Nitrogen‐, water‐ and radiation‐use efficiencies affected by sugarcane breeding in Argentina

This study aimed to identify whether and how sugarcane (Saccharum spp.) breeding in Argentina modified nitrogen‐use efficiency (NUE), water‐use efficiency (WUE) and radiation‐use efficiency (RUE). Thirteen varieties were grown in two consecutive seasons. Trends in different traits were estimated by fitting the data to linear or bilinear regression models. There was a linear increase in NUE and WUE with the year of release throughout the 70‐year span, whereas water use was not modified by sugarcane breeding. There was a positive and strong (r > 0.90; P < 0.01) association between NUE and WUE and between sugar yield and NUE or WUE. Although RUE was not modified by sugarcane breeding, the amount of radiation intercepted by the crop increased with the year of release. Modern varieties had a higher maximum interception and needed fewer days to reach maximum interception than old varieties. This study suggests that applying ecophysiological knowledge would be instrumental in sugarcane breeding programmes in order to develop varieties with high resource‐use efficiency and capable to adapt to global climate change.     

Selection of families and parents of sugarcane (Saccharum spp.) through mixed models by joint analysis of two harvests

At present, the sugarcane (Saccharum spp.) breeding programs from around the world have practiced early family selection, because in the early stages of breeding the evaluated characteristics have shown low heritability. This study aimed to select through restricted maximum likelihood/best linear unbiased prediction also known “mixed models” the best families among the 78 that comprise the RB05 sugarcane series, stage T1, of Programa de Melhoramento Genético da Cana-de-açúcar of the Rede Interuniversitária para o Desenvolvimento do Setor Sucroalcoleiro, Brazil. The experiments, conducted during 2007/2008 and 2008/2009 in Paranavaí, PR were implemented using an incomplete-block design with five replicates of each family. Each plot was composed of two planting rows of 5 m, spaced from each other by 1.40 m and spacing between plants was of 0.50 m, containing ten seedlings for planting row, resulting in a total of 20 individuals per replicate of each family. The characteristics evaluated were Brix (°Brix), ton of stalk per hectare (TCH) and ton of °Brix per hectare (TBH). The joint analysis obtained from these three traits in both harvests favored the selection of 35–41 families. The analysis also favored the selection of the best parents. The top five families for Brix were: F41M60, F02M77, F41M82, F61M38 and F62M35; to TCH were F66M30, F35M06, F78M45, F70M30 and F57M46 and for TBH the five elites families were F66M30, F35M06, F78M45, F70M30 and F01M39. The five best sugarcane parents for Brix were RB835486, SP91-1049, SP80-3280, RB72454 and RB925211; to TCH were IAC87-3396, RB941531, RB855511, RB915141 and RB957689, and for TBH the elite were IAC87-3396, RB855511, RB957689, RB941531 and RB855563. As each harvest season had different order of the best families, the joint analysis proved to be a primordial tool for plant breeders.     

Genetic diversity in cultivated Osteospermum as revealed by random amplified polymorphic DNA analysis

A collection of 28 Osteospermum clones and cultivated varieties of different origin were evaluated by random amplified polymorphic DNA (RAPD) analysis. All the clones were identified by 12 decamers selected from a set of 30. This is the first characterization by molecular markers of the genetic material of Osteospermum. The level of genetic diversity among genotypes was assayed and all the accessions tested were then classified into six groups by UPGMA cluster analysis; the clustering of genotypes using the RAPD data proved to be in accordance with their breeding group origin. RAPD analysis can therefore be a useful tool for evaluating genetic variability in other Osteospermum germplasm collections for breeding purposes and for protecting intellectual property rights of improved varieties.     

Use of PCR-based methodologies for the determination of DNA diversity between Saccharum varieties

Summary In this study, two PCR-based methodologies were evaluated for potential use in the determination of DNA diversity between 20 commercial sugarcane hybrids and 6 outgroup varieties of S. spontaneum, S. officinarum and hybrids from early in the genealogy. The first method involved PCR amplification of sugarcane DNA in the presence of random, decamer primers (RAPDs), while the second protocol utilized specific microsatellite and telomere sequences as primers. A total of 41 RAPD primers (356 loci) were screened across the varieties of which 15 (160 loci) were used in the calculation of DNA diversity (expressed% similarity). This varied from 61 to 95%, with most of the commercial varieties showing more than 80% similarity in their DNA. The RAPD data indicated that there had been a gradual decline in DNA diversity (84% reduction) from the early inter-specific crosses to the commercial hybrids, probably as a result of backcrossing and in-breeding strategies used in the previous 5 to 6 generations of sugarcane breeding. The microsatellite and telomere data produced a much greater range in DNA similarity values (25–91%), probably due to the fact that these primers detect highly variable regions of the genome. It is suggested that these specific primers would not be suitable for determination of DNA diversity, but could be used more effectively in the development of a methodology for routine, rapid identification of sugarcane varieties.     

Identification of RAPD markers closely linked to the mlo-locus in barley

Developing resistance to powdery mildew, Erysiphe graminis f.sp. hordei, is a major goal of many barley breeding programmes. Several resistance genes have been tagged or mapped with molecular markers. The mlo gene confers durable resistance towards all known isolates of the pathogen. In this study, RAPD markers and bulked segregant analysis were used to determine PCR-based markers linked to the mlo-locus. Sixty doubled haploid lines from a cross between an isogenic line of ‘Ingrid’ carrying the mlo11 allele and a susceptible cv. ‘Pokko’ were used as plant material. Seven linked RAPD markers were found, the closest lying 1.6 cM away from the resistance gene. When eight barley varieties were assayed for the presence of this band, F4-980, it was found in the resistant varieties but not in the susceptible ones. The linked marker bands could be amplified from DNA-samples prepared by using three different methods, including a quick squash technique. PCR-based markers linked to the resistance gene can be used as tools for selection in breeding programmes.     

Molecular diversity and genome structure in modern sugarcane varieties

Summary RFLP analysis was performed on 40 sugarcane cultivated varieties. Twenty-two maize low copy DNA clones located on different regions of the 10 maize chromosomes were used as probes to survey variability among the sugarcane varieties. A total of 425 fragments, 411 of which were polymorphic, were identified for 22 probe/enzyme combinations. Each variety displayed an average of 7.28 fragments per combination, revealing the complex polyploid origin of modern sugarcane varieties. The average genetic similarity between sugarcane varieties was 0.61. Although cultivated varieties appear closely related to S. officinarum clones, the genes of S. spontaneum seem to constitute the principal component of varietal diversity. A very weak global structuring among the 40 varieties is observed, in agreement with the profuse exchanges of parental materials between sugarcane breeding stations. Traces of linkage disequilibrium can be attributed to the distribution of S. spontaneum chromosomes among sugarcane varieties. The possibility of using modern varieties as a population for detecting associations between molecular markers and agronomic traits is suggested.     

SCAR and RAPD markers associated with 18-carbon fatty acids in rapeseed, Brassica napus

Breeding rapeseed for enhanced oil quality includes the development of varieties with low linolenic acid content. The breeder also aims to develop varieties with a high linoleic acid content because of its nutritional value. Restriction fragment length polymorphism (RFLP) and random amplified polymorphic DNA (RAPD) markers have been developed for linolenic acid content, but they are not best suited for a direct application in marker-assisted selection. The RFLP technique is too complex and time-consuming and RAPD markers lack codominance, precluding the distinction of homozygous from heterozygous individuals. In this report the conversion of a RAPD marker to a codominant sequence characterized amplified region (SCAR) marker named L1L9 is described. One of the alleles consisting of an 899 bp fragment (allele A), is associated with low linolenic acid content. The other allele consists of an 641 bp fragment (allele B) and is associated with high linolenic acid content. This marker explains approximately 25% of the genetic variation for this trait. Linkage analysis in the mapping population indicates that the SCAR marker probably tags an ω-3 desaturase gene in B. napus. Two RAPD markers were found to be associated with oleic/linoleic acid content. Markers M14-350 and I06-650 explained approximately 10% and 7% of the genetic variation for linoleic acid content, respectively. These two markers were found linked at 12.3cM in the segregating B. napus F₂ progeny used for mapping. All the markers reported in this paper should be useful in breeding programmes for developing high linoleic and low linolenic acid rapeseed varieties.     

Spanish spelt: a separate gene pool within the spelt germplasm

The renewed interest in spelt (Triticum spelta L.) for wheat improvement programmes requires the study of the available genetic diversity. The purpose of this study was to assess genetic diversity within a Spanish spelt collection. Sixty‐six Spanish spelt accessions, 19 accessions of T. spelta and T. macha from different origins, three bread wheat cultivars (T. aestivum) and one accession of T. dicoccum were screened using simple sequence repeats (SSRs). The diversity observed within the Spanish group was comparable with that observed in the other wheat varieties, despite their broader geographical diversity. Indeed, the highest polymorphic information content value calculated with SSRs for Spanish material (0.90) is similar to that observed for the other wheat varieties (0.98). Principal component analysis explained 46.5% of the cumulative variation and confirmed the Spanish accessions as a separate group. This study showed the Spanish spelt collection to be a variable and unique genetic resource for wheat and spelt breeding programmes.     

不同基因型甘蔗甘露糖抗性筛选体系的建立

为明确不同基因型甘蔗再生过程中对甘露糖的敏感性,为甘蔗基因工程育种中转化子的离体筛选提供技术。使用甘露糖作为筛选底物,对甘蔗品种‘ROC22’、‘桂糖94-119’和‘桂糖96-44’外植体的再生进行研究。建立3个基因型甘蔗的甘露糖抗性筛选体系,在甘蔗愈伤组织继代增殖、分化和小苗生根3个阶段进行抗性筛选时,甘露糖占总糖的比例分别是60%、60%~70%和30%。通过统计分析,明确了不同再生阶段甘露糖对3个甘蔗基因型的影响不同,继代增殖阶段基因型间无明显差异,但在分化和生根阶段达极显著差异。     

Application of in vivo and in vitro mutation techniques for crop improvement

Summary Conventional mutation techniques have often been used to improve yield, quality, disease and pest resistance in crops, or to increase the attractiveness of flowers and ornamental plants. More than 1700 mutant varieties involving 154 plant species have been officially released. In some economically important crops, e.g. barley, durum wheat and cotton, mutant varieties occupy the majority of cultivated areas in many countries. Mutation techniques have become one of the major tools in the breeding of ornamentals such as alstroemeria, begonia, chrysanthemum, carnation, dahlia and streptocarpus. The use of in vitro techniques such as anther culture, shoot organogenesis, somatic embryogenesis and protoplast fusion can overcome some of the limitations in the application of mutation techniques in both seed and vegetatively propagated crops. In vitro culture in combination with induced mutations can speed up breeding programmes, from the generation of variability, through selection, to multiplication of the desired genotypes. The expression of induced mutations in the pure homozygote obtained through microspore, anther or ovary culture, can enhance the rapid recovery of the desired traits. In some vegetatively propagated species, mutations in combination with in vitro culture technique, may be the only method of improving an existing cultivar. Currently, many molecular studies rely on the induction and identification of mutants in model species for construction and subsequent saturation of genetic maps, understanding of developmental genetics and elucidation of biochemical pathways. Once identified and isolated, the genes that encode agronomically-important features can be either introduced directly into crop plants or used as probes to search for similar genes in crop species. It seems most likely that the recent developments based on these technologies will soon provide improved methods for selection of desired mutants.     

Molecular mapping of black rot resistance locus Xca1bo on chromosome 3 in Indian cauliflower (Brassica oleracea var. botrytis L.)

Black rot is the most devastating disease of cauliflower worldwide causing severe damage to crop. The identification of markers linked to loci that control resistance can facilitate selection of plants for breeding programmes. In the present investigation, F₂ population derived from a cross between ‘Pusa Himjyoti’, a susceptible genotype, and ‘BR‐161’, a resistant genotype, was phenotyped by artificial inoculation using Xcc race 1. Segregation analysis of F₂ progeny indicated that a single dominant locus governed resistance to Xcc race 1 in ‘BR‐161’. Bulk segregant analysis in resistant and susceptible bulks of F₂ progeny revealed seven differentiating polymorphic markers (three RAPD, two ISSR and two SSR) of 102 markers screened. Subsequently, these markers were used to genotype the entire F₂ population, and a genetic linkage map covering 74.7 cM distance was developed. The major locus Xca1bo was mapped in 1.6‐cM interval flanked by the markers RAPD 04₈₃₃ and ISSR 11₆₃₅. The Xca1bo locus was located on chromosome 3. The linked markers will be useful for marker‐assisted resistance breeding in cauliflower.     

Genetic relationships and hybrid vigour in olive (Olea europaea L.) by microsatellites

The olive (Olea europaea) is one of the most important oleaginous crops of the Mediterranean basin. Increased demand for olive oil creates a need for new olive varieties to help meet the requirements of the global market. However, olive breeding has been handicapped by such varied challenges as a prolonged juvenile period, agrotechnical problems and insufficient genetic knowledge. The use of DNA markers has the potential to overcome these problems and increase the effectiveness of classical breeding programmes. In this study, co‐dominant polymorphic simple sequence repeats (SSRs) were used as markers to analyse the genetic relationships between several local and other ‘non‐native’ olive cultivars. Cluster analysis revealed four major groups among the 15 cultivars examined in this study. Table and oil cultivars were clustered in different groups. However, the clusters did not differentiate between cultivars of different geographical origins. In addition, we used the data gathered to analyse genetic relationships to evaluate the effects of heterosis in agricultural traits. Genetic distances between cultivars were determined based on the SSR genotype data and were used for evaluating the possible effects of heterosis in various F₁ populations. Interestingly, phenotypic data of F₁ progenies from crosses between different cultivars indicated the potential effects of heterosis as expressed in several traits. Genetic distance between parents was significantly correlated to F₁ performance for three traits: percentage of dry fruit weight, oil content and commercial oil production. Thus, crosses between olive cultivars exhibiting relatively extensive genetic distances one from the other are expected to result in better progeny performance in future Olea breeding programmes. Our study linked assessment of biodiversity of commercial olive cultivars with the application of this information in olive breeding programmes for selection of specific parents to generate superior new cultivars.     

Assessment of natural and induced genetic variation in Alstroemeria using random amplified polymorphic DNA (RAPD) markers

Summary We have used random amplified polymorphic DNA (RAPD) markers to study genetic variation in Alstroemeria. The first objective was to examine the discriminatory power of RAPD markers in different genotypes of Alstroemeria obtained by traditional breeding. All genotypes examined, including commercial Alstroemeria varieties, could be distinguished on the basis of their RAPD profiles. Progeny plants could be distinguished from their parents. A second objective of this study was to investigate whether RAPD markers can be used as a routine tool to detect mutant plants, as an alternative to glasshouse testing. To address this objective, we analysed Alstroemeria plants that carried phenotypically visible mutations that either were induced by irradiation using X-rays or were the result of somaclonal variation. In eight out of a total of 13 mutant Alstroemeria plants obtained after irradiation or tissue culture we detected no polymorphisms when compared to control plants that were considered to be non-mutated. Only in five of the mutant plants analysed we detected one to two polymorphisms. These results suggest that frequent genome rearrangements had not occurred in the mutant plants analysed. These results also demonstrate that the RAPD technique is an inappropriate tool for the rapid screening of Alstroemeria for induced variation. It that the RAPD technique is an inappropriate tool for the rapid screening of Alstroemeria for induced variation. It seems probable that this conclusion would be equally applicable in other plant genera in which induced variation has occurred. However, the RAPD technique is a simple and effective tool for genetic fingerprinting of Alstroemeria varieties, provided their differences are due to sexual propagation.     

Feeding quality assessment of fresh maize stover by means of near-infrared spectroscopy with a new sample presentation unit

Stover quality traits are important in breeding programmes of maize cultivars. However, conventional procedures for evaluation of stover quality are expensive and thus limit the full exploitation of the available genetic variability. Our objective was to assess the potential of near‐infrared spectroscopy (NIRS) with a new sample presentation unit to determine feeding quality of maize stover. The plant material comprised maize inbreds and hybrids. The sample presentation unit was equipped with a near‐infrared diode‐array spectrometer. Coefficient of determination in cross‐validation ranged between 0.77 and 0.94 for contents of dry matter, nitrogen, neutral detergent fibre and in vitro fermentability expressed as gas volume after 24h incubation time, but it was lower for ash content. It was concluded that NIRS with the new sample presentation unit was less accurate than laboratory NIRS to predict the standard reference methods. However, the new sample presentation unit might be used as a fast and efficient technique to perform large screenings of maize stover accessions in breeding programmes.     

Phenotypic and genotypic analyses of diversity and breeding progress in European triticale (× Triticosecale Wittmack)

Knowledge of the extent of phenotypic variability, genetic diversity and the realized breeding progress is central for the optimum design of breeding programmes, but little information is available for triticale (× Triticosecale Wittmack). In this study, a collection of 885 diverse European triticale lines was evaluated in multilocation field trials in 2 years. We observed significant genotypic variances and high heritabilities for several agronomic and morphological traits and significant correlations among different traits including grain yield. Based on a subset of the population of 121 varieties registered in Europe between 1983 and 2014, we observed a substantial breeding progress for grain yield with a significant rate of increase of 53 kg/ha or 0.67% per year. All lines were genotyped by a genotyping‐by‐sequencing approach yielding 58 888 polymorphic markers. Our analyses revealed the absence of major population structure but a certain grouping of lines according to their origin. Taken together, our results on triticale germplasm and its breeding history provide important information for breeding programmes and future selection gain in this crop.     

Genetic analysis of forage grasses based on heterologous RFLP markers detected by rice cDNAs

Genetic polymorphism within and between three species of forage grasses, perennial ryegrass (Lolium perenne L), meadow fescue (Festuca pratensis Huds.) and tall fescue (Festuca arundinacea Schreb.), was analyzed using restriction fragment length polymorphism (RFLP) markers detected by rice cDNA probes developed at the Rice Genome Research Programme of Japan (RGP). One hundred and ninety‐seven rice cDNA clones were used for hybridization to genomic DNA of forage grasses. Many of the rice cDNA clones produced no visible band or only a smear with no discrete bands. Twenty‐three clones showed high efficiency cross‐hybridization to the genomic DNA of forage grasses. Genetic variation was evaluated for five varieties and one population of forage grasses using 12 polymorphic rice cDNA RFLP probes. Genetic variability within varieties as measured by Rogers’ genetic distance was considerably lower for the F. pratensis variety ‘Tomosakae’ than for the L. perenne and F. arundinacea varieties. To determine the genetic diversity between varieties of different species, cluster analysis was performed using data from the 12 RFLP probes. The two accessions of Lolium perenne were clustered more closely together than the three varieties of F. arundinacea. Two Japanese varieties of F. arundinacea were grouped in the same cluster. The variety‐specific RFLP markers were seen among six accessions of L. perenne, F. pratensis and F. arundinacea. Such variety‐specific RFLP markers would provide very useful tools for breeding programmes such as the intergeneric hybridization of Lolium and Festuca genera.     

10个甘蔗品种染色体核型分析与比较

通过10个甘蔗品种的核型分析,研究其染色体组成系统演化和血缘构成的基本特征,为甘蔗育种选配亲本提供细胞学依据。以10个甘蔗品种的根尖为材料,采用改良的酶解去壁低渗法制备染色体标本,再利用Ikaros软件进行核型分析。‘粤糖86-368’的核型公式为2n=111=2M+103m+4sm+2T,‘桂糖11’的核型公式为2n=106=96m+10sm,这2个品种的核型分类均属于1B型;‘新台糖10’的核型公式为2n=108=98m+10sm,‘新台糖16’的核型公式为2n=111=4M+101m+6sm,‘新台糖20’的核型公式为2n=110=98m+10sm+2st,‘新台糖22’的核型公式为2n=110=2M+96m+12sm,‘粤糖93-159’的核型公式为2n=108=2M+88m+18sm,‘闽糖69-421’的核型公式为2n=109=95m+14sm,‘云蔗89-151’的核型公式为2n=111=91m+20sm这7个品种的核型分类均属于2B型;‘云蔗99-91’的核型公式为2n=108=84m+22sm+2st,核型分类属于2C型。研究结果说明,‘粤糖86-368’、‘桂糖11’这2个品种的进化程度都还比较原始;‘云蔗99-91’、‘云蔗89-151’进化程度最高;其余6个品种进化程度中等。     

Identification of PCR-based markers linked to the powdery-mildew-resistance gene Pl1 from Malus robusta in cultivated apple

The availability of molecular markers linked to mildew resistance genes would enhance the efficiency of apple-breeding programmes. This investigation focuses on the identification of random amplified polymorphic DNA (RAPD) markers linked to the Pl₁ gene for mildew resistance, which has introgressed from Malus robusta into cultivated apples. The RAPD marker technique was combined with a modified ‘bulked seg-regant analysis’ mapping strategy. About 850 random decamer primers used as single primers or in combinations were tested by PCR analysis on the basis of resistant and susceptible DNA pools. Selected primers producing RAPD fragments were applied in an additional selection step to M. robusta and genotypes representing intermediate breeding stages of the breeding population 93/9, for which a 1:1 segregation could be observed for the resistance trait. Seven RAPD markers, all representing introgressed DNA sequences from M. robusta, were identified and arranged with the Pl₁ locus in a common linkage group. The two most tightly-linked RAPD markers, OPAT20₄₅₀ and OPD2₁₀₀₀ were mapped with a genetic distance of 4.5 and 5 cM, respectively, from the Pl₁ gene. Both markers are suitable for marker-assisted selection in apple breeding. The polymorphic DNA fragment OPAT20₄₅₀ was cloned and sequenced, and longer primers for the generation of a sequence-characterized amplified region (SCAR) marker have been constructed; this marker was easier to score than the original RAPD marker.     

Inter and intra-population variability of <Emphasis Type="Italic">Jatropha curcas</Emphasis> (L.) characterized by RAPD and ISSR markers and development of population-specific SCAR markers

Jatropha curcas (Euphorbiaceae) is an oil-bearing species with multiple uses and considerable potential as a bioenergy crop. The present investigation has been undertaken to assess the extent of genetic diversity in a representative set of 42 accessions of J. curcas encompassing different crop growing regions in India along with a non-toxic genotype from Mexico as a prelude for utilization of promising and genetically divergent materials in the breeding programmes. Molecular polymorphism was 42.0% with 400 RAPD primers and 33.5% with 100 ISSR primers between accessions indicating modest levels of genetic variation in the Indian germplasm. The within-population variation based on RAPD polymorphism was 64.0% and was on par with the inter-population variation. Polymorphic ISSR markers have been identified that could differentiate the Indian accessions from the Mexican genotype and two of them were converted to SCAR markers. The SCAR primer pair ISPJ1 amplified a 543 bp fragment in all the Indian populations, while ISPJ2 with a specific amplicon of 1,096 bp was specific to the Mexican genotype. Population-specific bands have been identified for the accession from Kerala (2 RAPD markers), Neemuch-1 from Rajasthan (1 each of RAPD and ISSR markers) and the non-toxic genotype from Mexico (17 RAPD and 4 ISSR markers), which serve as diagnostic markers in genotyping. The study indicates an immediate need for widening the genetic base of J. curcas germplasm through introduction of accessions with broader geographical background.     

Genetic diversity in European perennial ryegrass cultivars investigated with RAPD markers

Perennial ryegrass (Lolium perenne L.) is the most important grass species for temperate grassland agriculture. The genetic relationship and distance among cultivars is largely unknown but of great interest for breeding programmes. The objectives of this study were to (i) investigate the molecular variation and structure of population cultivars, (ii) describe the relationship among cultivars in terms of the modified Rogers’ distance, and (iii) determine the minimum sample size required for characterization of cultivars of L. perenne using random amplified polymorphic DNA (RAPD) markers. A total of 22 ryegrass cultivars, mainly of European origin, were investigated with RAPD markers. The minimum sample size required for the characterization of cultivars was about 20 individuals per population. An analysis of molecular variance (AMOVA) revealed a much larger genetic variation within cultivars (66%) than between them (34%).