Validation of male sex‐specific UBC‐8071200 ISSR marker and its conversion into sequence tagged sites marker in Jojoba: a high precision oil yielding dioecious shrub

There is a recent surge in the marker‐assisted selection of desired sex type among economically important dioecious crops. Simmondsia chinensis (Jojoba), a dioecious crop is of immense agricultural importance where only the female plants are preferred for commerce. DNA fingerprinting technology, ISSR analysis along with bulk segregant analysis (BSA), has been carried out on a diverse set of 17‐ to18‐year‐old mature male and female plants of Jojoba to validate a male sex‐specific ISSR marker, UBC‐807₁₂₀₀ on larger population that comprises 330 female and 255 male plants of Jojoba. This male sex‐specific DNA fragment of ~1200 bp was cloned and sequenced. The sequence was found to be 1120 bp in length and based on the sequence information, a pair of sequence tagged sites primers was developed that amplified a single ~800 bp band, consistently only in all the male populations while no amplification was seen in their female counterparts. The marker was named as Jojoba Male Sex Marker which was further validated on 330 female plants from 22 genotypes and 255 male plants from 17 genotypes.     

Application of inter simple sequence repeat (ISSR) polymorphism for detection of sex-specific molecular markers in hop (Humulus lupulus L.)

Summary Hop (Humulus lupulus L.) is dioecious species with female plants of commercial value. During breeding process it is desirable to identify the sex of hop plants at the stage of seedling. Twenty two inter simple sequence repeat (ISSR) primers were screened on female and male hop genotypes of Russian and European origin. Two ISSR primers revealed fragments specific for male plants of hop. Based on the sequences two pairs of primers were designed. These male specific sequence tagged site (STS) markers were tested on male hop accessions of Russian origin and female hop accessions of Russian, European and American origin. A high homology of male specific hop DNA sequences to expressed sequences from EMBL plants EST database was found, most of which code cell wall glycoproteins. The applicability of ISSR-PCR analysis for development of sex molecular markers in hop is discussed.     

ISSR and SCAR markers linked to the mungbean yellow mosaic virus (MYMV) resistance gene in blackgram [Vigna mungo (L.) Hepper]

A recombinant inbred line (RIL) mapping population (F₈) was generated by crossing Vigna mungo (cv. TU 94‐2) with Vigna mungo var. silvestris and screened for mungbean yellow mosaic virus (MYMV) resistance. The inter simple sequence repeat (ISSR) marker technique was employed to identify markers linked to the MYMV resistance gene. Of the 100 primers screened, 54 showed amplification of which 36 exhibited polymorphism between the parents TU 94‐2 (resistant) and V. mungo var. silvestris (susceptible). Individual plants from 53 RIL populations were analysed and one marker (ISSR811₁₃₅₇) was identified as tightly linked to the MYMV resistant gene at 6.8 cM. Both the phenotype as well as the ISSR811₁₃₅₇ marker segregated in a 1 : 1 ratio. The ISSR811₁₃₅₇ marker was sequenced and sequence characterized amplified region (SCAR) primers were designed (YMV1‐F and YMV1‐R) to amplify the marker. Screening for the SCAR marker in the RIL population distinguished the MYMV resistant and susceptible plants, agreeing well with the phenotypic data. The ISSR811₁₃₅₇ marker was validated using diverse blackgram genotypes differing in their MYMV reaction. The marker will be useful for the development of MYMV‐resistant genotypes in blackgram.     

Data to the sex determination in <Emphasis Type="Italic">Pistacia</Emphasis> species using molecular markers

Sex identification in Pistacia species during the long juvenile stage is an economically desirable objective. Due to the lack of morphological methods to identify sex at this stage, the application of molecular markers is expected to facilitate breeding programs. The aim of our study was to identify a marker closely linked to sex loci in Pistacia atlantica Desf subsp. mutica, P. khinjuk, and P. vera subsp. Sarakhs. Samples were collected from both male and female plants of each species, and their band patterns were analyzed according to the presence or absence of specific bands. Thirty random amplified polymorphic DNA (RAPD) primers and a pair of sequence characterized amplified region (SCAR) primers were tested as potential markers of sex in wild Pistacia species. Among the RAPD primers, only BC₁₂₀₀ was found to amplify a specific sex band present in female plants. Based on our analysis of all individual samples, a fragment of approximately 300 bp was amplified in female trees but absent in male ones. Although sex determination mechanisms in Pistacia are still unknown, they may be controlled by a single locus that acts as a trigger. The SCAR technique has proved to be a reliable technique in gender determination of pistachio genotypes at the seedling phenophase. This method could reduce both the time and costs associated with breeding programs.     

Occurrence and frequency of putatively Y chromosome linked DNA markers in Cannabis sativa L.

DNA from female and male hemp (Cannabis sativa L.) plants belonging to nine different varieties were screened with180 RAPD primers in a search for sex-associated DNA markers. About 1500bands were produced in total, nine primers were found yielding one or two DNA bands amplified in all nine male DNA bulks and absent in all female DNA bulks. These putatively male-associated markers were then scored in three different F1progenies, deriving from a cross between a common male parent and three different female plants. The sex of the progeny was accurately scored on the basis of the floral phenotype, and the presence of the nine male-associated markers was verified by RAPD analysis. In all three progenies examined, all the male plants showed the DNA markers previously identified by bulk segregant analysis (BSA) on the hemp varieties, while all the female plants lacked them. The fact that the association between these markers and the staminate phenotype is found when examining male plants of distantly related varieties, and that such linkage is never broken when different progenies are examined, strongly supports the hypothesis that the markers found are physically located on the Y chromosome, in a region excluded from recombination during meiosis. Another marker was shown to be present in the male parent, in all the male plants of each progeny, and in 50% of the female progenies, while it was absent in the female parent; the possible occurrence of markers deriving from multiple amplification sites of the genome is discussed. This revised version was published online in August 2006 with corrections to the Cover Date.     

Identification of a sequence-tagged site (STS) marker linked to a restorer gene for Ogura cytoplasmic male sterility in radish (Raphanus sativus L.) by non-radioactive AFLP analysis

To identify DNA markers linked to a fertility restorer (Rf) genefor Ogura cytoplasmic male sterility in radish (Raphanus sativus L.),a non-radioactive, amplified fragment length polymorphism (AFLP) analysiswas performed on bulked DNA samples from male-sterile and male-fertileradishes. Ten male-fertile and 10 male-sterile plants selected arbitrarilyfrom an F₂ population made by selfing of F₁ plant from a crossbetween a male-sterile (`MS-Gensuke') plant and a restorer (`Comet') plantwere used as material. Using 32 AFLP primer pairs, one AFLP fragment(AFLP190) which is specific to the bulked DNA samples from male-fertileF₂ plants was identified. AFLP190 was characterized by molecularcloning and nucleotide sequencing, and was converted to a sequence-taggedsite (STS) marker, STS190. A linkage analysis performed in 126individuals of two independent F₂ populations showed tight linkageof STS190 to the Rf gene. The rate of recombination between themarker and Rf was estimated to be less than 1%, making STS1901.2 cM from the gene.     

Genetic analysis of a CSR10 (indica) × Taraori Basmati F3 population segregating for salt tolerance using ISSR markers

Segregation for salinity tolerance and ISSR markers based molecular polymorphism were investigated in a F₃ plant population raised via single-seed descent method from a cross between salt-tolerant indica rice variety CSR10 and salt-susceptible premium traditional Basmati rice variety Taraori Basmati HBC19. A total of 130 F₃plants were evaluated individually for salinity tolerance on 1–9 scale on the basis of seedling growth parameters; the average score ranged between 1.7 to 8.3. Frequency distribution curve obtained using the salinity tolerance data of F₃ population and a chi-square analysis, showed a good fit to a normal distribution. Eleven plants each in the category of salt-tolerant and salt-susceptible were selected from the segregating F₃ population for ISSR marker analysis. A total of 149 bands (4–11 bands per primer) ranging from 200 to 3530 bp were scored for the two rice varieties and the selected CSR10 × HBC19 segregating F₃ plants using 26 ISSR primers. Of these, 89 were monomorphic and 60 were polymorphic. Of the 60 polymorphic bands,36 and 20 bands were specific to CSR10 andHBC19 respectively. The remaining four bands were amplified using UBC primers 810,848, 853 and 886 and present in only some of the CSR10 × HBC19 F₃ plants. Notably, ISSR primers with dinucleotide repeat motif and 5'-anchored end amplified more number of bands (7.0 bands/primer) compared to3'-anchored dinucleotide primers (5.4bands/primer), but 3'-anchored dinucleotide primers revealed higher level of polymorphism (2.6 polymorphic bands/primer) compared to 5'-anchoreddinucleotide primers (1.43 polymorphic bands/ primer). While distribution of majority of the polymorphic bands were more or less in the expected ratios in salt-tolerant and/or salt-sensitive F₃segregating plants, but some of the bands amplified using UBC ISSR primers 823, 825,826, 849, 853, 864, 866 and 884 showed highly skewed distribution. Such polymorphic bands stand greater chances of having a linkage with the genes/ QTLs for salinity tolerance and shall be the target for further studies. This revised version was published online in July 2006 with corrections to the Cover Date.     

Novel male-specific molecular markers (MADC5, MADC6) in hemp

Decamer RAPD primers were tested on dioecious and monoecious hemp cultivars to identify sex-specific molecular markers. Two primers (OPD05 and UBC354) generated specific bands in male plants. These two DNA fragments were isolated, cloned and sequenced. Both markers proved to be unique, since no sequence with significant homology to OPD05₉₆₁ and UBC354₁₅₁ markers were found in databases. These markers were named MADC3 (OPD05₉₆₁) and MADC4 (UBC354₁₅₁) (Male-Associated DNA from Cannabis sativa). The markers were converted into sequence-characterized amplified region (SCAR) markers. The SCAR markers correlated with the sex of the segregating F₂ population and proved the tight linkage to the male phenotype. Results of F₂ plant population analysis suggest these markers are to be linked to the Y chromosome. This revised version was published online in August 2006 with corrections to the Cover Date.     

A SCAR marker for the sex types determination in Colombian genotypes of Carica papaya

Sex type determination in papaya (Carica papaya L.) is very important for crop improvement processes because it accelerates the identification of the fruitful plants. The use of molecular technology provides a quick and reliable identification of sex types in plantlets growing in seedbeds. Random amplified polymorphic DNA (RAPD) markers were used to determine the sex types of Colombian cultivars of dioecious papaya genotypes. This species has three sex types (male, female and hermaphrodite) determined by a multiallelic locus. There are no morphological differences at the chromosome level; therefore the identification of sex types by chromosomal dimorphism is not possible. A RAPD marker of 900 bp was found in male plants, but not in females or hermaphrodites. From this RAPD marker a sequence characterized amplified region (SCAR) was developed and it was possible to amplify fragments from the genomes of male and hermaphrodite plants, but not the female ones. The results indicate that this new SCAR marker will be valuable to determine the sex type of papaya plants.     

Identification, characterisation, and chromosome locations of rye and wheat specific ISSR and SCAR markers useful for breeding purposes

Rye (Secale cereale L. and S. strictum) offers potential to increase the genetic variability and to introduce desirable characters for wheat improvements. Cytogenetic techniques have been used to screen wheat lines containing rye chromatin. These techniques are not adequate since they are highly technical and time consuming. They are not suitable for breeding programs that require rapid screening of large numbers of genotypes. The main objective of this study was to develop and characterize ISSR and SCAR markers that can distinguish wheat from rye genome. Total DNA from wheat, rye, and triticale accessions from different provenances were amplified with ISSR primers in PCR assays. Three wheat-diagnostic sequences were identified. In addition three rye-diagnostic ISSR markers of which, one marker specifically diagnostic for Secale strictum were characterized. Pairs of primers flanking these specific sequences were designed to produce SCAR markers. Two SCAR markers were rye genome-specific. One SCAR was present in all the seven rye chromosome, and another was specific to rye chromosomes two, three, four, and seven. These newly developed ISSR and SCAR markers should be useful to wheat breeders screening genotypes that may contain rye chromatins.     

Expression of S-locus inhibitor gene (Sli) in various diploid potatoes

Summary S-locus inhibitor gene (Sli), which can inhibit gametophytic self-incompatibility in diploid potatoes and alter self-incompatible to self-compatible plants, was introduced by crossing into 32 diploid genotypes as females and its expression in the F₁ and S₁ progenies was investigated. We found that the expression of self-compatibility in the F₁ hybrid progeny depended largely upon the female genotypes and partly upon the male genotypes (=Sli gene donor clones). Successful females produced hybrid plants, in which 67.1% of self-pollinated plants set S₁ seeds. By second selfing upon the S₁ plants, an average of 44.2% of self-pollinated plants were self-compatible. Unsuccessful females produced hybrids, most of which were self-incompatible or male-sterile. Restriction fragment patterns of chloroplast DNA (ctDNA) were able to distinguish successful females (S- or A-type ctDNA) from unsuccessful females (W- or T-type ctDNA). A ctDNA high-resolution marker analysis using seven microsatellites and H3 marker supported a higher degree of differentiation between the two groups of ctDNA types and implied a possible interaction between the cytoplasm and Sli gene function. However, it has been known that the cytoplasm having T-type ctDNA and that derived from Solanum demissum (haplotype 26 of W-type ctDNA) cause male sterility, and the present case with unsuccessful females were likely caused by male sterility rather than the failure of Sli gene function.     

Analysis of molecular variance and population structure in southern Indian finger millet genotypes using three different molecular markers

The genetic relationship among 42 genotypes of finger millet collected from different geographical regions of southern India was investigated using random amplified polymorphic DNA (RAPD), inter simple sequence repeats (ISSR), and simple sequence repeats (SSR) markers. Ten RAPD primers produced 111 polymorphic bands. Five ISSR primers produced a total of 61 bands. Of these, 23 bands were polymorphic. The RAPD and ISSR fingerprints revealed 71.3 and 37.4% polymorphic banding patterns, respectively. Thirty-six SSR primers yielded 83 scorable alleles in which 62 were found to be polymorphic. Out of 36 SSR primers used, 14 primers (46.6%) produced polymorphic bands. The SSR primer UGEP7 produced a maximum number of six alleles. Mean polymorphic information content (PIC) of RAPD, ISSR and SSR were 0.44, 0.28, and 0.14, respectively. Molecular variances among the population were 2, 11, and 1% for RAPD, ISSR, and SSR markers, respectively. SSR produced 99% molecular variance within individuals. RAPD and ISSR markers produced a low level of molecular variance within individuals. The STRUCTURE (model-based program) analysis revealed that the 42 finger millet genotypes could be divided into a maximum of four subpopulations. Based on the Bayesian statistics, each RAPD and SSR marker produced three subpopulations (K=3), while ISSR marker showed four subpopulations (K=4). This study revealed that RAPD and SSR markers could narrow down the analysis of population structure and it may form the basis for finger millet breeding and improvement programs in the future.     

An extended random primer amplified region (ERPAR) marker linked to a dominant male sterility gene in cabbage (Brassica oleracea var. capitata)

Similar to SCAR, an extended random primer amplified region (ERPAR) marker is a PCR amplified genomic DNA fragment at a single genetically defined locus. However, ERPAR uses specific primer pairs derived from RAPD primers by adding bases sequentially to their 3′-ends. As an example, an ERPAR marker was derived from a RAPD marker (OT11₉₀₀) linked to a dominant male sterility gene in cabbage (Brassica oleracea var. capitata). After two cycles of base adding and primer pair screening, a primer pair (5′-TTCCCCGCGACT-3′and 5′-TTCCCCGCGAGA-3′) amplified a single intense band with the same size as OT11₉₀₀. The identity of the new marker and OT11₉₀₀ was verified by segregation analysis. The new marker amplified by this extended primer pair was named as EPT11₉₀₀. The development of ERPAR exploits the importance of 3′-end bases of primers in PCR ERPAR shares advantages of SCAR, but eliminates the need for cloning and sequencing. It is a fast and universal way of converting RAPD markers into stable markers. This revised version was published online in July 2006 with corrections to the Cover Date.     

Appraisal of RAPD and ISSR markers for genetic diversity analysis among cowpea (Vigna unguiculata L.) genotypes

The genetic variability and relationships among 11 cowpea genotypes representing two cultivars and nine elite genotypes were analyzed using 22 random amplified polymorphic DNA (RAPD) and nine inter-simple sequence repeat (ISSR) markers. ISSR markers were more efficient than RAPD assay with regards to polymorphism detection. But the average numbers of polymorphic loci per primer and resolution power were found to be higher for RAPD than for ISSR. Also, the total number of genotype specific marker loci, Nei’s genetic diversity, Shannon’s information index, total heterozygosity, and average heterozygosity were prominent in RAPD as compared to ISSR markers. The regression test between the two Nei’s genetic diversity indices showed low regression (0.3733) between ISSR and RAPD + ISSR-based similarities but maximum (0.9823) for RAPD and RAPD + ISSR-based similarities. The RAPD- and ISSR-generated cultivar- or genotype-specific unique DNA fingerprints able to identify the most diverse genotypes. A dendrogram constructed based on RAPD and ISSR combined data indicated a very clear pattern of clustering according to the groups (cultivars and elite genotypes). The results of principal coordinate analysis were comparable to the cluster analysis. Cluster analysis showed that most diverse genotypes (GP-125 — small size with good seed quality; GP-129, GP-90L — big size with poor seed quality) were separated from moderately diverse cultivars and genotypes. The genetic closeness among GP-129 and GP-90L, JCPL-42, and JCPL-107 could be explained by the high degree of commonness in these genotypes.     

Sex identification in Encephalartos natalensis (Dyer and Verdoorn) using RAPD markers

All cycads are strictly dioecious with a long juvenile stage. Currently, there is no method available to determine the sexuality of seedlings prior to the onset of cone formation. This study aimed to develop a sex specific Random Amplified Polymorphic DNA (RAPD) marker for Encephalartos natalensis. Initially, 140 primers were used to amplify the bulk DNA of five individuals each of known male and female sexuality. While a high degree of polymorphism was observed in the amplification profiles of male and female plants, only primer OPD-20 generated a specific band (∼850 bp) in female DNA. To validate this observation, this primer was re-tested with 69 individuals of E. natalensis. The 850 bp DNA band was present in all 38 female individuals tested and it was consistently absent in all 31 male plants tested. The result offers a rapid and simple test to determine sexuality of E. natalensis seedlings at early stages of development, before the onset of reproductive maturity thereby saving time and economic resources when cultivating these specimens.Electronic Supplementary Material Supplementary material is available to authorised users in the online version of this article at .     

Identification of a SCAR marker linked to a recessive male sterile gene (Tems) and its application in breeding of marigold (Tagetes erecta)

In marigold, an F₂ segregation population of 167 plants was constructed from a cross of a line (M525A) carrying the male sterility trait × an inbred line (f53f). In line M525A, the male sterility trait was controlled by the recessive gene, Tems . The intersimple sequence repeat (ISSR) and sequence-related amplified polymorphism (SRAP) techniques combined with bulked segregant analysis were used to develop markers linked to the trait. From a survey of the 38 ISSR primers and 170 SRAP primer combinations, only one SRAP marker that was closely linked to the target trait was identified and successfully converted into sequence characterized amplified region (SCAR) marker that was located within 2.4 cM from Tems locus. The marker was validated with five other two-type lines and in each case the male fertile plants were reliably identified. This SCAR marker therefore permits the efficient marker-assisted selection of male sterile individuals in breeding programmes of marigold and will greatly facilitate the breeding of F₁ cultivars.     

DNA typing of hops (Humulus lupulus) through application of RAPD and microsatellite marker sequences converted to sequence tagged sites (STS)

Summary Both random amplified polymorphic DNA and microsatellite repeat sequences were investigated as DNA markers for distinguishing hop cultivars. Microsatellite sequences converted to STS markers proved to be most successful. The relative abundance of microsatellite repeat sequences in the hop genome varied depending on the sequence class. Of the repeat types investigated the dinucleotide repeats (GA)_n and (GT)_n are the most highly represented in the hop genome. Microsatellite repeat sequences in hops have been shown to be highly polymorphic and are very informatives as STS molecular markers. A DNA typing system using sequence-tagged microsatellite site markers has been developed which will not only be useful for hop cultivar identification but also marker assisted breeding and quality control purposes.     

Fingerprinting in cucumber and melon (<Emphasis Type="Italic">Cucumis</Emphasis> spp.) Genotypes using morphological and ISSR markers

In the present investigation, 13 Cucumis genotypes from different geographical areas of India were screened for genetic diversity using 19 morphological traits and 15 ISSR primers. The analysis of morphological traits grouped the accessions into six clusters. Cluster V contained the maximum number of genotypes namely Kanivellari, Long Green, Andaman Local, Perundurai Local, and Sempatti Local. Clusters I and VI contained the minimum number of genotypes. Among all the characters, the highest mean value was observed in fruit length (23.38) and the lowest mean value was observed in stripes on the blossom end (1.31). The 15 ISSR primers generated 109 polymorphic alleles. The average number of ISSR alleles generated was 8.3 per primer and the level of polymorphism was 87.20%. The ISSR primer UBC 825 was highly informative with a PIC value of 0.8934. The 13 genotypes were grouped into six clusters based on ISSR markers. Cluster III contained the maximum number of genotypes, namely Kanivellari, Sankagiri Local, Perundurai Local, Long Melon, and Sempatti Local, while Clusters I, II, IV, and V (Karur Local, Andaman Local, Edapaddi Local, and N 78, respectively) contained the minimum number of genotypes. The ISSR profile generated genotypes specific allele namely, UBC 812_700bp and UBC 812_1000bp for Cluster VI which contained Cucumis genotypes collected from the northern part of India. Similarly, UBC 808 produced specific allele UBC 808_650bp formed in Cluster III which contained genotypes collected from Tamil Nadu and Kerala.     

Male and female genetic linkage map of hops, Humulus lupulus

A male and female linkage map of hop has been constructed using 224 DNA polymorphisms (106 amplified fragment length polymorphisms (AFLPs), three random amplified polymorphic DNAs (RAPDs), one RAPD‐sequence‐tagged‐site (STS), and three microsatellite (STSs) segregating in an F₁ population of the English cultivar ‘Wye Target’‐the German male breeding line ‘85/54/15’. Linkage between these loci was estimated using JOINMAP Version 2.0. The final map for the female parent consisted of 110 loci assigned to eight linkage groups covering a distance of 346.7 cM. For the male map, 57 loci could be mapped on nine linkage groups spanning over 227.4 cM. One of these male linkage groups (Gr09‐M) presumably represents the Y chromosome, since all markers assigned (10 AFLPs, three RAPDs and one STS) were closely linked to the male sex (M). Because of their sex‐specific segregation, 10 doubly heterozygous AFLPs spanning a distance of 18.7 cM could be identified as markers describing the X chromosome, which is part of the male and female map. Three STMSs, which had already proved useful in hop genotyping, could be integrated as codominant locus‐specific markers and thus allowed to produce reliable allelic bridges between the female and male counterparts.     

Fingerprinting temperate <Emphasis Type="Italic">japonica</Emphasis> and tropical <Emphasis Type="Italic">indica</Emphasis> rice genotypes by comparative analysis of DNA markers

This paper describes the relative efficiency of three marker systems, RAPD, ISSR, and AFLP, in terms of fingerprinting 14 rice genotypes consisting of seven temperatejaponica rice cultivars, three indica near-isogenic lines, three indica introgression lines, and one breeding line of japonica type adapted to high-altitude areas of the tropics with cold tolerance genes. Fourteen RAPD, 21 ISSR, and 8 AFLP primers could produce 970 loci, with the highest average number of loci (92.5) generated by AFLP. Although polymorphic bands in the genotypes were detected by all marker assays, the AFLP assay discriminated the genotypes effectively with a robust discriminating power (0.99), followed by ISSR (0.76) and RAPD (0.61). While significant polymorphism was detected among the genotypes of japonica and indica through analysis of molecular variance (AMOVA), relatively low polymorphism was detected within the genotypes of japonica rice cultivars. The correlation coefficients of similarity were significant for the three marker systems used, but only the AFLP assay effectively differentiated all tested rice lines. Fingerprinting of backcross-derived resistant progenies using ISSR and AFLP markers easily detected progenies having a maximum rate of recovery for the recurrent parent genome and suggested that our fingerprinting approach adopting the ‘undefined-element-amplifying’ DNA marker system is suitable for incorporating useful alleles from the indica donor genome into the genome of temperate japonica rice cultivars with the least impact of deleterious linkage drag.