Determining genetic origins of aberrant progeny from facultative apomictic Kentucky bluegrass using a combination of flow cytometry and silver-stained RAPD markers

Seeded plants that reproduce through facultative apomixis produce two types of progeny: (1) apomictic progeny genetically identical to the maternal genotype, and (2) aberrant progeny genetically different from the maternal genotype. Aberrant progeny have at least nine different genetic origins depending on gametic ploidy level and whether fertilization was self, cross, or absent. Multiple genetic origins of aberrant progeny complicate the results of basic and applied genetic studies. Determining the genetic origin of progeny plants using traditional techniques, such as cytology, embryology, and segregational studies, is technically difficult in Kentucky bluegrass. We have found that two relatively new techniques, flow cytometry and silver-stained RAPD (ssRAPD) markers, are powerful tools for rapidly determining the genetic origins of aberrant Kentucky bluegrass progeny. Our application of these techniques demonstrate that (1) flow cytometry accurately distinguishes progeny ploidy levels, and (2) ssRAPD markers distinguish progeny resulting from cross-fertilization. Therefore, a combination of flow cytometry and ss-RAPD data would be useful for most genetic studies of aberrant individuals. Moreover, ssRAPD s were found to be of value for measuring the loss of genetic markers from polyhaploids and quantifying the inheritance of parental genomes in polydiploid B_n (n+n) and polytriploid B_III (2n+n) hybrids. Quantifying shared ss-RAPD markers may also be useful for determining genetic relatedness between varieties and germplasm sources.     

Biochemical and molecular markers for investigating the mode of reproduction in the facultative apomict Poa pratensis L.

Isozymes and random amplified polymorphic DNA (RAPD) markers were used for precocious identification of non-maternal plants in progenies of the facultative apomict Poa pratensis. Four progenies obtained from controlled crosses that showed different degrees of apomixis on isozyme analysis of phospho-gluco-isomerases, esterases and peroxidases were chosen for RAPD analysis to generate genomic fingerprints using species-specific primers. At an advanced vegetative stage, a morphological analysis was also performed and characteristics related to growth habit and leaf morphology were observed and recorded. On the basis of the isozyme and RAPD electrophoretic pattern and the morphological appearance, each plant was classified as maternal or aberrant. All three classes of genetic markers employed were able to identify plants that exhibited aberrant traits in the four progenies. Overall, the results of RAPD analysis supported those of isozyme and morphology studies. However, in each progeny, some plants which both isozyme and morphological analyses distinguished as of maternal origin were aberrant according to RAPD analysis. Therefore, the RAPD method proved the most precise screening technique. The greater cost of the molecular approach was offset by its higher accuracy. The use of either three isozyme systems or six primers for PCR amplification seems to be sufficient for reliable estimation of the degree of apomixis. Histological analyses were carried out and the aposporic development of the plant material studied.     

High-frequency plant regeneration from seed-derived callus cultures of Kentucky bluegrass (Poa pratensis L.)

Shoot regeneration from seed-derived callus cultures of Kentucky bluegrass (Poa pratensis L.) was tested on MS basal medium supplemented with four different growth regulators. Regeneration frequencies for medium supplemented with 10 M 2,4-dichlorophenoxyacetic acid (2,4-D), 60 M 4amino-3, 5,6-picolinic acid (picloram), or 30 M 3,6dichloro-o-anisic acid (dicamba) ranged from 0.4 to 4%. Medium supplemented with 30 M dicamba plus 10 M 6-benzylaminopurine (BA) resulted in regeneration of shoots from 20% of the calli tested. Higher rates of growth regulators (60 or 90 M dicamba, 20 M BA) resulted in regeneration of shoots from 45% of calli of the cultivar Baron. In a subsequent study, the response of 12 North American cultivars grown on these media was cultivar-specific, with mean frequencies of regeneration ranging from 4% to 40%.Abbreviations 2,4-D 2,4-dichlorophenoxyaceticacid - dicamba 3,6-dichloro-o-anisic acid - picloram 4-amino-3,5,6-picolinic acid - BA 6-benzylaminopurine     

Generation of large numbers of transgenic Kentucky bluegrass (Poa pratensis L.) plants following biolistic gene transfer

A very efficient transformation system, using biolistic bombardment, has been developed for the production of transgenic plants of Kentucky bluegrass (Poa pratensis L.). Embryogenic calli, initiated from immature embryos, were transformed either with pAct1IHPT-4 containing the hygromycin phosphotransferase (hpt) gene or with pDM803 containing the phosphinothricin acetyltransferase (bar) gene and the β-glucuronidase (uidA) gene. In total 119 independent transgenic plants were recovered from 153 hygromycin-resistant lines. Bialaphos selection yielded a total of 99 bialaphos-resistant lines and from these 34 independent transgenic plants were recovered. Southern blot analysis demonstrated the independent nature of the transgenic plants and also revealed a complex transgene integration pattern with multiple insertions. The first two author contributed equally to this work     

Development of SCAR markers linked to self-incompatibility in <Emphasis Type="Italic">Brassica napus</Emphasis> L.

‘SI1300’ is a self-incompatible Brassica napus line generated by introgressing an S haplotype from B. rapa ‘Xishuibai’ into a rapeseed cultivar ‘Huayou No. 1’. Five S-locus specific primer pairs were employed to develop cleaved amplified polymorphic sequences (CAPS) markers linked the S haplotype of ‘SI1300’. Two segregating populations (F₂ and BC₁) from the cross between ‘SI1300’ and self-compatible European spring cultivar ‘Defender’, were generated to verify the molecular markers. CAPS analysis revealed no desirable polymorphism between self-incompatible and self-compatible plants. Twenty primer pairs were designed based on the homology-based candidate gene method, and six dominant sequence characterized amplified region (SCAR) markers linked with the S-locus were developed. Of the six markers, three were derived from the SRK and SP11 alleles of class II B. rapa S haplotypes and linked with S haplotype of ‘SI1300’. The other three markers were designed from the SLG-A10 and co-segregated with S haplotype of ‘Defender’. We successfully combined two pairs of them and characterized two multiplex PCR markers which could discriminate the homozygous and heterozygous genotypes. These markers were further validated in 24 F₃ and 22 BC₁F₂ lines of ‘SI1300 × Defender’ and another two segregating populations from the cross ‘SI1300 × Yu No. 9’. Nucleotide sequences of fragments linked with S-locus of ‘SI1300’ showed 99% identity to B. rapa class II S-60 haplotype, and fragments from ‘Defender’ were 97% and 94% identical to SLG and SRK of B. rapa class I S-47 haplotype, respectively. ‘SI1300’ was considered to carry two class II S haplotypes and the S haplotype on the A-genome derived from B. rapa ‘Xishuibai’ determines the SI phenotype, while ‘Defender’ carry a class I S haplotype derived from B. rapa and a class II S haplotype from B. oleracea. SCAR markers developed in this study will be helpful for improving SI lines and accelerating marker-assisted selection process in rapeseed SI hybrid breeding program.     

Identification of Prunus armeniaca cultivars by RAPD and SCAR markers

Nineteen cultivars of apricot (Prunus armeniaca) were distinguished using random amplified polymorphic DNA (RAPD) markers. One decamer out of 44 used was useful to differentiate cultivars of the Campania Region from those of Northern Italy, North America and Greece. A sequence characterized amplified region (SCAR) marker was obtained. The results provide a protocol to fingerprint DNA of apricots as an efficient way to quality control and fraud prevention.     

Stable transformation of a recalcitrant kentucky bluegrass (Poa pratensis L.) cultivar using mature seed-derived highly regenerative tissues

Summary An efficient method to produce highly regenerative tissues from seeds of a previously recalcitrant cultivar of Kentucky bluegrass (Poa pratensis L. ev. Kenblue) was established under dim-light conditions (10–30 μE m⁻²s⁻¹, 16-h light) using media supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D; 4.5 or 9.0 μM), 6-benzylaminopurine (BA; 0.44 or 2.2 μM), and a high level of cupric sulfate (5.0 μM). The tissues were co-transformed with three plasmids containing the genes for hygromycin phosphotransferase (hpt), β-glucuronidase (uidA; gus), and a synthetic green fluorescent protein gene [sgfp(S65T)]. From 463 individual explants bombarded, 10 independent transgenic events (2.2%) were obtained after a 3–4-month selection period for hygromycin resistance using 30–100 mg l⁻¹ hygromycin B; of the 10 independent events, seven (70%) were regenerable. Stable integration of the transgene(s) in transgenic plants was confirmed by polymerase chain reaction and DNA blot hybridization analyses. Coexpression frequency of all three genes was 20%; for two transgenes, either hpt/uidA or hpt/sgfp(S65T), coexpression frequency was 30–40%.     

Discovery of a new fragrance allele and the development of functional markers for the breeding of fragrant rice varieties

The recessive fgr gene on chromosome 8 is associated with rice fragrance. It has been reported that this gene is a non-functional badh2 allele and that the functional Badh2 allele encoding putative betaine aldehyde dehydrogenase (BADH2) could render rice non-fragrant. Here we report the discovery of a new badh2 allele and the development of functional markers for the badh2 locus. A total of 24 fragrant and ten non-fragrant rice varieties were studied and sequenced for their Badh2/badh2 loci. Of the 24 fragrant rice varieties, 12 were found to have the known badh2 allele (badh2-E7), which has an 8-bp deletion and three single nucleotide polymorphisms (SNPs) in exon 7; the others had a novel null badh2 allele (badh2-E2), which has a sequence identical to that of the Badh2 allele in exon 7, but with a 7-bp deletion in exon 2. Both null badh2 alleles are responsible for rice fragrance. Based on sequence divergence amongst the functional Badh2 and two null badh2 alleles, we developed functional markers which can be easily used to distinguish non-fragrant from fragrant rice and to differentiate between two kinds of fragrant rice. These functional markers will find their usefulness in breeding for fragrant rice varieties via marker-assisted selection. Weiwei Shi and Yi Yang contributed equally to this work.     

Use of the SSLP-based method for detection of rare apomictic events in a sexual AtSERK1 transgenic Arabidopsis population

Here we present a screening method to evaluate the potential of genes to transfer aspects of apomixis into sexual crop plants. Based on the assumption that an apomictic progeny is an exact genetic replica of the mother plant we employed a set of single sequence length polymorphism (SSLP) markers to identify individuals displaying heterozygosity fixation in segregating sexual populations as an indication of rare apomictic events. Here we present the results of such a study using the Arabidopsis thaliana SOMATIC EMBRYOGENESIS RECEPTOR KINASE 1 (AtSERK1) gene expressed under the control of the AtLTP1 promoter in sexual Arabidopsis plants. In one of the three tested F2 transgenic populations expressing the AtLTP1::AtSERK1 construct we observed two plants with heterozygosity maintenance for the full set of SSLP markers indicating a possible clonal inheritance. However, as their offspring revealed a close to binomial segregation for a number of heterozygous loci, it was concluded that these two putative apomictic plants either lost their clonal ability in the next generation or resulted from incidental recombination events displaying the genotype of the parent. Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.     

Identification of AFLP fragments linked to hydroxysafflor yellow A in Flos Carthami and conversion to a SCAR marker for rapid selection

Hydroxysafflor yellow A (HSYA), an important active compound in treating focal cardiac and cerebral ischemia, is uniquely present in flower petals of Carthamus tinctorius. In this study, inheritance and molecular marker analyses for HSYA trait in safflower were carried out. HSYA contents in parents, cross hybridized F₁ and F₂ individuals were analyzed by high performance liquid chromatography. Results revealed that the presence/absence of HSYA was controlled by one major nuclear gene termed HSya. A total of 48 AFLP primer combinations were screened, and bulked segregant analysis was performed by preparing two pools of 10 present-HSYA and ten absent-HSYA plants selected from the 498 individuals of the F₂ segregating population. Four AFLP markers, AFLP-5, AFLP-7, AFLP-15 and AFLP-16, were identified to be closely associated with HSya. Of those, AFLP-16 was the closest to HSya, estimated at about 9.4 cM in genetic distance. The dominant AFLP-16 marker was converted into a simple sequence characterized amplified region marker based on the sequence information of the cloned flanking regions of the AFLP fragment and was designated as SCM16. Our result has direct application for marker-assisted selection of quality breeding in safflower.     

Identification of SSR markers for a broad-spectrum blast resistance gene Pi20(t) for marker-assisted breeding

The Pi20(t) gene was determined to confer a broad-spectrum resistance against diverse blast pathotypes (races) in China based on inoculation experiments utilizing 160 Chinese Magnaporthe oryzae (formerly Magnaporthe grisea) isolates, among which isolate 98095 can specifically differentiate the Pi20(t) gene present in cv. IR24. Two flanking and three co-segregating simple sequence repeat (SSR) markers for Pi20(t), located near the centromere region of chromosome 12, were identified using 526 extremely susceptible F₂ plants derived from a cross of Asominori, an extremely susceptible cultivar, with resistant cultivar IR24. The SSR OSR32 was mapped at a distance of 0.2 cM from Pi20(t), and the SSR RM28050 was mapped to the other side of Pi20(t) at a distance of 0.4 cM. The other three SSR markers, RM1337, RM5364 and RM7102, co-segregated with Pi20(t). RM1337 and RM5364 were found to be reliable markers of resistance conditioned by Pi20(t) in a wide range of elite rice germplasm in China. As such, they are useful tags in marker-assisted rice breeding programs aimed at incorporating Pi20(t) into advanced rice breeding lines and, ultimately, at obtaining a durable and broad spectrum of resistance to M. oryaze. Wei Li and Cailin Lei contributed equally to this work.     

Irregular meiosis in a somatic hybrid between S. bulbocastanum and S. tuberosum detected by species-specific PCR markers and cytological analysis

A system of randomly amplified polymorphic DNA (RAPD) markers was developed to facilitate the transfer of S. bulbocastanum (blb) genes into the S. tuberosum (tbr) genome by hybridization and backcrossing. DNA from tbr, blb and the hexaploid hybrid was used as a template for polymerase chain reaction (PCR) amplification. Polymorphic RAPD products, originating from 10-mer primers, specific for blb were cloned and sequenced at their ends to allow the synthesis of 18-mer primers. The 18-mer primers allowed a more reproducible assay than the corresponding RAPDs. Of eight 18-mer primer pairs, four amplified the expected products specific for blb. However, the stringency of the primer annealing conditions needed to be carefully optimized to avoid amplification of the homeologous tbr product, suggesting that the original RAPD polymorphisms were due to single base-pair changes rather than deletions or insertions. Two primers used for amplification of backcross 2 progeny segregated in a 11 (presence:absence) ratio; the other two were unexpectedly absent. The most likely explanation for the loss of these markers is irregular meiosis in the original hexaploid hybrid and subsequent elimination of chromosomes. Cytological analysis of the meiosis in the hybrid demonstrated widespread irregular pairing and the presence of lagging univalents. In addition, the first backcross individual used as the parent for the second backcross had 54 chromosomes instead of the predicted 60. In conclusion, our results demonstrate that PCR technology can be used for the efficient isolation of taxon-specific markers in Solanum. Furthermore, by the use of these markers we detected the loss of chromosomes that was subsequently shown by cytological analysis to be caused by irregular meiosis of the somatic hybrid.     

Molecular characterization of RAPD and SCAR markers linked to the Tm-1 locus in tomato

We have cloned and sequenced six RAPD fragments tightly linked to the Tm-1 gene which confers tomato mosaic virus (ToMV) resistance in tomato. The terminal ten bases in each of these clones exactly matched the sequence of the primer for amplifying the corresponding RAPD marker, except for one in which the 5-endmost two nucleotides were different from those of the primer. These RAPD clones did not cross-hybridize with each other, suggesting that they were derived from different loci. From Southern-hybridization experiments, five out of the six RAPD clones were estimated to be derived from middle- or high-repetitive sequences, but not from any parts of the ribosomal RNA genes (rDNA), which are known to be tightly linked with the Tm-1 locus. The remaining clone appeared to be derived from a DNA family consisting of a few copies. These six RAPD fragments were converted to sequence characterized amplified region (SCAR) markers, each of which was detectable using a pair of primers having the same sequence as that at either end of the corresponding RAPD clone. All pairs of SCAR primers amplified distinct single bands whose sizes were the same as those of the RAPD clones. In four cases, the SCAR markers were present in the line with Tm-1 but absent in the line without it, as were the corresponding RAPD markers. In the two other cases, the products of the same size were amplified in both lines. When these SCAR products were digested with different restriction endonucleases which recognize 4-bp sequences, however, polymorphisms in fragment length were found between the two lines. These co-dominant markers are useful for differentiating heterozygotes from both types of homozygote.     

SCAR,RAPD and RFLP markers linked to a dominant gene (Are) conferring resistance to anthracnose in common bean

Anthracnose, caused by the fungusColletotrichum lindemuthianum, is a severe disease of common bean (Phaseolus vulgaris L.) controlled, in Europe, by a single dominant gene,Are. Four pairs of near-isogenic lines (NILs) were constructed, in which theAre gene was introgressed into different genetic backgrounds. These pairs of NILs were used to search for DNA markers linked to the resistance gene. Nine molecular markers, five RAPDs and four RFLPs, were found to discriminate between the resistant and the susceptible members of these NILs. A backcross progeny of 120 individuals was analysed to map these markers in relation to theAre locus. Five out of the nine markers were shown to be linked to theAre gene within a distance of 12.0 cM. The most tightly linked, a RAPD marker, was used to generate a pair of primers that specifically amplify this RAPD (sequence characterized amplified region, SCAR).The research was supported by the CNRS and the Ministère Français de l'Education Nationale     

Development of SCAR markers linked to male sterility and very high linoleic acid content in safflower

Practically no molecular tools have been developed so far for safflower (Carthamus tinctorius L.) breeding. The objective of the present research was to develop molecular markers for the closely linked genes Li, controlling very high linoleic acid content, and Ms, controlling nuclear male sterility (NMS). A mapping population of 162 individuals was developed from the NMS line CL1 (64–79% linoleic acid) and the line CR-142 (84–90%), and phenotyped in the F₂ and F₃ generations. Bulked segregant analysis with random amplified polymorphic (RAPD) markers revealed linkage of five RAPD bands to the Li and Ms genes. RAPD fragments were converted into sequence-characterized amplified region (SCAR) markers. A linkage map including the five SCAR markers and the Li and Ms genes was constructed. SCAR markers flanked both loci at minimum distances of 15.7 cM from the Li locus and 3.7 cM from the Ms locus. These are the first molecular markers developed for trait selection in safflower.     

Poa annua L. in Antarctic: searching for the source of introduction

The level of polymorphism, genetic variability and relatedness of a population of Poa annua L. from South Shetlands Islands was studied and compared with results obtained for populations from two potential sources of introduction (Argentina—Ushuaia and Poland—Dziekanów Leśny) using the amplified fragment length polymorphism (AFLP) approach. Five primer pairs used for AFLP profiling amplified 226 scoreable DNA fragments that were used for Clustral and Factorial analyses. The level of molecular variability among all individuals from all the analysed populations reaches 30%. Clustral and Factorial analyses show that all populations formed clear-cut uniform groups according to their locations. However, population from King George Island show high variability. High genetic diversity may be related with escalated human activity at the area of Arctowski Station, favouring introductions of P. annua from many different sources and by many different vectors.     

Development of a multiallelic SCAR marker for the scab resistance gene Vr1/Vh4/Vx from R12740-7A apple and its utility for molecular breeding

A major scab resistance gene initially called Vr1 was identified in the apple cultivar “Regia” derived from the Malus scab resistance source R12740-7A (Russian seedling, RS). A codominant, multiallelic sequence characterized amplified region (SCAR) marker was developed from a random amplified polymorphic DNA marker identified by bulked-segregant analysis. Additional alleles of the AD13 marker locus proved to be informative for the analysis of genetic relationships within Malus including putative relatives of RS. Separate linkage maps were created for the two families derived from crosses with “Regia”. Using phenotypic data from the greenhouse scab tests, the recombination frequency between Vr1 and AD13-SCAR was between 6 and 17%. The Vr1 locus appeared to be closely linked to the Vx [Hemmat et al. J Am Soc Hortic Sci, 127:365–370, 2002], Vr2 [Patocchi et al. Theor Appl Genet, 109:1087–1092, 2004], and the Vh4 gene [Bus et al. Mol Breed, 15:103–116, 2005a]. Our linkage analysis of the molecular markers identified by Hemmat et al. [J Am Soc Hortic Sci, 127:365–370, 2002] for two scab resistance factors from RS (Vr and Vx) indicate that both genes are separated by a large distance on apple linkage group 2 [Boudichevskaia et al. Acta Hortic, 663:171–175, 2004]. This is in agreement with the results of Bus et al., [Mol Breed, 15:103–116, 2005a] who concluded that (1) the RS-derived gene Vh2 is identical to Vr, (2) the RS-derived gene Vh4 is identical to Vx and Vr1, (3) Vh2/Vr and Vh4/Vr1/Vx map on opposite sides of LG 2. One of our main goals was the verification of the Vr1-SCAR within a practical apple-breeding program. The utility of the AD13-SCAR was evident after 2 years under natural scab infection conditions in both families investigated. This is the first report about the confirmation of a molecular marker for a RS resistance factor in a 2-year field experiment. A multiplex polymerase chain reaction assay based on two codominant SCARs for Vf and Vr1 was tested in an apple progeny segregating for both genes. The result of the two-marker approach is discussed with respect to scab races, which are able to overcome the Vf resistance gene.     

Identification of AFLP markers in soybean linked to resistance to Meloidogyne javanica and conversion to Sequence Characterized Amplified Regions (SCARs)

Meloidogynejavanica is the most widely spread nematode pest on soybean in SouthAfrica. Only a few registered commercial South African cultivars are poor hostsof this nematode species and there is an urgent need for an efficient breedingprogramme for resistant cultivars of all maturity groups. However, breeding ishampered by laborious screening procedures for selection of poor host cultivarsand/or lines. The objective of this study was to develop an economically viablemolecular marker system for application in selection procedures. BothRestriction Fragment Length Polymorphism (RFLP) and Amplified Fragment LengthPolymorphism (AFLP) screening techniques identified markers linked togall-indexvariation in a segregating population of 60 F₂ progeny from a crossbetween a resistant cultivar (Gazelle) and a highly susceptible variety(Prima).A codominant RFLP marker( B212) was linked significantly to M.javanica resistance and explained 62% of the variation ingall-index.Seven AFLP markers were linked significantly to the resistance trait, of whichfour were linked in repulsion phase and three in coupling phase. All seven AFLPmarkers mapped to LG-F (Linkage Group F) on the public soybean molecular map.The major quantitative trait locus (QTL) for resistance mapped between markersE-ACC/M-CTC2(SOJA6) (linked in coupling phase), B212 and E-AAC/M-CAT1(SOJA7)(linked in repulsion phase). These two AFLP markers bracketing the majorresistance QTL were successfully converted to SCARs (Sequence CharacterizedAmplified Regions). Marker E-ACC/M-CTC2 was converted to a codominant SCARmarker SOJA6, which accounted for 41% of variation in gall-index in the mappingpopulation. Marker E-AAC/M-CAT1 was converted to a dominant SCAR marker (SOJA7)and explained 42% of gall-index variation in the mapping population. These twomarkers mapped approximately 3.8 cM and 2.4 cMrespectively from the resistance QTL. This study represents the first report ofthe development of PCR-based sequence specific markers linked to M.javanica resistance in soybean.     

Marker-assisted introgression of 4 Phytophthora capsici resistance QTL alleles into a bell pepper line: validation of additive and epistatic effects

The aim of the present study was to transfer resistance to P. capsici alleles at four quantitative trait loci (QTLs) from a small fruited pepper into a bell pepper recipient line using markers. The marker-assisted selection program was initiated from a doubled-haploid line issued from the mapping population and involved three cycles of marker-assisted backcross (MAB). Two populations, derived by selfing the plants selected after the first selection cycle, were genotyped and evaluated phenotypically for their resistance level. The additive and epistatic effects of the four resistance factors were re-detected and validated in these populations, indicating that introgression of 4 QTLs in this MAB program was successful. A decrease of the effect for the moderate-effect QTLs and of the epistatic interaction was observed. Phenotypic evaluations of horticultural traits were performed on sample of each backcross generation. The results indicated an efficient return to the recipient phenotype using this MAB strategy.