Identifying the markers and tagging a leucine-rich repeat receptor-like kinase gene for resistance to anthracnose disease in vegetable cowpea [Vigna unguiculata (L.) Walp.]

Anthracnose caused by the fungus Colletotrichum lindemuthianum is the most destructive disease of cowpea. Field-type cowpeas show various levels of resistance, whereas pole-type vegetable cowpeas are highly susceptible. Transfer of resistance available in field types to vegetable types is a major breeding objective in cowpea. This paper details the development of an F₂ mapping population by crossing field-type cowpea variety Kanakamony (Vigna unguiculata ssp. cylindrica) with pole-type vegetable cowpea variety Sharika (Vigna unguiculata ssp. sesquipedalis), screening this population with artificial inoculation and Bulked Segregant Analysis (BSA) with random marker systems Random Amplified Polymorphic DNA (RAPD) and Inter-Simple Sequence Repeats (ISSR); the objective is to identify the markers linked with major resistance-contributing genes. RAPD primer OPA02 has yielded marker at 850 bp in susceptible genome, whereas ISSR primers UBC810 and UBC811 have yielded markers at 1.4 kb and 1.5 kb respectively in resistant genomes. These markers were reproducible and their linkage with resistance and suitability in marker assisted selection (MAS) were confirmed through co-segregation analysis in F₃ population. UBC811 marker was eluted, cloned on pGEM-T, and sequenced. The sequence had shown that this marker is anchored on LRR receptor-like serine/t\hreonine protein kinase gene which could be involved in the resistance mechanism.     

葫芦科作物果实品质性状的分子标记与定位研究进展

近年来,人们对果实品质的要求不断提高,而品质性状基因的表达易受环境因素的影响,在一定程度上制约了品质育种的发展。随着分子标记技术的应用,分子标记辅助选择育种成为提高果实品质的有效途径。根据有关文献结合作者课题组的研究成果,综述了葫芦科作物果实品质性状的分子标记与定位研究进展,并对未来葫芦科果实品质育种的研究方向进行了展望,以期为提高果实品质提供参考。     

Utility of RAPD,ISSR, IRAP and REMAP markers for the genetic analysis of Citrus spp.

The application and informativeness of RAPD, ISSR, IRAP and REMAP markers to study the genetic diversity and relationship among the Citrus and its relative genotypes were investigated. High levels of polymorphism were observed for all four marker systems. The RAPD technique generated the highest number of polymorphic bands and average number of polymorphic band per assay unit. Average limit of the discriminating power was very close to its actual discriminating power of each marker. The highest and the lowest values of effective number of patterns were obtained from the marker REMAP (5.94) and RAPD (4.48), respectively. Correlation between the genetic similarities matrices were estimated from all four markers using the Mantel matrix correspondence test, and results showed significant correlations among the RAPD, ISSR, IRAP and REMAP similarity matrices. The highest correlations were found comparing RAPD and ISSR markers, whereas RAPD and REMAP (r = 0.143) markers were poorly correlated. To assess the usefulness of the overall information provided by these marker data for establishing phylogenetic relationships and Citrus germplasm classification, cluster analysis was performed. All four techniques, solely or in combination, discriminated the genotypes very efficiently and generated a high similarity in dendrogram topologies, although some differences were observed. The linkage analysis was conducted based on the segregation of 38 RAPD, 13 ISSR, 19 IRAP and 9 REMAP loci in 96 progeny of an intergeneric cross Citrus sinensis and Poncirus trifoliata. Among the 81 studied loci 65 loci distributed on five linkage groups. Comparing the result obtained with RAPD, ISSR, IRAP and REMAP markers in this study, IRAP and REMAP proved to be as a reliable molecular marker for fingerprinting, mapping and diversity study of Citrus and its relatives.     

ISSR及其在果树上的应用

ISSR是一种基于微卫星序列发展起来的新的分子标记,具有简便、稳定、DNA多态性高等优点。ISSR标记呈孟德尔式遗传,大部分ISSR标记为显性标记。综合有关文献就ISSR的原理、操作及其在果树种质鉴定、遗传多样性检测、亲缘关系分析和遗传图谱构建等方面的应用和进展进行简要的阐述。     

南瓜作物分子标记辅助育种研究进展

现代分子育种中,利用分子辅助育种改良植物品种的重要经济性状已成为主要途径。综述了RFLP、RAPD、AFLP、SSR、ISSR等分子标记在南瓜种质亲缘关系及种质资源遗传多样性、遗传图谱构建、杂交种纯度鉴定及优势分析和QTL定位等方面的研究进展,并展望了南瓜分子标记辅助育种的前景。     

Genetic diversity of Dianthus accessions as assessed using two molecular marker systems (SRAPs and ISSRs) and morphological traits

Dianthus chinensis, Dianthus barbatus and Dianthus superbus are members of the Caryophyllaceae and are grown widely as ornamental plants. Information about relative genetic relationships can facilitate breeding programs. Here, we have compared two polymerase chain reaction (PCR)-based systems (sequence-related amplified polymorphisms (SRAPs) and inter-simple sequence repeats (ISSRs)) and morpological trait measurements for their relative effectiveness in estimating the genetic diversity found between 22 Chinese pink (D. chinensis) inbred-lines, one accession of D. barbatus and one accession of D. superbus. Interspecific differences were readily detected but the markers were less reliable in distinguishing the accessions according to their region of origin or in separating the wild species from the cultivars. Morphological traits were found to be the least effective genetic markers. The relative effectiveness of the three systems as markers for genetic diversity was concluded to be SRAP > ISSR > morphological traits, but the combined data from ISSR + SRAP analyses was superior to all three. The information generated by the SRAP marker system correlated more closely with morphological variability than did the ISSR marker system. The morphological markers of plant height/crown size ratio, lower leaf length, ovary shape index and calyx length showed strong correlations with the genetic diversity index (GD_ij, PPB(II) and PSB) as generated by the percentage of polymorphic bands and percentage of special bands of the PCR-based markers.     

Phylogenetic study and molecular identification of 31 Dendrobium species using inter-simple sequence repeat (ISSR) markers

The genetic diversity of the genus Dendrobium is not well known and the phylogenetic relationship of Dendrobium species are mainly determined by studies of the comparative vegetative anatomy and plant systematics. In the present study, we used the technique of inter-simple sequence repeats (ISSRs) to evaluate genetic diversity and phylogenetic relationship among 31 Dendrobium species from Yunnan region of China. In total, 2368 bands were amplified by 17 ISSR primers, resulting from 278 ISSR loci with 100% polymorphism at genus level. Thirty-one species were unequivocally distinguished based on ISSR fingerprinting. Species-specific ISSR markers were identified in nine of 31 tested Dendrobium species. Unweighted pair-group mean analysis (UPGMA) showed that 31 Dendrobium species were grouped into six clusters, indicating the genus was polyphyletic with several well-supported lineages. The high polymorphism and reliable amplification across species demonstrated the utility of ISSR marker for species diagnosis and genetic diversity study of the genus Dendrobium.     

A preliminary genetic linkage map of chrysanthemum (Chrysanthemum morifolium) cultivars using RAPD,ISSR and AFLP markers

A genetic linkage map of chrysanthemum (Chrysanthemum morifolium) was constructed by genotyping 142 F₁ progeny of the bi-parental cross ‘Yuhualuoying’ × ‘Aoyunhanxiao’ with a combination of RAPD, ISSR and AFLP markers in a double pseudo-testcross mapping strategy. A total of 567 polymorphic markers, including 153 RAPDs, 61 ISSRs and 353 AFLPs, were used in linkage mapping. 336 of 567 (60%) markers were grouped on the two parental maps, leaving 231 (40%) markers unlinked. In the ‘Yuhualuoying’ linkage map, 210 markers including 116 testcross and 94 intercross markers were placed in 12 major and 32 minor (8 triplets and 24 doublets) linkage groups, covering 1034 cM with an average map distance of 6.2 cM between adjacent markers. In ‘Aoyunhanxiao’ linkage map, 190 markers consisting of 113 testcross and 77 intercross markers were resolved into 9 major and 24 minor linkage groups, with genome coverage of 1095 cM and a mean inter-marker separation of 6.9 cM between adjacent markers. Six pairs of homologous linkage groups were established on the basis of 64 intercross markers shared by the two parental maps. The maps lay a foundation for further quantitative traits loci (QTL) mapping and marker-assisted breeding of chrysanthemum.     

大白菜耐热性QTL定位与分析

 应用352个标记位点的大白菜AFLP和RAPD图谱，采用复合区间作图的方法对控制大白菜耐热性的数量性状基因位点(QTL)进行定位和遗传效应研究。用苗期热害指数进行耐热性表型鉴定，共检测到5个耐热性Q几位点，分布于3个连锁群上。ht-1、ht-3和ht-5表现为增效加性效应，ht-2和舡4表现为减效加性效应。ht一2对大白菜耐热性的遗传贡献率最大，其次是ht-5。发现了与5个 I8紧密连锁的侧连分子标记，它们与Q几间的距离为0．1 2．4 cM。这为大白菜耐热性分子标记辅助选择提供了理论基础。关键词：中图分类号：     

Genetic diversity of ash gourd [Benincasa hispida (Thunb.) Cogn.] inbred lines based on RAPD and ISSR markers and their hybrid performance

Ten inbred lines of ash gourd [Benincasa hispida (Thunb.) Cogn.] were crossed to produce 45 F₁ hybrids (without reciprocal) which were evaluated along with the parents for 20 growth- and yield-related traits, in a replicated field trial. High level of heterosis was observed among the hybrids for most of the traits examined, including yield. These inbred lines were analysed by using 42 RAPD primers those produced 282 DNA marker bands. A total of 130 RAPD markers were obtained with a mean of 3.1 per primer, which in combination discriminated all the inbreds from each other. Pair-wise genetic distance measurements ranged from 0.07 to 0.31, suggesting a wide genetic diversity for these inbreds. These inberds were also analysed with five ISSR primers of which four were informative. Twenty-six ISSR marker bands were generated of which 11 were polymorphic with an average of 2.80 per primers. The percentage of polymorphic bands produced were higher in ISSR markers (>80%) than generated through RAPD markers (46%). Although the results indicated significant positive correlations of genetic distance with hybrid performance and heterosis, the RAPD based genetic distance measures and use of limited ISSR markers in this present study could not effectively predict hybrid performance in this crop. The genetic variation among ash gourd inbred lines examined, herein, defined a marker array (combined ISSR and RAPD) for the development of a standard reference for further genetic analyses, and the selection of potential parents for predicting hybrid performance and heterosis.     

Genetic structure of mulberry from different ecotypes revealed by ISSRs in China: An implications for conservation of local mulberry varieties

Mulberry is a perennial and economically important plant that has traditionally been used for feeding the silkworm. Evaluating genetic relationship is important for long-term improvement in mulberry yield, quality and resistance, and for germplasm conservation and identification. Population structure and genetic diversity of 8 mulberry populations from different ecotypes in China were analyzed by ISSR markers. Twelve ISSR primers generated a total of 83 amplification products, of which 50 were polymorphic, revealing 60.24% polymorphism among 66 mulberry local varieties, the mean PIC value was 0.1469. The total heterozygosity (H_T), heterozygosity within population (H_S), diversity between populations (D_ST) were 0.1600, 0.0851 and 0.0749, respectively. The coefficient of population differentiation (G_ST) was 0.4683, indicating that the variations among populations and those within populations contributed 46.8% and 53.2% to the total heterozygosity, respectively. The gene flow (N_m) was 0.5678, suggesting that genetic drift between populations can caused local genetic differentiation and therefore, population divergence. The mean genetic similarity coefficient was 0.8456, genetic similarity coefficient among 8 mulberry populations ranged from 0.8441 to 0.9640, indicating that genetic diversity of different populations existed variation. A dendrogram of all 66 local varieties of mulberry based on the genetic similarity using ISSR markers was generated by UPGMA cluster method. In the dendrogram, most varieties from the same ecotype clustered together.     

Genetics and molecular mapping of gynoecious (F) locus in cucumber (Cucumis sativus L.)

Gynoecious is an important economic trait of cucumber for determinant of earliness and yield, yet genetic mechanism is not well understood for this trait. The experiment was conducted using F₂ mapping population by crossing of PPC-2, a gynoecious and parthenocarpic line with Pusa Uday (monoecious and non-parthenocarpic cultivar). Out of 179 SSR markers screened, 39 markers differentiated the gynoecious and monoecious parents. However, only 17 markers were segregating with F₂ mapping population, those were used for genotyping and linkage map analysis and these markers were placed along with F locus on chromosome 6 covering a total distance of 100.4cM. The SSR markers, SSR13251 and UW020605 were found to be closely linked to gynoecious (F) locus at 1.0 and 4.5 cM, respectively. The segregation of F₂ population of PPC-2 × Pusa Uday and GPC-1 × Punjab Naveen and test crosses for sex type herein suggested that single dominant gene controlled the gynoecious sex expression in cucumber particularly in gynoecious genotypes PPC-2 and GPC-1. Therefore, the monogenic dominant nature of gynoecious sex identified in the present experiment and SSR markers closely linked to the F locus will be useful in marker-assisted backcross breeding for transfering gynoecious trait into horticulturally desirable varieties.     

Genetic diversity and classification of Nelumbo germplasm of different origins by RAPD and ISSR analysis

In this study, RAPD and ISSR markers were used to investigate the genetic diversity and genetic relationships among different germplasm of Nelumbo including 70 Chinese ornamental cultivars, 7 wild Thai genotypes, 2 Nelumbo lutea genotypes and 8 hybrids of Nelumbo nucifera and N. lutea. High genetic diversities of 96.4% and 91.2% respectively were detected in the Nelumbo accessions using RAPD and ISSR markers. A dendrogram based on both RAPD and ISSR clustering data indicated that: (1) the genotypes of N. nucifera and N. lutea from different geographical origins were clustered into different groups. This indicated significant genetic differentiation attributed to extensive periods of geographical isolation and lack of gene exchange; (2) the Thai wild genotypes were separated from Chinese genotypes. This indicated genetic divergence between germplasm from Southeast Asia and that from China. Geographical location appears to have affected genetic diversity due to adaptation of the plants to the different environments. A new Southeastern Asia Lotus category is suggested as an addition to the current lotus cultivars classification system; (3) data on three morphological traits (namely: plant size, petal shape and flower color), showed that only the data on plant size was consistent with the dendrogram constructed from molecular data. This finding suggests that using data on genetic relationships in combination with morphological characteristics would serve to improve the classification system of lotus cultivars currently in use. The finding of previously unknown germplasm in this study indicated the potential of RAPD and ISSR techniques in identifying and managing lotus resources. Both marker techniques are potentially useful in improving the current strategies in breeding and germplasm conservation to enhance the ornamental and economic value of lotus.     

The advancement of research on litchi and longan germplasm resources in China

Litchi and longan are famous southern subtropical fruits in China. Besides closely related to each other taxonomically, litchi and longan share many similarities in origin, cultivation history and environmental requirements; furthermore, the developments of research on litchi and longan germplasm resources are somewhat similar and comparable. This paper reviews the literature on origin and distribution, collection and conservation, classification and identification, selection and breeding of litchi and longan germplasm resources in China. The classification of litchi and longan can be divided into three stages: classification based on morphological traits, classification by isozyme analysis and DNA markers. The expression of morphological traits is often affected by environmental conditions; the number of loci that isozyme analysis can examine is small, cultivars, which are closely related often have identical isozyme patterns. The limitations of classification based on morphological traits and isozyme analysis are then overcome by the utilization of molecular markers. At present, most new litchi and longan cultivars are selected from superior seedlings obtained after open-pollination or sport; germplasm with desirable characteristics such as aborted seeds, rather big-fruit and rather late-bearing have not been fully exploited. In order to achieve the goal of improving fruit quality and agronomic characteristics, it is suggested that additional studies of genetic diversity on litchi and longan germplasm should be carried out with more powerful markers such as SSR and ISSR; marker assisted selection (MAS) should be employed to improve litchi and longan's cross breeding efficiency.     

SSR技术及其在果树上的应用

SSR(Simple sequence repeat)技术以其丰富的多态性、共显性遗传、重复性好和操作简便等优点日益受到重视,已成为植物遗传和育种研究中不可缺少的分子标记。对SSR技术的原理和特点作了简要的介绍,较详细地分析了如何获得SSR引物,特别是综述了果树上SSR引物的研究现状,同时将其与其它几种主要的分子标记进行了比较分析,认为SSR标记检测的位点多态性水平明显高于RFLP,而且重复性优于RAPD;着重介绍了SSR技术在果树种质资源和构建果树遗传图谱及基因定位等研究中的应用现状;指出SSR技术将在果树科研上起到重要的作用。     

Quantitative trait loci for photosynthetic pigment concentration in a doubled-haploid population of Pak Choi (Brassica rapa ssp. chinensis var. communis Tsen et Lee)

Summary

Quantitative trait loci (QTL) mapping and marker development are essential steps in any molecular breeding programme. In this study, QTL for photosynthetic pigment concentration were identified using a doubled-haploid (DH) population of 112 lines derived from an F₁ hybrid of Pak Choi, ‘Shulv’, obtained from a cross between Brassica rapa lines SW-13 and SU-124 through microspore culture. QTL mapping was carried out using a modification to the map of ‘Shulv’. Alignment of the linkage groups was carried out with those of reference B. rapa maps, allowing the assignment of these groups as A01 – A10. In total, 11 QTL were associated with photosynthetic pigment concentration and identified on linkage groups A05, A08, and A10. The maximum phenotypic variance for a single QTL was 38.0%. Markers closely linked to these QTL could assist in the development of Pak Choi cultivars with increased yield, due to having improved photosynthetic capabilities.     

Identification of quantitative trait loci for bolting and flowering times in Chinese kale (Brassica oleracea var. alboglabra) based on SSR and SRAP markers

SUMMARY

Expressed sequence tag-simple sequence repeat (EST-SSR), simple sequence repeat (SSR), and sequence-related amplified polymorphism (SRAP) markers were used to construct the first intra-specific genetic linkage map for B. oleracea var. alboglabra. In addition, QTL locations were determined for bolting and flowering traits. A total of 189 polymorphic marker loci were detected in the two parents, with 45 marker loci not located on any linkage group (LG). The remaining 144 loci defined ten LGs and included 69 EST-SSR loci, three SSR loci, and 72 SRAP loci. The length of the map was 1,173.8 cM, the average distance between loci was 8.15 cM, and the proportion of segregation distortion loci was 25.7%. Three QTLs controlling bolting time and two QTLs controlling flowering time were detected in the F2:3 generation, which explained 37.07% and 18.44% of the phenotypic variation, respectively. Two QTLs controlling bolting time and two QTLs controlling flowering time were detected in the F2 generation, which explained 27.90% and 25.59% of the phenotypic variation, respectively. In both generations, QTLs ftB.1 and ftA.1 were located on LG5, and the distance between the two QTLs was only 1.0 cM, suggesting that they could be at an identical locus. These results provide a useful reference for future QTL studies on bolting and flowering time in B. oleracea var. alboglabra, and can be used to identify markers linked to these QTLs for marker-assisted selection in commercial breeding programmes.     

Analysis of Genetic Relationships Between Wild Roses (<Emphasis Type="Italic">Rosa</Emphasis> L. Spp.) Growing in Turkey

Rosa L. taxa are dominantly used for horticulture, food, medicinal, ornamental and aesthetic purposes. Rosa is represented with approximately 200 wild species in the world. The number of natural Rosa species in Turkey is reported as about 31. Rosa species are widely distributed in all regions in Turkey. In this study, RAPD and ISSR markers were applied to assess genetic diversity and genetic relationships among 27 species of Rosa. The UPGMA cluster was constructed using a combination of data from RAPD and ISSR markers. The dendrogram revealed two main clusters. Each cluster was divided into subgroups. This investigation showed that genetic distance was relatively significant among the species. As the results of the study close genetic relationships were found between evolutionary relations, morphplogical properties, and phytogeographical distributions of the Rosa species, The results also propose that RAPD and ISSR markers are useful tools for indicating genetic relationships among Rosa genotypes.     

A comparative analysis of the genetic diversity between inbred lines of Zinnia elegans using morphological traits and RAPD and ISSR markers

Genetic diversity within Zinnia elegans is key to the genetic improvement of this important ornamental species. Here, morphological traits and RAPD and ISSR molecular markers were used to assess levels of polymorphism across 20 inbred lines. Thirty-four morphological traits were scored and also 147 RAPD marker-fragments, as amplified by 12 arbitrary primers, and 128 ISSR marker-fragments as generated by 9 primers. The number of polymorphic loci, the percentage of polymorphic loci, Shannon's Information index (I) and the effective number of alleles (Ne) were calculated from the RAPD data as 100, 68.03%, 0.3559 and 1.4169, respectively. From the ISSR data, these respective statistics were calculated as 97, 76.38%, 0.4013 and 1.4728. Thus, ISSR markers were considered slightly superior to RAPD markers for assessing genetic diversity between the accessions; however, Mantel's test indicated significant correlation (R = 0.733) of the RAPD and ISSR results. By contrast, the morphological matrix showed low correlation with both RAPD and ISSR data matrices (R = 0.3814 and 0.3765, respectively). Cluster analysis showed that groupings of the accessions according to all three methods correlated well with their geographic region of origin, but flower color was not strongly associated with the genetic classification of these inbred lines of Z. elegans.     

利用重组自交系群体构建番茄AFLP 遗传连锁图谱

 以普通栽培番茄（Solanum lycopersicum）99165-30为母本,野生多毛番茄（Solanum habrochaites）LA1777为父本进行杂交,通过单粒传得到了含有80个F_5︰6家系的重组自交系分离群体,利用荧光AFLP分子标记技术构建番茄分子遗传连锁图谱。AFLP标记采用MseⅠ和EcoRⅠ两种内切酶及荧光标记（IRD-700或IRD-800）的E + 3和非荧光标记的M + 3引物组合进行选择性扩增,扩增结果经95 ℃预变性后在6%变性聚丙烯酰胺凝胶上电泳2.5 h,运用LICOR 公司的NEN Global Edition IR² DNA Analyzer（Model 5200 LI-COR Biosciences,Lincoln,NE）荧光扫描检测DNA多态性。对RILs群体中产生分离的274个AFLP标记运用JoinMap 3.0软件分析,得到一张番茄分子遗传连锁图谱,图谱总长度为662 cM,共包括18个主要连锁群,125个多态性分子标记。每条连锁群上的标记数在3 ~ 22个之间,连锁群的长度在14.0 ~ 58.0 cM的范围内,平均图距在 2.27 ~ 13.3 cM。总平均距离5.3 cM,本研究中构建的番茄永久遗传图谱,为番茄分子辅助育种及重要农艺性状的定位奠定了基础。