A modifier locus affecting the expression of the S-RNase gene could be the cause of breakdown of self-incompatibility in almond

Self-compatibility has become the primary objective of most almond (Prunus amygdalus Batsch) breeding programmes in order to avoid the problems related to the gametophytic self-incompatibility system present in almond. The progeny of the cross ‘Vivot’ (S ₂₃ S _fa) × ‘Blanquerna’ (S ₈ S _fi) was studied because both cultivars share the same S _f allele but have a different phenotypic expression: active (S _fa) in ‘Vivot’ and inactive (S _fi) in ‘Blanquerna’. In addition, the microscopic observation of pollen tube growth after self-pollination over several years showed an unexpected self-incompatible behaviour in most seedlings of this cross. The genotypes of this progeny showed that the S _fi pollen from ‘Blanquerna’ was not able to grow down the pistils of ‘Vivot’ harbouring the S _fa allele, confirming the active function of this allele against the inactive form of the same allele, S _fi. As self-compatibility was observed in some S ₈ S ₂₃ and S ₈ S _fa individuals of this progeny, the S _f haplotype may not always be linked to the expression and transmission of self-compatibility in almond, suggesting that a modifier locus may be involved in the mechanism of self-incompatibility in plants.     

Negative inbreeding effects in tree fruit breeding: self-compatibility transmission in almond

Inbreeding depression has been observed in most fruit trees, negatively affecting the offspring of related parents. This problem is steadily increasing due to the repeated utilization of parents in breeding programmes. In almond, self-compatibility transmission from ‘Tuono’ to its offspring remains partially unexplained due to deviations from the expected genotype ratios. In order to test if these deviations could be due to inbreeding, the S-genotypes of the seedlings of four almond families, ‘Tuono’ (S ₁ S _f ) × ‘Ferragnès’ (S ₁ S ₃ ), ‘Tuono’ (S ₁ S _f ) × ‘Ferralise’ (S ₁ S ₃ ) and reciprocal crosses were studied. The S-genotype determination of each seedling by separation of stylar S-RNases and by S-allele-specific PCR amplification gave identical results. The ratio of S-genotypes of the family ‘Tuono’ × ‘Ferralise’ was the one least adjusted to the expected 1:1 ratio, because the number of self-compatible seedlings (S _f S ₃ ) was less than a half the number of self-incompatible ones (S ₁ S ₃ ). A mechanism acting against inbreeding would favour cross-breeding in the following generation to increase heterozygosity. This fact stresses the need to avoid crosses between related parents in fruit breeding programmes.     

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.     

Molecular mapping of the blast resistance gene, Pi44(t), in a line derived from a durably resistant rice cultivar

 A recombinant inbred line derived from a cross between CO39 and ‘Moroberekan’, RIL276, was found to be resistant to lineage 44 isolates of Pyricularia grisea in the Philippines. One hundred F₂ individuals were obtained from a backcross of RIL276 and CO39. Phenotypic analysis showed that RIL276 carries a single locus, tentatively named Pi44(t), conferring complete resistance to lineage 44 isolates of P. grisea. RFLP probes, STS primers and AFLP markers were applied to identify DNA markers linked to Pi44(t). Neither RFLP nor STS-PCR analysis gave rise to DNA markers linked to the locus. Using bulk segregant AFLP analysis, however, two dominant AFLP markers (AF₃₄₈ and AF₃₄₉) linked to Pi44(t) were identified. AF₃₄₉ and AF₃₄₈ were located at 3.3±1.5 cM and 11±3.5 cM from Pi44(t), respectively. These markers were mapped on chromosome 11 using an F₂ population derived from a cross between ‘Labelle’ and ‘Black Gora’. The location of AF₃₄₈ on chromosome 11 was confirmed using another F₂ mapping population derived from IR40931-26-3-3-5/ PI543851. DNA products at the loci linked to Pi44(t) were amplified from RIL276, ‘Labelle’ and PI543851 using the same primer pairs used to amplify AF₃₄₉ and AF₃₄₈. Sequence analysis of these bands showed 100% identity between lines. This result indicates that these AFLP markers could be used for the comparison of maps or assignment of linkage groups to chromosomes. Received: 12 May 1998 / Accepted: 13 November 1998     

Quantitative trait loci for resistance to spot blotch caused by Bipolarissorokiniana in wheat (T.aestivum L.) lines ‘Ning 8201’ and ‘Chirya 3’

Spot blotch caused by Bipolaris sorokiniana is a destructive disease of wheat in warm and humid wheat growing regions of the world. To identify quantitative trait loci (QTLs) for spot blotch resistance, two mapping populations were developed by making the crosses between common susceptible cultivar ‘Sonalika’ with the resistant breeding lines ‘Ning 8201’ and ‘Chirya 3’. Single seed descent derived F₆, F₇, F₈ lines of the first cross ‘Ning 8201’ × ‘Sonalika’ were evaluated for resistance to spot blotch in three blocks in each of the 3 years. After screening of 388 pairs of simple sequence repeat primers between the two parents, 119 polymorphic markers were used to genotype the mapping population. Four QTLs were identified on the chromosomes 2AS, 2BS, 5BL and 7DS and explained 62.9% of phenotypic variation in a simultaneous fit. The QTL on chromosome 2A was detected only in 1 year and explained 22.7% of phenotypic variation. In the second cross (‘Chirya 3’ × ‘Sonalika’), F₇ and F₈ population were evaluated in three blocks in each of the 2 years. In this population, five QTLs were identified on chromosomes 2BS, 2DS, 3BS, 7BS and 7DS. The QTLs identified in the ‘Chirya 3’ × ‘Sonalika’ population explained 43.4% of phenotypic variation in a simultaneous fit. The alleles for reduced disease severity in both the populations were derived from the respective resistant parent. The QTLs QSb.bhu-2B and QSb.bhu-7D from both populations were placed in the same deletion bins, 2BS1-0.53-0.75 and 7DS5-0.36-0.61, respectively. The closely linked markers Xgwm148 to the QTL on chromosome 2B and Xgwm111 to the QTL on chromosome 7D are potentially diagnostic markers for spot blotch resistance.     

Use of the multi-allelic self-incompatibility gene in apple to assess homozygocity in shoots obtained through haploid induction

 To obtain homozygous genotypes of apple, we have induced haploid development of either the female or the male gametes by parthenogenesis in situ and anther culture, respectively. Of the shoots obtained, which were mainly of a non-haploid nature, some could be derived from fertilised egg cells or from sporophytic anther tissue. In order to select the shoots having a true haploid origin, and thus homozygotes, we decided to use the single multi-allelic self-incompatibility gene as a molecular marker to discriminate homozygous from heterozygous individuals. The rationale behind this approach was that diploid apple cultivars contain 2 different alleles of the S-gene and therefore the haploid induced shoots obtained from them should have only one of the alleles of the single parent. The parental cultivars used were ‘Idared’ (parthenogenesis in situ) and ‘Braeburn’ (androgenesis), and their S-genotypes were known, except for 1 of the ‘Braeburn’S-alleles. To stimulate parthenogenetic development ‘Idared’ styles were pollinated with irradiated ‘Baskatong’ pollen, the S-alleles of the latter (2n) cultivar were also unknown. The cloning and sequence analysis of these 3 unidentified S-alleles, 1 from ‘Braeburn’ and 2 from ‘Baskatong’ is described, and we show that they correspond to the S ₂₄ -, S ₂₆ - and S ₂₇ -alleles. We have optimised a method for analysis of the S-alleles of ‘Idared/Baskatong’- or ‘Braeburn’-derived in vitro plant tissues and have shown that this approach can be applied for the screening of the in vitro shoots for their haploid origin. Received: 18 August 1997 / Accepted: 10 September 1997     

A linkage map of the pea (Pisum sativum L.) genome containing cloned sequences of known function and expressed sequence tags (ESTs)

 A linkage map of the pea (Pisum sativum L.) genome is presented which is based on F₂ plants produced by crossing the marrowfat cultivar ‘Primo’ and the blue-pea breeding line ‘OSU442-15’. This linkage map consists of 209 markers and covers 1330 cM (Kosambi units) and includes RFLP, RAPD and AFLP markers. By mapping a number of anchor loci, the ‘Primo’בOSU442-15’ map has been related to other pea linkage maps. A feature of the map is the incorporation of 29 loci representing genes of known function, obtained from other laboratories. The map also contains RFLP loci detected using sequence-characterized cDNA clones developed in our laboratory. The putative identities of 38 of these cDNA clones were assigned by examining public-sequence databases for protein or nucleotide-sequence similarities. The conversion of sequence-characterized pea cDNAs into PCR-amplifiable and polymorphic sequence-tagged sites (STSs) was investigated using 18 pairs of primers designed for single-copy sequences. Eleven polymorphic STSs were developed. Received: 18 June 1997 / Accepted: 11 August 1997     

Fine mapping of <Emphasis Type="Italic">Sta1</Emphasis>, a quantitative trait locus determining stele transversal area,on rice chromosome 9

The stele (root vascular cylinder) in plants plays an important role in the transport of water and nutrients from the root to the shoot. A quantitative trait locus (QTL) on rice chromosome 9 that controls stele transversal area (STA) was previously detected in an F₃ mapping population derived from a cross between the lowland cultivar ‘IR64’, with a small STA, and the upland cultivar ‘Kinandang Patong’, with a large STA. To identify the gene(s) underlying this QTL, we undertook fine mapping of the locus. We screened eight plants from BC₂F₃ lines in which recombination occurred near the QTL. Progeny testing of BC₂F₄ plants was used to determine the genotype classes for the QTL in each BC₂F₃ line. Accordingly, the STA QTL Sta1 (Stele Transversal Area 1) was mapped between the InDel markers ID07_12 and ID07_14. A candidate genomic region for Sta1 was defined more precisely between markers RM566 and RM24334, which delimit a 359-kb interval in the reference cultivar ‘Nipponbare’. A line homozygous for the ‘Kinandang Patong’ allele of Sta1 had an STA approximately 28.4% larger than that of ‘IR64’. However, Sta1 did not influence maximum or total root length, suggesting that this QTL specifically controls STA.     

Identification and mapping of a locus conferring plum pox virus resistance in two apricot-improved linkage maps

Sharka disease, caused by the plum pox virus (PPV), is one of the major limiting factors for stone fruit crops in Europe and America. In particular, apricot is severely affected suffering significant fruit losses. Thus, PPV resistance is a trait of great interest for the apricot breeding programs currently in progress. In this work, two apricot maps, earlier constructed with the F₁ ‘Goldrich × Currot’ (G×C) and the F₂ ‘Lito × Lito’-98 (L×L-98) populations, have been improved including 43 and 37 new simple sequence repeat (SSR) loci, respectively, to facilitate PPV resistance trait mapping. Screening of PPV resistance on the segregating populations classified seedling phenotypes into resistant or susceptible. A non-parametric mapping method, based on the Kruskal–Wallis (KW) rank sum test, was initially used to score marker–trait association, and results were confirmed by interval mapping. Contrary to the putative digenic model inferred from the phenotypic segregations, all significant markers for the KW statistic (P < 0.005) mapped in a unique region of ~21.0 and ~20.3 cM located on the upper part of the G1 linkage group in ‘G×C’ and ‘L×L-98’ maps, respectively. According to the data, PPV resistance is suggested to be controlled by at least one major dominant locus. The association between three SSRs distributed within this region and the PPV resistance was tested in two additional populations (‘Goldrich × Canino’ and ‘Lito × Lito’-00) and breeding program parents. The marker ssrPaCITA5 showed the highest KW value (P < 0.005) in all cases, pointing out its usefulness in marker-assisted selection. Electronic Supplementary Material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Identification of quantitative trait loci associated with self-compatibility in a Prunus species

Self-compatibility in Rosaceous fruit species is based on a single-locus qualitative trait. However, the evidence observed in different species has indicated the presence of modifier genes outside the S locus affecting the expression of self-compatibility/self-incompatibility. The study of a progeny obtained from the cross of the almond genotypes ‘Vivot’× ‘Blanquerna’ has allowed the construction of a genetic map based on microsatellite markers and the identification for the first time in the Rosaceae family of two additional loci located outside the S locus and affecting the expression of self-compatibility/self-incompatibility. A quantitative trait locus (QTL) was located relatively close to the S locus, on linkage group 6 (G6), whereas the second one was located on G8. These QTLs appear to be involved in conferring self-compatibility to genotypes not possessing the S _f allele. These results are consistent with almond being a self-incompatible species with a genetic background of pseudo-self-compatibility controlled by modifier genes. The effect of the S _f allele and the two QTLs may contribute to explain the wide range of fruit sets observed when self-pollinating different almond genotypes.     

Determination of self-locompatibility genotypes of Korean apple cultivars based on S-RNase PCR

To prevent self-fertilization, apple has a gametophytic self-incompatibility mechanism, part of a widespread intraspecific system, that is controlled by a multi-allelic locus. This attribute has been exploited in breeding programs for new cultivars. Likewise, many apple orchards depend on artificial pollination. Therefore, molecular analysis and early identification of the self-incompatibility (S) genotype could greatly improve breeding schemes and pollen donors selection. Here, we PCR-amplified the S-RNase PCR fragments from a total of 14 cultivars and parents, using new primers (ASPF3+ASPR3) common to 23 S-alleles in apple. The S-genotypes were determined for the following: ‘Hongro’ (S₁S₃), ‘Gamhong’ (S₁S₉), ‘Saenara’ (S₁S₃), ‘Chukwang’ (S₃S₉), ‘Hwahong’ (S₃S₉), ‘Seokwang’ (S₃S₃), ‘Hwarang’ (S₁S₉), ‘Sunhong’ (S₃S₉), ‘S.E.B.’ (S₁S₁₉), ‘S.G.D.’ (S₂S₃), and ‘Mollie’s Delicious’ (S₃S₇). We also confirmed the characteristics of the S-genotypes for eight Korean apple cultivars by PCR-Southern blot analysis, using seven S-RNases as probes.     

Inheritance of reduced linolenic acid content in soybean seed oil

 Linolenic acid is the unstable component of soybean [Glycine max (L.) Merr.] oil that is responsible for the undesirable odors and flavors commonly associated with poor oil quality. Two mutants, M-5 and KL-8, have been identified that have lower linolenic acid levels in the seed oil than the ‘Bay’ cultivar. Our objective was to determine the relationships between the genetic systems controlling linolenic acid in these mutants. Reciprocal crosses were made between the mutants and ‘Bay’, and between the two mutants. No maternal effect for linolenic acid content was observed from the analysis of F₁ seeds in any of the crosses. The data for linolenic acid content in F₂ seeds of M-5בBay’ and KL-8בBay’ crosses satisfactorily fit a 1 : 2 : 1 and 3 : 1 ratio, respectively. For the M-5×KL-8 cross, segregation observed from the analysis of F₂ seeds for linolenic acid content satisfactorily fit a ratio of 3 more than either mutant: 12 within the range of the two mutants: 1 less than either mutant. The segregation ratio of F₂ seeds and the segregation of F₃ seeds from F₂ plants indicated that M-5 and KL-8 have alleles at different loci that control linolenic acid content. The allele in KL-8 has been designated as fanx (KL-8) to distinguish it from fan (M-5). The low linolenic acid segregates with the genotype fanfanfanxfanx provide additional germplasm to reduce the linolenic acid content from the seed oil of soybean. Received: 18 December 1995 / Accepted: 12 July 1996     

Development and utility of cleaved amplified polymorphic sequences (CAPS) and restriction fragment length polymorphisms (RFLPs) linked to the Fom-2 fusarium wilt resistance gene in melon (Cucumis melo L.)

Fusarium wilt, caused by Fusarium oxysporum Schlecht f. sp. melonis Snyder & Hans, is a worldwide soil-borne disease of melon (Cucumis melo L.). Resistance to races 0 and 1 of Fusarium wilt is conditioned by the dominant gene Fom-2. To facilitate marker-assisted backcrossing with selection for Fusarium wilt resistance, we developed cleaved amplified polymorphic sequences (CAPS) and restriction fragment length polymorphisms (RFLP) markers by converting RAPD markers E07 (a 1.25-kb band) and G17 (a 1.05-kb band), respectively. The RAPD-PCR polymorphic fragments from the susceptible line ’Vedrantais’ were cloned and sequenced in order to construct primers that would amplify only the target fragment. The derived primers, E07SCAR-1/E07SCAR-2 from E07 and G17SCAR-1/G17SCAR-2 from G17, yielded a single 1.25-kb fragment (designated SCE07) and a 1.05-kb fragment (designated SCG17) (the same as RAPD markers E07 and G17), respectively, from both resistant and susceptible melon lines, thus demonstrating locus-specific associated primers. Potential CAPS markers were first revealed by comparing sequence data between fragments amplified from resistant (PI 161375) and susceptible (’Vedrantais’) lines and were then confirmed by electrophoresis of restriction endonuclease digestion products. Twelve restriction endonucleases were evaluated for their potential use as CAPS markers within the SCE07 fragment. Three (BclI, MspI, and BssSI) yielded ideal CAPS markers and were subsequently subjected to extensive testing using an additional 88 diverse melon cultigens, 93 and 119 F₂ individuals from crosses of ’Vedrantais’ x PI 161375 and ’Ananas Yokneam’×MR-1 respectively, and 17 families from a backcross BC₁S₁ population derived from the breeding line ’MD8654’ as a resistance source. BclI- and MspI-CAPS are susceptible-linked markers, whereas the BssSI-CAPS is a resistant-linked marker. The CAPS markers that resulted from double digestion by BclI and BssSI are co-dominant. Results from BclI- and MspI-CAPS showed over 90% accuracy in the melon cultigens, and nearly 100% accuracy in the F₂ individuals and BC₁S₁ families tested. This is the first report of PCR-based CAPS markers linked to resistance/susceptibility for Fusarium wilt in melon. The RFLP markers resulting from probing with a clone-derived 1.05-kb SCG17 PCR fragment showed 85% correct matches to the disease phenotype. Both the CAPS and RFLP markers were co-dominant, easier to score, and more accurate and consistent in predicting the melon phenotype than the RAPD markers from which they were derived. Received: 28 July 1998 / Accepted: 7 December 1998     

An intervarietal molecular marker map in Triticum aestivum L. Em. Thell. and comparison with a map from a wide cross

 An intervarietal molecular marker map covering most of the nuclear genome was developed in Triticum aestivum. One hundred and six androgenetic-derived doubled haploid lines obtained from the F₁ between monosomics of ‘Chinese Spring’ and ‘Courtot’ were analysed for genetic mapping. The map covered 18 of the 21 chromosomes with an identical distribution of markers in the A and B genome, and only small segments of the D genome. Distorted markers were mapped using Bailey’s 2-point method and revealed skewed regions on 1A, 1DS, 2A, 2B, 4AS and 6B. Comparison with a wide cross [‘Opata’×Synthetic hexaploid (T. tauschii/‘Altar 84’)] showed colinearity for markers on homologous chromosomes, but revealed a large proportion (25%) of markers mapped on non-homoeologous chromosomes, i. e. heterologous markers. The origin of the material and distortion segregation are discussed with particular emphasis on investigations of D-genome markers. Received: 2 May 1996 / Accepted: 2 August 1996     

Localization of the rice stripe disease resistance gene, Stv-bi, by graphical genotyping and linkage analyses with molecular markers

 We used graphical genotyping and linkage analyses with molecular markers to determine the chromosomal location of the rice stripe disease resistance gene, Stv-b ⁱ . The stripe resistance gene from the indica rice (Oryza sativa) cv ‘Modan’ was introgressed into several Japanese rice varieties. We found 4 RFLP markers in ‘Modan’, five susceptible parental rice varieties (‘Norin No. 8’, ‘Sachihikari’, ‘Kanto No. 98’, ‘Hokuriku No.103’ and ‘Koganebare’) and four resistant progeny varieties (‘St. No. 1’, ‘Aichi No. 6’, ‘Aoisora’ and ‘Asanohikari’). Graphical genotyping of the resistant progeny revealed a chromosomal segment ascribable to ‘Modan’ and associated with stripe resistance. The chromosomal segment from ‘Modan’ was located at 35.85 cM on chromosome 11. Linkage analysis using 120 F₂ individuals from a cross between ‘Koshihikari’ (susceptible) and ‘Asanohikari’ (resistant) revealed another 8 RFLP markers in the same chromosome. We performed a bioassay for rice stripe resistance in F₃ lines of the F₂ individuals using infective small brown planthoppers and identified an 1.8-cM segment harboring the rice stripe disease resistance gene, Stv-b ⁱ , between XNpb220 and XNpb257/ XNpb254. Furthermore, Stv-b ⁱ was linked by 0.0 cM to a RFLP marker, ST10, which was developed on the basis of the results of RAPD analysis. These DNA markers near the Stv-b ⁱ locus may be useful in marker-assisted selection and map-based cloning of the Stv-b ⁱ gene. Received: 26 September 1997 / Accepted: 4 November 1997     

Genomic regions involved in productivity of two interspecific poplar families in Europe. 1. Stem height, circumference and volume

Interspecific hybrids of Populus species are known for their superior growth. In this study, we examined the effect of the genetic background and contrasting environmental conditions on growth and searched for quantitative trait loci (QTL) affecting growth traits. To this end, two hybrid poplar families resulting from controlled crosses, Populus deltoides ‘S9-2’ × P. nigra ‘Ghoy’ (D × N, 180 F₁) and P. deltoides ‘S9-2’ × P. trichocarpa ‘V24’ (D × T, 182 F₁), were grown at two contrasting sites, Northern Italy and Central France. At the end of the second growing season, tree dimensions (stem height, circumference, and volume) were assessed. The performances of both families significantly differed within and between sites. Tree volume was significantly larger at the Italian site as compared to the French site. Genotype by environment interactions were significant but low for both families and for all growth traits. Tight correlations among the individual growth traits indicated that there may be a common genetic mechanism with pleiotropic effects on these growth traits. In line with previous studies, linkage groups I, VII, IX, X, XVI, XVII, and XIX appeared to have genomic regions with the largest effects on growth traits. This study revealed that (1) both families have high potential for selection of superior poplar hybrids due to the pronounced heterosis (hybrid vigor) and the large genetic variability in terms of growth and (2) the choice of site is crucial for poplar cultivation. Dillen and Storme contributed equally to the work. An erratum to this article can be found at     

Effect of enzyme concentrations on protoplast isolation and protoplast culture of <Emphasis Type="Italic">Spathiphyllum</Emphasis> and <Emphasis Type="Italic">Anthurium</Emphasis>

Vital protoplasts from Spathiphyllum wallisii ‘Alain’ and Anthurium scherzerianum ‘238’ were isolated from both somatic embryos and leaves. The highest yields were obtained when 1.5% cellulase, 0.5% macerase and 0.5% driselase were used for Spathiphyllum wallisii leaves and 0.5% cellulase, 0.3% macerase and 0.5% driselase for Anthurium scherzerianum embryos. About 1 × 10⁶ protoplasts g⁻¹ and 1 × 10⁵ protoplasts g⁻¹ could be isolated from leaves and embryos, respectively. For protoplast fusion Spathiphyllum wallisii ‘Alain’ and Anthurium scherzerianum ‘238’ were mixed in a 1:1 ratio in a fusion solution containing 1 mM CaCl₂·2H₂O, 1 mM MES and 0.5 M mannitol. Fusion was performed by protoplast alignment under 500 V cm⁻¹ alternating current for 60 s and subsequent generation of two pulses of 4500 V cm⁻¹ direct current during 50 μs. Development until colony stage was achieved using agarose beads for protoplast culture.     

Development of 1,030 genomic SSR markers in switchgrass

Switchgrass, Panicum virgatum L., a native to the tall grass prairies in North America, has been grown for soil conservation and herbage production in the USA and recently widely recognized as a promising dedicated cellulosic bioenergy crop. A large amount of codominant molecular markers including simple sequence repeats (SSRs) are required for the construction of linkage maps and implementation of molecular breeding strategies to develop superior switchgrass cultivars. The objectives of this study were (1) to identify SSR-containing clones and to design PCR primer pairs (PPs) in SSR-enriched genomic libraries, and (2) to validate and characterize the designed SSR PPs. Five genomic SSR enriched libraries were constructed using genomic DNA of ‘SL93 7 × 15’, a switchgrass genotype selected in an Oklahoma State University (OSU) southern lowland breeding population. A total of 3,046 clones from four libraries enriched in (CA/TG)n, (GA/TC)n, (CAG/CTG)n and (AAG/CTT)n SSR repeats were sequenced at the OSU Core Facility. From the sequences, we isolated 1,300 unique SSR-containing clones, from which we designed 1,398 PPs using SSR Locator V.1 software. Among the designed PPs, 1,030 (73.7%) amplified reproducible and strong bands with expected fragment size, and 802 detected polymorphic alleles, in SL93 7 × 15 and ‘NL94 16 × 13’, two parents of one mapping population. All of the four libraries contained a high rate of perfect SSR repeat types, ranging from 62.7 to 76.2%. Polymorphism of the effective SSR markers was also tested in two lowland and two upland switchgrass cultivars, encompassing ‘Alamo’ and ‘Kanlow’, and ‘Blackwell’ and ‘Dacotah’, respectively. The developed SSR markers should be useful in genetic and breeding research in switchgrass.