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.     

Identification of polymorphic DNA markers in cultivated peanut (Arachis hypogaea L.)

The detection of DNA polymorphism in cultivated peanut (Arachis hypogaea L.) is reported here for the first time. The DNA amplification fingerprinting (DAF) and amplified fragment length polymorphism (AFLP) approaches were tested for their potential to detect genetic variation in peanut. The AFLP approach was more efficient as 43% of the primer combinations detected polymorphic DNA markers in contrast to 3% with the DAF approach. However, the number of polymorphic bands identified using primers selected in both approaches was comparable. In the DAF study, when 559 primers of varying types were screened, 17 (mostly 10-mer types) detected polymorphism producing an average of 3.7 polymorphic bands per primer with a total of 63 polymorphic markers. In the AFLP study, when 64 primer combinations (three selective nucleotides) corresponding to restriction enzymes Eco RI and Mse I were screened, 28 detected polymorphism. On an average, 6.7% of bands obtained from these 28 primer pairs were polymorphic resulting in a total of 111 AFLP markers. Our results demonstrate that both AFLP and DAF approaches can be employed to generate DNA markers in peanut and thus have potential in the marker-assisted genetic improvement and germplasm evaluation of this economically important crop. This revised version was published online in July 2006 with corrections to the Cover Date.     

Classification of Southeast Asian mints (Mentha spp.) based on simple sequence repeat markers

Mentha is a complex genus encompassing many species as a consequence of their interspecific hybridization and polyploidy. Southeast Asian mints have been poorly distinguished though they are widely used for culinary and medical purposes. In this study, we have analyzed Southeast Asian mints and known varieties as well as a related Lamiaceae species (Nepeta sp.) using simple sequence repeat (SSR) markers and leaf morphology. Two types of mints were clearly distinguished based on their venation pattern and leaf shape index. We developed 12 SSR markers that allowed good amplification in the Mentha and another Lamiaceae species. In the SSR-based phylogram, the Mentha lines could be delimited into groups I–VI. The Southeast Asian mints divided into groups I and II, and the phylogram separated most of the available species, with groups I and II containing the known species M. × cordifolia and M. arvensis, respectively. The separation of the two groups was supported by a population structure analysis. The SSR markers developed in this study enabled the simultaneous classification of mints and will help improve our understanding of the genetic composition of known mint varieties and as yet unclassified Southeast Asian mints.     

Genetic linkage map of Chinese native variety faba bean (Vicia faba L.) based on simple sequence repeat markers

Simple sequence repeat (SSR) marker is a powerful tool for construction of genetic linkage map which can be applied for quantitative trait loci (QTL) and marker‐assisted selection (MAS). In this study, a genetic map of faba bean was constructed with SSR markers using a 129 F₂ individuals population derived from the cross of Chinese native variety 91825 (large seed) and K1563 (small seed). By screening 11 551 SSR primers between two parents, 149 primer pairs were detected polymorphic and used for F₂ population analysis. This SSR‐based genetic linkage map consisted of 15 linkage groups with 128 SSR. The map encompassed 1587 cM with an average genetic distance of 12.4 cM. The genetic map generated in this study will be beneficial for genetic studies of faba bean for identification of marker‐locus‐trait associations as well as comparative mapping among faba bean, pea and grasspea.     

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.     

Development of genomic and EST-SSR markers in radish (Raphanus sativus L.)

Radish (Raphanus sativus L.) belongs to Brassicaceae family and is a close relative of Brassica. This species shows a wide morphological diversity, and is an important vegetable especially in Asia. However, molecular research of radish is behind compared to that of Brassica. For example, reports on SSR (simple sequence repeat) markers are limited. Here, we designed 417 radish SSR markers from SSR-enriched genomic libraries and the cDNA data. Of the 256 SSR markers succeeded in PCR, 130 showed clear polymorphisms between two radish lines; a rat-tail radish and a Japanese cultivar, ‘Harufuku’. As a test case for evaluation of the present SSRs, we conducted two studies. First, we selected 16 SSRs to calculate polymorphism information contents (PICs) using 16 radish cultivars and four other Brassicaceae species. These markers detected 3–15 alleles (average = 9.6). PIC values ranged from 0.54 to 0.92 (average = 0.78). Second, part of the present SSRs were tested for mapping using our previously-examined mapping population. The map spanned 672.7 cM with nine linkage groups (LGs). The 21 radish SSR markers were distributed throughout the LGs. The SSR markers developed here would be informative and useful for genetic analysis in radish and its related species.     

河南省审定花生品种的指纹图谱构建

利用14个SSR标记构建了河南省2015年之前选育并审定的90个花生品种的DNA指纹图谱,用14个SSR标记产生的95个多态性位点可将90个花生品种完全区分开,其中84个品种间有≥2个位点的差异,在剩余的3对品种中,每对仅有1个差异位点。聚类分析结果表明,在遗传相似系数0.98处,90个花生品种被聚集成88类,有2对品种分别聚集在一起,是由于它们每一个品种分别以另一个品种作亲本选育而成,仅有1个差异SSR位点,表明所构建的指纹图谱是有效的。以遗传相似系数0.95为划分标准,有74.4%的品种具有特异性,与其他作物相比,河南省育成花生品种总体上亲缘关系相对较近。根据60个SSR标记的群体结构分析,90个花生品种可以分为3个亚群,与根据分枝开花习性和荚果类型的分类相吻合,亚群划分情况与聚类分析结果基本一致。     

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。     

Intercropping Studies in Peanut (Arachis hypogaea L.)

With a view to study the effect of intercropping and plant geometries in peanut cv. VRI-1 , a held experiment was conducted at Area Agronomic Centre, Tamil Nadu Co-operative Oilseeds Growers' Federation Limited, Neyveli, India during winter season 1989, (November 89–April 90) under irrigated condition. Three intercrops viz., pigeonpea, sunflower and finger millet were tested at two plant geometries viz., paired rows of 40/20 cm and 45/15 cm. The study indicated that pigeonpea was compatible with peanut and their combination resulted in higher peanut kernel equivalent yield and higher profit. Sunflower and finger millet depressed the base crop yield and found to be unsuitable for intercropping in peanut. Base crop yield was not influenced by the plant geometries.     

花生EST-SSR分子标记的开发

利用NCBI的Genbank数据库中公布的花生53177条EST序列以及本实验室创造的花生栽培品种E12(Arachis hypogaea L.)所构建的Unigene文库中的4 074条EST序列,对这些序列进行前期处理(去除冗余序列,对含有重叠区域的EST序列进行拼接),总共获得非冗余且拼接较长的序列11 260条.通过软件分析发现两个EST库中共包含有1 323个SSR位点,主要是2个和3个核苷酸重复,除此之外也有少量的4核苷酸重复以及复合重复.这些EST-SSR平均长度为18.88 bp,平均每8.5条EST序列就包含有一个SSR位点.其中AG/TC、CTY/GAA重复出现的频率最大,分别占到2个核苷酸重复和3个核苷酸重复的39.3%和22.6%.     

花生巢式群体的脂肪含量遗传分析

巢式群体可以利用多个亲本解析复杂性状的遗传机制。本研究利用1个共同亲本与6个基础亲本所配置巢式组合F_2:3家系的种子脂肪含量数据,分析了花生脂肪含量的遗传模型,旨在探明不同的基础亲本组合中脂肪含量性状的遗传差异,为制定脂肪含量遗传改良的亲本选配和后代选择策略提供依据。6个组合的共同亲本为高脂肪含量的普通型大果品种豫花15号,其他6个基础亲本为不同脂肪含量和不同植物学类型的品种。结果表明,在不同杂交组合中脂肪含量的遗传模式有所不同,6个组合分别符合无主基因模型、1对主基因加性显性模型和2对主基因等显性模型3种遗传模式。各种遗传效应的估计值也各不相同,主基因遗传力从32%到80%,说明不同杂交组合中,控制脂肪含量的基因位点差异及其重组和分离方式不同。高脂肪含量双亲杂交后代的高脂肪含量个体较多,但主基因遗传力较低,不宜在早代实施表型选择;双亲脂肪含量差异较大的后代脂肪含量变异幅度更大,能够选择到不同脂肪含量的类型。本研究也表明,巢式组合具有较丰富的脂肪含量变异类型,揭示出脂肪含量性状遗传的复杂性和多基因调控的特点,为较全面地了解脂肪含量的遗传提供了基础。该巢式群体也将有助于进一步开展脂肪含量的QTL定位研究。     

花生荚果发育过程中的microRNA鉴定与表达分析

花生(Arachis hypogaea L.)是典型“地上开花、地下结果”的作物,为从转录后调控水平解析此独特的果实发育现象,本文应用small RNA测序技术研究荚果发育11个时期果壳及种子中的microRNA及其靶基因。通过测序分别获得212个已知的microRNA和112个新microRNA,其中,已知microRNA包括197个保守microRNA和15个花生特异microRNA,新microRNA来自62个新的microRNA前体序列。表达分析发现,67个microRNA及其靶基因在荚果发育的11个时期存在时空特异性表达,部分microRNA的表达量积累阶段性调节果壳与种子的发育,表明microRNA参与了花生荚果暗发育的整个过程。此外,对28个microRNA与30个靶基因进行荧光定量PCR验证发现,microRNA和靶基因的表达趋势与测序结果基本一致。本研究通过对花生荚果不同发育时期的果壳和种子进行small RNA测序,鉴定参与调控花生荚果膨大相关的microRNA,为研究黑暗条件下植物果实发育的调控机制与花生遗传改良奠定理论基础。     

花生栽培种SSR遗传图谱的构建

花生栽培种品种间分子多态性相对缺乏, 至今未构建出较完整的分子遗传图谱。本研究以粤油13和阜95-5为亲本, 通过杂交构建包含184个F6重组自交系的遗传作图群体。采用652对genomic-SSR引物和392对EST-SSR引物对亲本进行多态性检测, 从中筛选出121对多态性引物, 在亲本中共检测到123个多态性位点。利用作图群体对多态性SSR位点进行遗传连锁分析, 获得包含108个SSR标记(102个genomic-SSR标记和6个EST-SSR标记), 涉及20个连锁群, 总长568 cM, 平均图距为6.45 cM的花生栽培种遗传图谱。与前人构建的花生野生种(A. duranensis × A. stenosperma, AA genome)SSR遗传图谱比较, 初步确定本研究构建的遗传图谱中有11个连锁群与野生种遗传图谱的6个连锁群存在同源关系。     

花生栽培种EST-SSRs分布特征及应用研究

利用自行开发的20 160条花生栽培种荚果EST, 通过序列拼接, 获得8 289条无冗余EST。经搜索, 共检测出740个SSR位点, 分布于651条EST中, 发生频率为7.8%, 平均每6.8 kb EST序列含一个SSR位点。功能注释结果表明具生物过程、分子功能和细胞组分的EST分别为73、111和56条。在花生荚果EST-SSR中, 三核苷酸重复类型出现频率最高, 占总SSR的62.8%, 其次是二核苷酸重复类型, 占总SSR的33.6%。在出现的26类重复基序中, AG/TC重复基序出现频率最高, AAG/TTC次之。利用Primer premier 5从651条含有SSR的EST中共设计引物233对, 从中随机选取100对引物检测EST-SSR在花生栽培种中的多态性及在野生种中的可转移性。结果表明, 有86对引物在供试的22个花生栽培品种中得到有效扩增, 其中10对在栽培种中具有多态性, 每对引物检测出的等位基因数2~3个, 平均2.2个。可扩增引物在野生种中的可转移率为12.5%~100%,平均96%。在野生种间检测出多态性的引物76对,每对引物检测出等位基因2~9个, 平均4.06个。     

The role of mutations in intraspecific differentiation of groundnut (Arachis hypogaea L.)

Summary Based on morphological diversity, cultivated groundnut (Arachis hypogaea L.) is classified into two subspecies (fastigiata and hypogaea) and further into four botanical types (Spanish bunch, Valencia, Virginia bunch and Virginia runner). In a cross between two Spanish cultivars belonging to ssp. fastigiata, a true breeding variant (Dharwad early runner) sharing some characters of both the subspecies was isolated. The variant, on mutagenesis with ethyl methane sulphonate (EMS) yielded a very high frequency of mutants resembling all four botanical types. Some of the mutants produced germinal reversions to Dharwad early runner in later generations indicating genetic instability. While most of the revertants bred true, some of the mutants continued to segregate, wherein each botanical group of mutants produced all other botanical types. A detailed analysis of the breeding behaviour of mutants revealed several unusual features (such as homozygous mutations, mutation outbursts, segregation distortions, somatic mutations and multiple character mutations) that could not be explained through conventional mutation theory. In the light of these findings, the role of mutations in evolutionary differentiation of the crop and the probable mode of their origin have been discussed.     

Segregation distortion in doubled haploid lines of barley (Hordeum vulgare L.) detected by simple sequence repeat (SSR) markers

A total of 147 simple sequence repeat (SSR) markers (including86 barley and 61 wheat microsatellite markers) were tested for their segregation in a doubled haploid (DH) and an F₂ population of barley. The DH population consisted of 71 doubled haploid lines, developed from F₁ plants of a cross between Tadmor and WI2291using isolated microspore culture technique. A genetic linkage map consisting of 43 microsatellite markers was constructed using the DH population. Particularly on chromosome 4H microsatellite markers showed distorted segregation ratios. Segregation of DH lines based on molecular markers were compared with segregation of 92 F₂ lines from the same cross. The proportion of loci deviating from the expected monogenic segregation ratios in the DH population was significantly higher (19/43loci, 44%) than in the F₂ population (7/43 loci, 16%). The deviation was biased towards the WI2291 parent alleles. In line with this observation, WI2291 was found to perform better than Tadmor in regenerating green plantlets with the isolated microspore-culture technique. This revised version was published online in July 2006 with corrections to the Cover Date.     

The classification of varieties of groundnut (Arachis hypogaea L.)

Summary An expanded scheme of groundnut variety classification is presented which is revised in the light of recently collected West African material and is designed in concordance with the most recent taxonomic treatment of the species Arachis hypogaea L. This species consists of two subspecies ssp. hypogaea and ssp. fastigiata Waldron. Each subspecies contains two botanical varieties. Those of ssp. hypogaea are var. hypogaea and var. hirsuta Kohler and those of ssp. fastigiata Waldron are var. fastigiata and var. vulgaris Harz. Each infraspecific taxon contains a morphologically distinct group of cultivated varieties.Grain Legume Research Laboratory, Agricultural Research Council of Malawi, POB215, Lilongwe, Malawi.Department of Agricultural Botany, University of Reading, Reading, England.