Identification of RAPD markers linked to nut weight and plant stature in cashew

Cashew is an important edible nut crop of tropics. Bulk segregant analysis (BSA) was carried out on DNA bulks constituted from F₂ population and germplasm in order to link or associate molecular markers with economic characters. In all 458 RAPD, 31 ISSR and 21 pairs of SSR primers were used and identified polymorphic markers between parents. Though screening F₂ bulks with these markers identified markers polymorphic between the bulks but none could be validated with the individuals of their bulks. Hence screening with germplasm bulks was carried out and could identify four RAPD markers polymorphic between the bulks for nut weight and plant stature and also between the individuals of their bulks. Of the four, three markers were associated with nut weight amplifying at 775, 475, 275, bp region in primers OPN 14, UBC 184 and UBC 185 respectively. Out of these three, two markers were specific to low nut weight and one marker was specific to high nut weight and their bands were present in greater frequency (50–77.8% and 75%) of individuals constituting the respective bulks. Similarly, the another marker UBC 185₂₇₅ was detected which was specific to low plant stature and was present in 66.7% and 10% individuals constituting short and tall bulks respectively. Markers identified with bulks and with the individuals of bulks were validated further with more individuals of F₂ and germplasm.     

Variety identification and comparative analysis of genetic diversity in yardlong bean (Vigna unguiculata spp. sesquipedalis) using morphological characters,SSR and ISSR analysis

Genetic diversity and relatedness of 23 yardlong bean (Vigna unguiculata spp. sesquipedalis) accessions and 7 accessions of a hybrid between cowpea (V. unguiculata spp. unguiculata) and yardlong bean (dwarf yardlong bean) in Thailand were estimated using morphological characters, simple sequence repeat (SSR) and inter-simple sequence repeat (ISSR) markers. In addition, two mungbean (Vignaradiata (L.) Wilczek) and two blackgram (Vigna mungo (L.) Hepper) accessions were also used as outgroup species for molecular analysis. Five morphological characters were diverse among most accessions. However, five groups of 2–3 accessions could not be distinguished from one another based on these morphological characters alone. Unweighted pair-group arithmetic average (UPGMA) analysis of these characters separated these 30 accessions into 2 major groups; the yardlong bean group and the dwarf yardlong bean group. Eleven of the sixteen SSR primers yielded clear SSRs, ten of which were polymorphic (90.91% polymorphism), detecting a total of 54 alleles with an average of 4.91 alleles per locus. These 10 polymorphic SSR markers successfully distinguished 28 yardlong bean and dwarf yardlong bean accessions. The polymorphic information content (PIC) among genotypes varied from 0.251 to 0.752 with an average of 0.597. Among the 16 ISSR primers used, a total of 312 ISSR fragments were amplified for these three Vigna species, revealing the polymorphism percentage of 91.03%. The average ISSR PIC value (0.197) with the range of 0.137–0.276 was lower than that of SSR. Nevertheless, the average marker index of this multilocus marker was 3.495, which was higher than that of SSR (0.669), owing to the differences in the effective multiplex ratio. In addition, Mantel test cophenetic correlation coefficient was higher for ISSR (0.566) than that of SSR (0.198). These results indicated higher efficiency of ISSR for estimating the levels of genetic diversity and relationships among yardlong beans and dwarf yardlong beans in this study. Pair-wise coefficients of SSR- and ISSR-based genetic similarity among all yardlong bean and dwarf yardlong bean accessions averaged 0.87 and 0.91, respectively, suggesting a narrow genetic base that emphasizes the need to broaden genetic diversity to ensure continued breeding success. Clustering of genotypes within groups was not similar when SSR and ISSR derived dendrograms from UPGMA analysis were compared. It appeared that ISSR was the most effective marker system in determining the genetic variability and relationships among yardlong bean and dwarf yardlong bean accessions and differentiating three Vigna species. In addition, ISSR was also most useful for variety identification since all 30 yardlong beans and dwarf yardlong bean accessions can be effectively distinguished by only four ISSR primers with the highest PIC values.     

与黄瓜矮生基因连锁的ISSR标记及其SCAR转换

以黄瓜矮生品系D8与蔓生品系JIN5杂交并自交得到的124株F₂代为试材,应用ISSR分子标记技术和BSA（混合群体分组分析法）法寻找与黄瓜矮生基因连锁的分子标记。从80条ISSR引物中筛选到一个黄瓜矮生性状特异的多态性引物UBC818,经F₂代单株验证,UBC818的扩增片段在蔓生黄瓜JIN5植株中稳定出现,而在矮生黄瓜D8植株中没有。连锁关系分析表明,标记与黄瓜矮生基因间的遗传距离为11.1 cM。根据对该片段的序列分析结果重新设计了1对引物,将ISSR标记成功转化成了SCAR标记, 并命名为UBC818-S。     

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.     

Assessment of genetic purity of tomato (Lycopersicon esculentum L.) hybrid using molecular markers

Three DNA molecular marker systems, RAPD, ISSR and SSR, were used to test seed genetic purity of two commercial hybrid tomato (Lycopersicon esculentum L.) cultivars ‘Hezuo 903’ and ‘Sufen No. 8’. Genomic DNA from the two F₁ hybrid cultivars and their corresponding parental lines was screened with 218 RAPD decamer primers, 54 ISSR primers and 49 SSR primers. Among the 321 primers, 4 primers for ‘Hezuo 903’ and 3 for ‘Sufen No. 8’, which could produce both female and male parent-specific markers, were selected for testing the genetic purity. A total of 210 hybrid individuals of each cultivar were analyzed using the identified primers. The combined results of the marker analysis showed that eight of the 210 F₁ plants in ‘Hezuo 903’ and 13 of 210 in ‘Sufen No. 8’ were false hybrids, and the overall genetic purity of the two F₁ hybrid seed lots was 96.2 and 93.8%, respectively. This study showed that RAPD and SSR markers could provide a practical and efficient tool in quality control of the tomato commercial hybrid seeds.     

Genetic diversity of Indonesian cauliflower cultivars and their relationships with hybrid cultivars grown in Australia

The objectives of this study were to investigate genetic variation and relationships between Indonesia-, Australian- and European-based cultivars and to evaluate variation within Indonesia cultivars as all cultivars are open-pollinated. Eight cauliflower cultivars collected from three production regions in Indonesia and four F₁ hybrids cultivars grown in Australia were evaluated using RAPD and ISSR markers. DNA polymorphisms generated from 10 polymorphic RAPD primers were used to construct a dendogram using the unweighted pair-group method with arithmetic averages (UPGMA) and to generate a fingerprinting key. ISSR marker analysis using 14 primers were attempted but DNA polymorphisms could not be clearly identified. The RAPD technique indicated that variation occurred both within and between Indonesian cultivars. Comparison between Indonesian-, Australian- and European-based cultivars showed that Indonesian cultivars have unique genotypes and would be good sources of genes for future crop improvement.     

Race-specific genetics of resistance to black rot disease [Xanthomonas campestris pv. campestris (Xcc) (Pammel) Dowson] and the development of three random amplified polymorphic DNA markers in cauliflower

Summary

In order to understand the genetics of resistance to black rot disease caused by Xanthomonas campestris pv. campestris (Xcc) (Pammel) Dowson in cauliflower (Brassica oleracea var. botrytis L.) and to identify random amplified polymorphic DNA (RAPD) markers that segregate with the resistance genes, susceptible (‘Pusa Himjyoti’, female parent) and resistant (‘BR-161’, pollen parent) plants were crossed. Six generations of plants (30 P₁, 30 P₂,30 F₁, 120 F₂, 90 B₁, and 90 B₂) were evaluated for the presence or absence of black rot disease in a randomised block design with three replications. The pattern of segregation of resistance was tested by the χ² test at the 5% level of significance. All F₁ progeny plants were resistant, and the segregation of resistant and susceptible plants in the F₂ and two backcross generations (B₁ and B₂) showed that a single dominant gene caused resistance to the black rot pathogen in ‘BR-161’. Three polymorphic RAPD markers (OPO-04₈₃₃, OPAW-20₂₅₃₈, and OPG-25₆₂₅) were found by bulk segregant analysis, which produced unique amplicons 833 bp, 2,538 bp, and 625 bp in length, respectively. These markers were associated in coupling phase to the resistance allele. Best fit ratios of 3:1 (resistant:susceptible) in the F₂ plants with the three RAPD markers, suggested that the markers were linked to the single gene controlling black rot resistance. These markers will be useful to identify more closely-linked markers and to develop black rot-resistant hybrid cauliflower varieties.     

Genetic diversity analysis in date palm (Phoenix dactylifera L.): a comparative assessment using ISSR and RAPD marker assays

SUMMARY

A comparative study was conducted to evaluate genetic diversity in 45 genotypes of date palm (Phoenix dactylifera L.), including both male and female plants, employing RAPD and ISSR marker systems. The data were analysed to calculate the total number of bands, the number of polymorphic bands, the percentage polymorphism, the average number of bands per primer, the effective multiplex ratio (EMR), the polymorphic information content (PIC), the marker index (MI), and genetic similarity coefficients. The 37 RAPD and 53 ISSR primers used generated 363 and 608 scorable amplified products, respectively, of which 95.0% and 90.9% were polymorphic. The ISSR markers produced more information than the RAPD markers due to their higher EMR and MI values. Jaccard similarity values among male plants, female plants, and between all male and all female plants varied between 0.72 – 0.80. The results indicate the effectiveness of these two marker systems for demonstrating genetic relationships among date palm genotypes.     

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.     

Breaking the intergeneric hybridization barrier in Carica papaya and Vasconcellea cauliflora

The present investigation was undertaken to develop PRSV (Papaya ringspot virus) resistant hybrids through intergeneric hybridization. Intergeneric hybridization was done involving nine Carica papaya cultivars as female and Vasconcellea cauliflora as male. To break the intergeneric hybridization barrier, various nutrient combinations were used. Among the combinations used, sucrose 5%, sucrose 5% + boron 0.5% and sucrose 5% + CaCl₂ 0.5% improved the fruit set and seed set percentage. A total number of 1197 flowers were pollinated and 308 fruits were obtained. On extraction, 721 seeds were obtained from CO 7, Pusa Nanha and CP 50. Out of 721 F₀ seeds (crossed seeds) sown, 419 seeds germinated and artificial screening for PRSV was carried out 27 days after sap inoculation. Out of 29 F₁ hybrid plants from CO 7 x V. cauliflora cross, only six plants namely CO 7V1 to CO 7V6 were found free from PRSV symptoms. Similarly, out of 55 F₁ hybrids from cross involving Pusa Nanha x V. cauliflora only 23 plants namely PNV1 to PNV23 were found free from the symptoms and 70 plants namely CPV1 to CPV70 out of 335 plants of CP50 x V. cauliflora cross were found free from PRSV symptoms. Among the crosses, Pusa Nanha x V. cauliflora had higher yield under PRSV infected conditions, however, total soluble solids and total sugars were found lesser than the CO 7 x V. cauliflora cross. The hybridity of the progenies were confirmed by using ISSR (Inter Simple Sequence Repeats) primers by the amplification of DNA from progenies and their parents. ISSR primers UBC 856, UBC807 and ISSR primer combinations UBC 856-817, UBC 810-817, UBC 861-817, UBC 856-810, UBC 861-810 and UBC 856-817 clearly amplified specific bands of the male parent, which were present in F₁ progenies, but it was absent in female parents.     

SSHscreen and SSHdb,generic software for microarray based gene discovery: application to the stress response in cowpea

Background  

Suppression subtractive hybridization is a popular technique for gene discovery from non-model organisms without an annotated genome sequence, such as cowpea (Vigna unguiculata (L.) Walp). We aimed to use this method to enrich for genes expressed during drought stress in a drought tolerant cowpea line. However, current methods were inefficient in screening libraries and management of the sequence data, and thus there was a need to develop software tools to facilitate the process.     

Development of ISSR- and RAPD-derived SCAR markers for identification of Gladiolus germplasm

Gladiolus is an economically important ornamental crop, cultivated for its beautiful flowers throughout the world. The correct genotype identification of plant material is very significant for the floriculture industry. The aim of this study was to develop sequence-characterised amplified region (SCAR) markers from RAPD and ISSR fragments for identification and authentication of Gladiolus germplasm. The SCAR markers developed could be easily employed as valuable tools to identify newly developed Gladiolus cultivars. The SCAR markers, viz. ScG12, ScG34, and ScG36, are specific to the DNA from all 62 Gladiolus cultivars, as they did not amplify the DNA of other taxa of the family Iridaceae, including Iris, Amaryllis, and Narcissus. All three SCAR markers distinguished Gladiolus from other taxa of the family Iridaceae, whereas marker ScAm was specific to the ‘Amethyst’ cultivar. Our results confirmed that this particular SCAR marker distinguished the ‘Amethyst’ cultivar from the other 62 Gladiolus cultivars investigated in the present study. This development of SCAR markers based on RAPD and ISSR markers seems to be the maiden attempt for Gladiolus cultivars.     

Association analysis of fruit traits in mulberry species (Morus L.)

Improvement of fruit traits is an important objective in current mulberry breeding programs. In this study, 93 mulberry accessions of diverse origin were genotyped using 15 ISSR markers to identify marker–trait associations with fruit traits. Fifteen ISSR primers generated a total of 104 amplification products, of which 94 were polymorphic, revealing 90.38% polymorphism; the mean PIC value was 0.2698. UPGMA cluster analysis showed clear genetic relationships between the 93 mulberry cultivars, and the major clusters were related to known pedigree relationships and their ecotype. The mean r² value for all intra-chromosomal loci pairs was 0.0210. Marker–trait associations were investigated using the unified mixed-model approach, considering both population structure (Q) and kinship (K). In total, 24 marker–trait associations (p < 0.01) were identified using different ISSR markers. The results suggest that association mapping in mulberry is a viable alternative to quantitative trait loci mapping, and detection of associations between markers and mulberry fruit traits will also provide important information for marker-assisted breeding.     

番茄抗晚疫病基因Ph-3的RAPD标记

 以番茄抗晚疫病材料CLN2037和感病材料T2-03杂交的F₂分离群体的147个单株为试材, 通过抗病性鉴定, 选择高抗单株和高感单株分别建抗、感病池。采用BSA法筛选了230条RAPD引物, 其中引物CCPB272-03 (序列为GGTCGATCTG) 在抗、感病池扩增出多态性片段CCPB272-03₇₄₀ , 通过F₂单株验证后证明CCPB272-03₇₄₀与抗晚疫病基因Ph-3紧密连锁, 遗传距离为518 cM。     

Assessment of the mating system of Camellia sinensis in biclonal seed orchards based on PCR markers

Summary

The mating system parameters of tea (Camellia sinensis) were determined in four isolated biclonal seed orchards using two PCR-based marker technologies, Randomly Amplified Polymorphic DNA (RAPD) and Inter-Simple Sequence Repeats (ISSR), in an open-pollinated array of 180 progeny from six genotypes. The genotypes consisted of the three cultivated varieties of tea, namely China, Assam and Cambod, with 60 progeny of each. High-value multi-locus (t_m = 0.995 ± 0.094) and single-locus (t_s = 0.971 ± 0.011) estimates of out-crossing rates indicated predominant out-crossing and suggested maintenance of adequate genetic variability within families. The t_m estimated from RAPD markers (0.988 ± 0.051) was higher than that estimated based on ISSR markers (0.936 ± 0.022). The difference between t_m – t_s (0.024 ± 0.094) was low, indicating negligible biparental in-breeding. Likewise, the estimated correlation of paternity (r_p = 0.070) was low, indicating that many of the offspring did not share the same paternal genitor, despite the isolation of the two putative parents, which is an indication of high pollen contamination rate and long-distance mating. These results suggest that a review of the current isolation distances between orchards is necessary, to increase the pollination efficiency between two isolated breeding cultivars.     

Cultivar identification and genetic diversity analysis of broccoli and its related species with RAPD and ISSR markers

A total of 18 genotypes of broccoli were subjected to random amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR) marker analysis. Seventy-four RAPD and eight ISSR primers generated 344 and 67 polymorphic bands, respectively. All broccoli genotypes could be distinguished with two-primer combinations, indicating that RAPD and ISSR markers can be used to efficiently identify broccoli cultivars. These 18 broccoli genotypes could be separated into two major sub-groups. The first major sub-group (A) included 13 genotypes and the second major sub-group (B) was comprised of five genotypes belonging to early-maturating cultivars. Genetic diversity analysis was performed on the 18 broccoli genotypes, one radish genotype, and six related Brassica accessions. All accessions could be clustered into two groups (radish and Brassica) based on the unweighted pair group of arithmetic average (UPGMA) cluster analysis. The UPGMA analysis indicated that broccoli is most closely related to cauliflower, than to cabbage and Chinese cabbage.     

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.     

番茄耐热性相关的SSR和RAPD标记筛选

 以耐热性差异较大的热敏感材料 01137和耐热材料CLN2001A杂交产生的256个F₂单株为作图群体,应用SSR和RAPD 分子标记技术筛选了与番茄耐热性数量性状相关的分子标记,得到与耐热性相关的1个SSR标记和1个RAPD标记,对其耐热性遗传的贡献率分别为5.2%和7.7%。利用单标记法在第3 和第7连锁群上各检测到1个与耐热性有关的QTL。利用筛选出的与番茄耐热性显著相关2个分子标记对43份番茄耐热、热敏种质资源进行SSR和RAPD分析,将43份番茄种质资源分成2组,耐热组的分组准确率为93.3%,热敏组为96.5%。     

A SCAR marker linked to the N gene for resistance to root knot nematodes (Meloidogyne spp.) in pepper (Capsicum annuum L.)

The root-knot nematodes (Meloidogyne spp.) are important nematode pests and cause serious diseases in pepper in the world. No molecular markers linked to the nematodes resistance N gene have been reported. In this paper, ‘Carolina Wonder’ (Capsicum annuum L.), a sweet pepper line resistant to root-knot nematode with N gene, ‘20080-5-29’ (C. annuum L.), an inbred line susceptible to root-knot nematode with good horticultural characteristics, and their F₂ progeny with 320 individuals were used as materials. Evaluation of resistance and susceptibility of parental lines, F₁ and F₂ progeny inoculated with root-knot nematodes (Meloidogyne incognita) were carried out. ‘Bulked segregant analysis’ method was used to search for polymorphic markers from 512 pairs of AFLP primers. Based on the assessment of resistance and susceptibility and polymorphism of the AFLP marker in F₂ population, the genetic linkage distance between the AFLP marker and the N gene was estimated. One AFLP marker E39/M41-339 was obtained and transferred to a SCAR marker amplifying a 315 bp DNA fragment linked to the N resistant allele and a 331 bp fragment linked to the N⁺ susceptible allele. The distance between the molecular marker and the nematodes resistance N gene is 6.3 cM. This research delivered a valuable tool for the marker assisted selection of nematodes resistance in pepper.     

利用多种分子标记构建龙眼高密度分子遗传图谱

 以龙眼特优质品种‘凤梨朵’为母本, 大果型主栽品种‘大乌圆’为父本, 杂交创建了‘凤梨朵’ ב大乌圆’F₁群体。从该杂种群体中随机选用94个单株作为作图群体, 连同两个亲本品种, 进行了RAPD、ISSR、SRAP和AFLP分子标记分析, 并运用JoinMap3.0进行连锁分析, 分别构建了‘凤梨朵’和‘大乌圆’的分子遗传图谱, 其中, ‘凤梨朵’的遗传图谱为21个连锁群, 包含183个标记位点,覆盖总图距965.1 cM, 位点间平均遗传距离为5.84 cM; ‘大乌圆’的遗传图谱为22个连锁群, 包含251个标记位点, 覆盖总图距1 064.8 cM, 位点间平均遗传距离为4.65 cM。这是龙眼上首次报道的高密度分子遗传图谱, 为后续的基因定位及辅助选择奠定了良好的基础。