A comparison of molecular markers for genetic analysis of macadamia

Various marker systems exist for genetic analysis of horticultural species. Isozymes were first applied to the woody perennial nut crop, macadamia, in the early 1990s. The advent of DNA markers saw the development, for macadamia, of STMS (sequence-tagged microsatellite site), RAPD (randomly amplified polymorphic DNA), and RAF (randomly amplified DNA fingerprinting). The RAF technique typically generates dominant markers, but within the dominant marker profiles, certain primers also amplify multi-allelic co-dominant markers that are suspected to be microsatellites. In this paper, we confirm this for one such marker, and describe how RAF primers can be chosen that amplify one or more putative microsatellites. This approach of genotyping anonymous microsatellite markers via RAF is designated RAMiFi (randomly amplified microsatellite fingerprinting). Several marker systems were compared for the type, amount, and cost-efficiency of the information generated, using data from published studies on macadamia. The markers were also compared for the way they clustered a common set of accessions. The RAMiFi approach was identified as the most efficient and economical. The availability of such a versatile tool offers many advantages for the genetic characterisation of horticultural species.

Using molecular markers to assess the effect of introgression on quantitative attributes of common bean in the Andean gene pool

Progress in bean breeding programs requires the exploitation of genetic variation that is present among races or through introgression across gene pools of Phaseolus vulgaris L. Of the two major common bean gene pools, the Andean gene pool seems to have a narrow genetic base, with about 10% of the accessions in the CIAT core collection presenting evidence of introgression. The objective of this study was to quantify the degree of spontaneous introgression in a sample of common bean landraces from the Andean gene pool. The effects of introgression on morphological, economic and nutritional attributes were also investigated. Homogeneity analysis was performed on molecular marker data from 426 Andean-type accessions from the primary centres of origin of the CIAT common bean core collection and two check varieties. Quantitative attribute diversity for 15 traits was studied based on the groups found from the cluster analysis of marker prevalence indices computed for each accession. The two-group summary consisted of one group of 58 accessions (14%) with low prevalence indices and another group of 370 accessions (86%) with high prevalence indices. The smaller group occupied the outlying area of points displayed from homogeneity analysis, yet their geographic origin was widely distributed over the Andean region. This group was regarded as introgressed, since its accessions displayed traits that are associated with the Middle American gene pool: high resistance to Andean disease isolates but low resistance to Middle American disease isolates, low seed weight and high scores for all nutrient elements. Genotypes generated by spontaneous introgression can be helpful for breeders to overcome the difficulties in transferring traits between gene pools.

Development of PCR-based markers for detection of Leifsonia xyli subsp. xyli in fibrovascular fluid of infected sugarcane plants

DNA of Leifsonia xyli subsp. xyli (Lxx), the causal agent of ratoon stunting disease of sugarcane, was detected in the fibrovascular fluid of sugarcane plants using random amplified polymorphic DNA PCR-based amplification using two 10-mer oligonucleotide primers. The primers OPC-02 and OPC-11 produced Lxx-specific markers of approximately 800 bp and 1000 bp, respectively. A cloned DNA fragment from the 800 bp PCR product (pSKC2-800) hybridised to a single genomic DNA fragment from Lxx when used as a probe in Southern hybridisation. This cloned fragment did not hybridise to L. xyli subsp. cynodontis (Lxc), or L. xyli-like bacteria isolated from grasses in Australia, indicating the usefulness of this DNA fragment as a specific probe for Lxx. A cloned fragment from the 1000 bp PCR product ( pSKC11-1000) hybridised to three genomic fragments in Lxx isolates, one genomic fragment in two of the four isolates of L. xyli-like bacteria, and in two of the four isolates of Lxc isolated from the USA. These results indicate that L. xyli-like bacteria are more likely to be related to Lxc than Lxx. These probes did not hybridise to the DNA from strains of the species of Clavibacter, Rathayibacter, Acidovorax, Ralstonia, Pseudomonas and Xanthomonas tested. Two oligonucleotide primers (21-mer) designed from the pSKC2-800 sequences specifically amplified template DNA from Lxx and detected as few as 5 x 10(4) cells/mL in fibrovascular fluid from sugarcane plants infected with Lxx.

Molecular introduction to head and neck cancer (HNSCC) carcinogenesis

Of all human cancers, HNSCC is the most distressing affecting pain, disfigurement, speech and the basic survival functions of breathing and swallowing. Mortality rates have not significantly changed in the last 40 years despite advances in radiotherapy and surgical treatment. Molecular markers are currently being identified that can determine prognosis preoperatively by routine tumour biopsy Leading to improved management of HNSCC patients. The approach could help decide which early stage patient should have adjuvant neck dissection and radiotherapy, and whether Later stage patients with operable lesions would benefit from resection and reconstructive surgery or adopt a conservative approach to patients with poor prognosis regardless of treatment. In the future, understanding these basic genetic changes in HNSCC would be important for the management of HNSCC. (C) 2004 The British Association of Plastic Surgeons. Published by Elsevier Ltd. All rights reserved.

Markers associated with stalk number and suckering in sugarcane colocate with tillering and rhizomatousness QTLs in sorghum

Two important factors influencing sugar yield, the primary focus of sugarcane plant breeding programs, are stalk number and suckering. Molecular markers linked to both of these traits are sought to assist in the identification of high sugar yield, high stalk number, low-suckering sugarcane clones. In this preliminary mapping study, 108 progeny from a biparental cross involving two elite Australian sugarcane clones were evaluated at two sites for two years for both stalk number and suckering. A total of 258 DNA markers, including both restriction fragment length polymorphisms (RFLPs) and radio-labelled amplified fragments (RAFs), were scored and evaluated using single-factor analysis. Sixteen (7 RFLPs and 9 RAFs) and 14 (6 RFLPs and 8 RAFs) markers were identified that were significantly associated (P < 0.01) with stalk number and suckering, respectively, across both years and sites. The seven and six RFLP markers associated with stalk number and suckering, respectively, were generated by eight different RFLP probes, of which seven had been mapped in sorghum and (or) sugarcane. Of significant interest was the observation that all seven RFLP probes could be shown to be located within or near QTLs associated with tillering and rhizomatousness in sorghum. This observation highlights the usefulness of comparative mapping between sorghum and sugarcane and suggests that the identification of useful markers for stalk number and suckering in sugarcane would be facilitated by focussing on sorghum QTLs associated with related traits.

A genetic linkage map in autotetraploid lucerne adapted to northern Australia, and use of the map to identify DNA markers linked to resistance to Phytophthora medicaginis

Phytophthora root rot, caused by Phytophthora medicaginis, is a major limitation to lucerne ( Medicago sativa L.) production in Australia and North America. Quantitative trait loci (QTLs) involved in resistance to P. medicaginis were identified in a lucerne backcross population of 120 individuals. A genetic linkage map was constructed for tetraploid lucerne using 50 RAPD ( randomly amplified polymorphic DNA), 104 AFLP (amplified fragment length polymorphism) markers, and one SSR ( simple sequence repeat or microsatellite) marker, which originated from the resistant parent (W116); 13 markers remain unlinked. The linkage map contains 18 linkage groups covering 2136.5 cM, with an average distance of 15.0 cM between markers. Four of the linkage groups contained only either 2 or 3 markers. Using duplex markers and repulsion phase linkages the map condensed to 7 homology groups and 2 unassigned linkage groups. Three regions located on linkage groups 2, 14, and 18, were identified as associated with root reaction and the QTLs explained 6 - 15% of the phenotypic variation. The research also indicates that different resistance QTLs are involved in conferring resistance in different organs. Two QTLs were identified as associated with disease resistance expressed after inoculation of detached leaves. The marker, W11-2 on group 18, identified as associated with root reaction, contributed 7% of the phenotypic variation in leaf response in our population. This marker appears to be linked to a QTL encoding a resistance factor contributing to both root and leaf reaction. One other QTL, not identified as associated with root reaction, was positioned on group 1 and contributed to 6% of the variation. This genetic linkage map provides an entry point for future molecular-based improvement of lucerne in Australia, and markers linked to the QTLs we have reported should be useful for marker-assisted selection for partial resistance to P. medicaginis in lucerne.

Evaluation of candidate markers for the peritubular myoid cell lineage in the developing mouse testis

Despite the importance of peritubular myoid (PM) cells in the histogenesis of the fetal testis, understanding the origin and function of these cells has been hampered by the lack of suitable markers. The current study was aimed at identifying molecular markers for PM cells during the early stages of testis development in the mouse embryo. Expression of candidate marker genes was tested by section in situ hybridisation, in some instances followed by immunofluorescent detection of protein products. Collagen type-1, inhibin beta A, caldesmon 1 and tropomyosin 1 were found to be expressed by early-stage PM cells. These markers were also expressed in subsets of interstitial cells, most likely reflecting their common embryological provenance from migrating mesonephric cells. Although not strictly specific for PM cells, these markers are likely to be useful in studying the biology of early PM cells in the fetal testis.

Contrasting quantitative traits and neutral genetic markers for genetic resource assessment of Mesoamerican Cedrela odorata

We compared within-population variability and degree of population differentiation for neutral genetic markers (RAPDS) and eight quantitative traits in Central American populations of the endangered tree, Cedrela odorata. Whilst population genetic diversity for neutral markers (Shannon index) and quantitative traits (heritability, coefficient of additive genetic variation) were uncorrelated, both marker types revealed strong differentiation between populations from the Atlantic coast of Costa Rica and the rest of the species' distribution. The degree of interpopulation differentiation was higher for RAPD markers (F-ST 0.67 for the sampled Mesoamerican range) than for quantitative traits (Q(ST) = 0.30). Hence, the divergence in quantitative traits was lower than could have been achieved by genetic drift alone, suggesting that balancing selection for similar phenotypes in different populations of this species. Nevertheless, a comparison of pair-wise estimates of population differentiation in neutral genetic markers and quantitative traits revealed a strong positive correlation (r = 0.66) suggesting that, for C. odorata, neutral marker divergence could be used as a surrogate for adaptive gene divergence for conservation planning. The utility of this finding and suggested further work are discussed.

Identification of a major locus conferring resistance to powdery mildew (Erysiphe polygoni DC) in mungbean (Vigna radiata L. Wilczek) by QTL analysis

A major locus conferring resistance to the causal organism of powdery mildew, Erysiphe polygoni DC,, in mungbean (Vigna radiata L. Wilczek) was identified using QTL analysis with a population of 147 recombinant inbred individuals. The population was derived from a cross between 'Berken', a highly susceptible variety, and ATF 3640, a highly resistant line. To test for response to powdery mildew, F-7 and F-8 lines were inoculated by dispersing decaying mungbean leaves with residual conidia of E. polygoni amongst the young plants to create an artificial epidemic and assayed in a glasshouse facility. To generate a linkage map, 322 RFLP clones were tested against the two parents and 51 of these were selected to screen the mapping population. The 51 probes generated 52 mapped loci, which were used to construct a linkage map spanning 350 cM of the mungbean genome over 10 linkage groups. Using these markers, a single locus was identified that explained up to a maximum of 86% of the total variation in the resistance response to the pathogen.

Highly organized structure in the non-coding region of the psbA minicircle from clade C Symbiodinium

The chloroplast genes of dinoflagellates are distributed among small, circular dsDNA molecules termed minicircles. In this paper, we describe the structure of the non-coding region of the psbA minicircle from Symbiodinium. DNA sequence was obtained from five Symbiodinium strains obtained from four different coral host species (Goniopora tenuidens, Heliofungia actiniformis, Leptastrea purpurea and Pocillopora damicornis), which had previously been determined to be closely related using LSU rDNA region D1/D2 sequence analysis. Eight distinct sequence blocks, consisting of four conserved cores interspersed with two metastable regions and flanked by two variable regions, occurred at similar positions in all strains. Inverted repeats (IRs) occurred in tandem or 'twin' formation within two of the four cores. The metastable regions also consisted of twin IRs and had modular behaviour, being either fully present or completely absent in the different strains. These twin IRs are similar in sequence to double-hairpin elements (DHEs) found in the mitochondrial genomes of some fungi, and may be mobile elements or may serve a functional role in recombination or replication. Within the central unit (consisting of the cores plus the metastable regions), all IRs contained perfect sequence inverses, implying they are highly evolved. IRs were also present outside the central unit but these were imperfect and possessed by individual strains only. A central adenine-rich sequence most closely resembled one in the centre of the non-coding part of Amphidinium operculatum minicircles, and is a potential origin of replication. Sequence polymorphism was extremely high in the variable regions, suggesting that these regions may be useful for distinguishing strains that cannot be differentiated using molecular markers currently available for Symbiodinium.

Segregation distortion for seed testa color in mungbean (Vigna radiata L. Wilcek)

Genetic segregation experiments with plant species are commonly used for understanding the inheritance of traits. A basic assumption in these experiments is that each gamete developed from megasporogenesis has an equal chance of fusing with a gamete developed from microsporogenesis, and every zygote formed has an equal chance of survival. If gametic and/or zygotic selection occurs whereby certain gametes or zygotic combinations have a reduced chance of survival, progeny distributions are skewed and are said to exhibit segregation distortion. In this study, inheritance data are presented for the trait seed testa color segregating in large populations (more than 200 individuals) derived from closely related mungbean (Vigna radiata L. Wilcek) taxa. Segregation ratios suggested complex inheritance, including dominant and recessive epistasis. However, this genetic model was rejected in favor of a single-gene model based on evidence of segregation distortion provided by molecular marker data. The segregation distortion occurred after each generation of self-pollination from F-1 thru F-7 resulting in F-7 phenotypic frequencies of 151:56 instead of the expected 103.5:103.5. This study highlights the value of molecular markers for understanding the inheritance of a simply inherited trait influenced by segregation distortion.

Comparison of identity by descent and identity by state for detecting genetic regions under selection in a sorghum pedigree breeding program

Seventy sorghum inbred lines which formed part of the Queensland Department of Primary Industries (QDPI) sorghum breeding program were screened with 104 previously mapped RFLP markers. The lines were related by pedigree and consisted of ancestral source lines, intermediate lines and recent releases from the program. We compared the effect of defining marker alleles using either identity by state (IBS) or identity by descent (IBD) on our capacity to trace markers through the pedigree and detect evidence of selection for particular alleles. Allelic identities defined using IBD were much more sensitive for detecting non-Mendelian segregation in this pedigree. Only one marker allele showed significant evidence of selection when IBS was used compared with ten regions with particular allelic identities when IBD was used. Regions under selection were compared with the location of QTLs for agronomic traits known to be under selection in the breeding program. Only two of the ten regions were associated with known QTLs that matched with knowledge of the agronomic characteristics of the ancestral lines. Some of the other regions were hypothesised to be associated with genes for particular traits based on the properties of the ancestral source lines.