together with possible applications of AFLPs in sugar beet breeding.     

RAPD markers linked to a rust resistance gene in cultivated groundnut (Arachis hypogaea L.)

Groundnut rust (Puccinia arachidis Speg.) is an important air borne pathogen, which causes substantial losses in groundnut yield and quality. Although large numbers of accessions were identified as rust resistant in wild, interspecific derivative and cultivated groundnut species, transfer of resistance to well-adapted cultivars is limited due to linkage drag, which worsens yield potential and market acceptance. A F₂ mapping population comprising 117 individuals was developed from a cross between the rust resistant parent VG 9514 and rust susceptible parent TAG 24. Rust resistance was governed by single dominant gene in this cross. We identified 11 (out of 160) RAPD primers that exhibited polymorphism between these two parents. Using a modified bulk segregant analysis, primer J7 (5′CCTCTCGACA3′) produced a single coupling phase marker (J7₁₃₅₀) and a repulsion phase marker (J7₁₃₀₀) linked to rust resistance. Screening of the entire F₂ population using primer J7 revealed that the coupling phase marker J7₁₃₅₀ was linked with the rust resistance gene at a distance of 18.5 cM. On the other hand, the repulsion phase marker J7₁₃₀₀ was completely linked with rust resistance. Additionally, both J7₁₃₀₀ (P = 0.00075) and J7₁₃₅₀ (P < 0.00001) were significantly associated with the rust resistance. Marker J7₁₃₀₀ identified all homozygous rust resistant genotypes in the F₂ population and was present in all the eight susceptible genotypes tested for validation. Thus, J7₁₃₀₀ should be applicable for marker-assisted selection (MAS) in the groundnut rust resistance breeding programme in India. To the best of our knowledge this is the first report on the identification of RAPD markers linked to rust resistance in groundnut.     

Molecular markers for yellow stem borer Scirpophaga incertulas (Walker) resistance in rice

Breeding for yellow stem borer resistance in rice is difficult owing to the complex genetics of the trait and inherent difficulties in screening. Identification of molecular markers linked to the trait would enhance phenotypic evaluation for the trait. An F₂ population was developed using parents contrasting in their reaction to yellow stem borer resistance. Random Amplified Polymorphic DNA (RAPD) analysis,in conjunction with bulked segregant analysis, enabled us to identify four phenotype-specific RAPD markers. The markers C1₃₂₀ and K6₆₉₅ were linked with resistance and AH5₆₆₀ and C4₁₃₀₀ with susceptibility. The markers K6₆₉₅ and AH5₆₆₀ were linked to the gene(s) at distances of 12.8 cM and 14.9 cM, respectively. Scoring of these markers in a set of germplasm confirmed their reproducibility and their association with the trait. This revised version was published online in August 2006 with corrections to the Cover Date.     

Ribosomal DNA repeat unit polymorphism and heritability in peanut (Arachis hypogaea L.) accessions and related wild species

Repeat unit length and restriction site variation in ribosomal RNA geneclusters (rDNA) was surveyed in 77 Arachis accessions, includingsamples from 39 accessions of cultivated Arachis hypogaea(2n=4x=40), 36 accessions representing 15 related tetraploid and diploidwild species, and two synthetic amphidiploids. Total genomic DNA wasdigested with five restriction enzymes, and probed with three heterologousribosomal clones of wheat and broad bean. Four rDNA repeat unit lengthclasses were recognized in the Arachis species. Restriction site analysisshowed that some SacI, BamHI and TaqI cleavage sites in rDNA unit werehighly conserved. With few exceptions, the variable BamHI and EcoRV siteswere able to differentiate the taxonomic sections and species, respectively.Arachis hypogaea and A. duranensis accessions produced fourrDNA length classes. Among these, three were identical with those of otherArachis species. A SacI restriction site (s) from probe (Ver6-5) cangenerally distinguish the two subspecies A. hypogaea ssp. hypogaea and A. hypogaea ssp. fastigiata. Forty nine per centof bands were polymorphic across the A. hypogaea accessionsanalysed. This study does not support A. batizocoi to be a progenitorof A. hypogaea. For the gene array, the contribution from eachparental genome can be detected in the two synthetic amphidiploids.     

Molecular markers linked to genes for specific rust resistance and indeterminate growth habit in common bean

Bulked segregant analysis was utilized to identify random amplified polymorphic DNA (RAPD) markers linked to genes for specific resistance to a rust pathotype and indeterminate growth habit in an F2 population from the common bean cross PC-50 (resistant to rust and determinate growth habit) × Chichara 83-109 (susceptible to rust and indeterminate growth habit). Six RAPD markers were mapped in a coupling phase linkage with the gene ( Ur-9) for specific rust resistance. The linkage group spanned a distance of 41 cM. A RAPD marker OA4.1050 was the most closely linked to the Ur-9 gene at a distance of 8.6 cM. Twenty-eight RAPD markers were mapped in a coupling phase linkage with the gene ( Fin) for indeterminate growth habit. The linkage group spanned a distance of 77 cM. RAPD markers OQ3.450 and OA17.600 were linked to the Fin allele as flanking markers at a distance of 1.2 cM and 3.8 cM, respectively. The RAPD markers linked to the gene for specific rust resistance of Andean origin detected here, along with other independent rust resistance genes from other germplasm, could be utilized to pyramid the different genes into a bean cultivar for durable rust resistance.     

Molecular diversity and association of SSR markers to rust and late leaf spot resistance in cultivated groundnut (Arachis hypogaea L.)

Molecular diversity and association of simple sequence repeat (SSR) markers with rust and late leaf spot (LLS) resistance were detected in a set of 20 cultivated groundnut genotypes differing in resistance against both diseases. Out of 136 bands amplified from 26 primers, 104 were found polymorphic (76.5%). Cluster analysis (UPGMA) revealed two main clusters separated at 52% Jaccard's similarity coefficient according to disease reaction to rust and LLS. Based on the Kruskal–Wallis one-way anova and simple regression analysis three and four SSR alleles were found associated with rust and LLS resistance, respectively.     

利用RAPD标记对我国主栽的汕优杂交稻和其亲本进行区别和鉴定

利用RAPD技术, 从500个随机寡核苷酸引物(10聚体)中筛选出8个引物能在3个主栽的汕优系统杂交稻组合汕优63、 汕优64 和汕优晚3 及其亲本之间稳定地扩增出12个强的多态性标记。 利用这些多态性标记能够有效地区别汕优63、 汕优64 和汕优晚3 及其亲本。     

Identification and inheritance of male sterility in groundnut (Arachis hypogaea L.)

Summary Some plants without pods but with gynophores were observed in two F₄ progenies of two crosses of goundnut (Arachis hypogaea L.). The flowers on these plants had translucent white anthers with no or a few sterile pollen grains. Three such plants in the succeeding generation were hand pollinated with pollen from a short-duration Indian cv. JL 24. The resulting F₁ hybrid plants (male sterile x JL 24) were normal. Chi-square tests for segregation for male fertile and male sterile plants in F₂ and F₃ generations indicated that the male sterility in these crosses of groundnut is governed by two recessive genes. We designate these genes as ms₁ and ms₂ with ms₁ms₁ms₂ms₂ being a male sterile genotype.Submitted as ICRISAT J. A. No. 1812.     

花生黄曲霉侵染抗性的AFLP标记

本研究利用抗、感黄曲霉菌侵染的花生品种为亲本配制杂交组合“J11×中花5号”,以其F₂分离群体为研究材料，采用AFLP技术和BSA分析方法，获得了与花生黄曲霉菌侵染抗性连锁的2个分子标记，标记与抗性间的遗传距离分别为8.8 cM和6.6 cM;利用获得的分子标记对抗、感黄曲霉的花生种质资源进行了分子鉴定，实验结果表明分子标记与抗性鉴定结果具有较高的一致性，证实了两标记应用于研究群体之外的育种潜力。该抗侵染分子标记的建立为开展花生抗黄曲霉辅助选择育种提供了有效的筛选技术。     

DNA amplification fingerprinting and marker screening for pseudo-testcross mapping of flowering dogwood ( Cornus florida L.)

DNA amplification fingerprinting (DAF) with arbitrary oligonucleotide primers was used to study genetic relationships between cultivars of flowering dogwood (Cornus florida L.), evaluate extent of plant hybridization, and generate markers in pseudo-testcross mapping at the intraspecific level. Modified Taguchi optimization methods defined a robust DAF system based on high annealing temperature (48–52 °C) and primer concentration (typically 8 μM) that was used to study genetic diversity of representative dogwood cultivars and hybrids. Phenetic analysis using cluster and numerical methods showed that: (1) cultivars were relatively conserved at the genetic level; (2) their hybridization could be identified in the F1 progeny in the absence of phenotypic or physiological markers; (3) several cultivars grouped according to their recorded ancestry; and (4) dogwood anthracnose-resistant lines originally selected in Catoctin Mountain Park (Maryland) grouped separately from those of southern origin. The DAF protocol was also tested in pseudo-testcross mapping of dogwood at the intraspecific level. A preliminary screening of parents ‘Pink Sachet’ and ‘Fragrant Cloud’ and 7 F1 segregants with 22 octamer primers produced 703 amplified loci, 30 and 39 of which were male and female markers segregating at 1:1 ratios with 98.6% confidence levels in pseudo-testcross configuration. Overall results show that DAF generated markers very efficiently (3 per primer) despite the close relatedness of parental dogwood cultivars. This study constitutes the basis for a future genetic linkage mapping and marker-assisted selection (MAS) effort initially targeted to control important fungal diseases in dogwood. This revised version was published online in July 2006 with corrections to the Cover Date.     

RAPD-mapping of the distal portion of chromosome 3 of barley, including the BaMMV/BaYMV resistance gene ym4

Experiments were carried out in order to identify RAPD-markers for the BaMMV/BaYMV resistance gene ym4 to facilitate efficient marker-based selection in practical breeding programmes. Linkage analysis was carried out with 283 doubled haploid (DH) barley lines out of a cross between the BaMMV/BaYMV susceptible cv. ‘Igri’ and the resistant cv. ‘Franka’. Using bulked segregant analysis, 310 RAPD-primers were screened for polymorphism between the parents. Four of these primers gave rise to specific bands linked to the resistance gene ym4.     

工业大麻雄性相关RAPD和SCAR标记的筛选与鉴定

利用42条RAPD（Random Amplified Polymorphic DNA）随机扩增引物分析工业大麻品种“火麻一号”组成的雄性或雌性DNA池（DNA pools）,结果显示,引物OPV-08扩增得到一条大小为869bp与工业大麻雄性相关的特异条带。根据测序结果,合成了两条SCAR（Sequence Characterized Amplified Region）标记引物,该SCAR标记不仅可以对工业大麻雌雄异株材料花期已知性别的雌雄植株进行准确鉴定,还可以对幼苗期未知性别的大麻雌雄植株进行鉴定;也可对雌雄同株材料可能出现的雄化进行早期鉴定。这不仅为工业大麻早期性别鉴定提供基础,且为减少雌雄同株材料的雄化提供支撑。     

花生籽仁大小相关性状QTL定位

花生籽仁大小相关性状是决定花生产量的直接因素。为发掘与花生籽仁大小相关的QTL,本研究以中花16×J11构建的RIL群体为材料,得到了一张包含289个SSR标记、21个连锁群、覆盖长度为947.3cM的遗传连锁图谱。连续2年对籽仁大小相关性状鉴定表明,各性状在群体中变异广泛,呈典型正态分布,且大部分性状间显著相关。结合本研究构建的遗传图谱,利用WinCart2.5进行QTL定位分析,2年共检测到66个QTL,贡献率为3.23%~33.01%。与籽仁长(SL)、籽仁宽(SW)、籽仁长宽比(LWR)和百仁重(HSW)相关的QTL分别有18、16、18和14个。在这些QTL中,A05染色体上的区间A05A1500?A05A1530同时存在控制籽仁长(qSLA05.1和qSLA05.2)和百仁重的相关的QTL(qHSWA05.1);B06染色体上的区间A06B135?A06B113同时存在控制籽仁宽(qSWB06.2和qSWB06.4)和百仁重相关的QTL(qHSWB06.4),这些稳定存在的主效QTL将为花生产量相关性状的精细定位和分子育种奠定基础。     

格尔德霉素(GDA)能导致小麦基因组DNA多态性和甲基化修饰增加

刚露白的普通小麦(Triticum aestivum L.)种子用150μmol/L格尔德霉素溶液处理后,小麦根的生长受到抑制.随机扩增多态性DNA标记技术和双限制性内切酶酶切-随机扩增法检测显示,小麦根端分生细胞基因组DNA的多态性和甲基化比例均增加.本研究从分子生物学和表观遗传学水平探讨了格尔德霉素抑制小麦生长的机制.     

Genetic linkage map construction for white jute (Corchorus capsularis L.) using SRAP,ISSR and RAPD markers

White jute (Corchorus capsularis) and dark jute (Corchorus olitorius) are two important cultivated crops that are used for natural fibre production. Some genetic maps have been developed for dark jute, but the genetic map information for white jute (C. capsularis) is limited. In this study, a linkage map comprising 44 sequence‐related amplified polymorphisms (SRAPs), 57 intersimple sequence repeats (ISSRs) and 18 randomly amplified polymorphic DNA (RAPD) covering 2185.7 cM with a mean density of 18.7 cM per locus was constructed in an F₂ population consisting of 185 individuals derived from a cross between two diverse genotypes of ‘Xinxuan No. 1’ and ‘Qiongyueqing’ in white jute. These markers were evenly distributed in the linkage groups without any clustering. This genetic linkage map construction will facilitate the mapping of agronomic traits and marker‐assisted selection breeding in white jute.     

Promoter anchored amplified polymorphism based on random amplified polymorphic DNA (PAAP-RAPD) in cotton

Non-coding sequences account for a majority of the higher plant genome, some of which have important effects in gene regulation and plant development. In an effort to develop molecular marker systems to search for polymorphisms associated with high fiber yield and quality in cotton, we have developed a methodology that could specifically target the regulatory regions of the cotton genome. In this study we designed 10-nucleotide degenerate promoter primers based on conserved core promoter sequences and tested their applicability in PCR amplifications in combination with 10-mer random amplified polymorphic DNA (RAPD) primers. The amplified markers are called promoter anchored amplified polymorphism based on RAPD (PAAP-RAPD). Forty cotton genotypes with diverse genetic and geographical backgrounds were used to test the PAAP-RAPD system using polyacrylamide gel electrophoresis. Based on PAAP-RAPD markers amplified from 12 primer combinations, the 40 genotypes were classified into five distinctive groups: two Upland cotton (Gossypium hirsutum) groups from China, another two Upland cotton groups from the USA, and one group from American Pima cotton (G. barbadense). The groupings are in general consistent with their genetic and geographical origins. Thirty-six PAAP-RAPD and RAPD fragments were cloned and four of them were further subjected to sequence analysis. Signal scanning using software PLACE confirmed that they contained an array of cis-regulatory sequences in addition to the core promoter sequences. The results demonstrate the potential application of PAAP-RAPD as a new marker system specifically targeting regulatory regions of the plant genome.