RFLP analysis allows for the identification of alfalfa ecotypes

Three seed lots, obtained from different ‘foundation farms’, for each of the alfalfa, Medicago sativa L., ecotypes ‘Vogherese’ and ‘Maremmana’, together with the variety ‘Iside’ as a control, were studied to test the possibility of distinguishing them through restriction fragment length polymorphism analysis. Twenty plants per seed lot were analysed with 25 DNA probes that yielded 155 informative polymorphic fragments. Variance partitioning showed that within‐population variability accounted for nearly 98% of the total variance, while a small but significant contribution to the total variance was due to among‐lot variability.‘Vogherese’ was more homogeneous than ‘Maremmana’, as a consequence of the greater environmental homogeneity of the adaptation area of the former ecotype compared with the latter. Bulk analysis yielded eight variety‐specific bands, 12 bands present in both ecotypes and absent in the variety and one band specific of ‘Maremmana’. Results are discussed in relation to the practical use of molecular markers for alfalfa ecotype identification.     

Use of RAPD and microsatellite (SSR) variation to assess genetic relationships among populations of tetraploid alfalfa, Medicago sativa

The level of random amplified polymorphic DNA (RAPD) and microsatellite variation present in four ecotypes and two varieties of alfalfa (lucerne) from Italian and Egyptian germplasm sources was evaluated. A sample of 100 plants from 10 populations was analysed by means of 41 RAPD markers and 37 simple sequence repeat (SSR) markers. Both molecular approaches revealed a high degree of genetic diversity within each of the cultivated populations and enabled each of the plants considered to be uniquely fingerprinted. The genetic relationships among plants and populations were analysed by computing AMOVA (analysis of molecular variance) and F_ST analyses. RAPDs were able to separate the Italian populations from the Egyptian variety. SSRs allowed strong separation of the four Italian alfalfa ecotypes. It was concluded that RAPD and microsatellites could be useful and powerful tools for assessing genetic variation and genetic relationships in tetraploid alfalfa.     

Genetic structure of putative heterotic populations of alfalfa

Semi‐hybrids between genetically distant alfalfa (Medicago sativa subsp. sativa) populations may display heterosis whose extent is affected by the structure of genetic diversity across populations. This study aimed to assess the genetic diversity across three putative heterotic populations, one Italian, one Egyptian and one of semi‐erect germplasm from Eastern Europe, Canada and Spanish Mielga (EECM population). Each population was bred from ten parents after various selection cycles. Fifteen genotypes per population were characterized by 20 polymorphic SSR markers. The among‐population variance was over eightfold smaller than the average within‐population variance (2.05 vs. 17.24) and accounted for 10.6% of the total variation. G’_ST = .090 across markers indicated modest population differentiation. Various diversity measures, multidimensional scaling, and cluster analysis of the genetic structure indicated that the Italian population was more distant from the EECM population than the Egyptian one. The EECM and Egyptian populations were the most distant geographically and genetically. EECM displayed widest intrapopulation variation, accordingly to its constitutive geographical diversity. In conclusion, this study indicates modest genetic differentiation between alfalfa populations even for geographically distant germplasm.     

Variation among and within Italian alfalfa ecotypes by means of bio-agronomic characters and amplified fragment length polymorphism analyses

Three accessions, corresponding to different ‘foundation’ farms, of each of the alfalfa (lucerne) Italian ecotypes ‘Vogherese’ and ‘Marem‐mano’, and two varieties were studied in order to evaluate: (1) the among‐ and within‐ecotype variation; and (2) the possibility of distinguishing the populations by means of bio‐agronomic characters and molecular markers (amplified fragment length polymorphisms, AFLPs). The two ecotypes could be distinguished by the differences in persistency after 15 cuttings in 3 years cultivation and by the seasonal distribution of the dry matter yield. Within‐ecotype variation was always of minor importance as compared with the among‐ecotype differences. The AFLP analyses generated 20 polymorphic fragments in ‘bulk’ samples: none was able to distinguish the two ecotypes, while variety‐specific fragments were found. The same analysis on an individual plant basis (485 polymorphic fragments) confirmed the absence of differences among ecotypes, while 10% of the total variance was attributable to variation among accessions within ecotype. The results of the analyses using agronomic traits and molecular markers are discussed in relation to the possibility of ecotype identification.     

Alfalfa germplasm from a Sahara oasis: characterization by means of bio-agronomic traits and SSR markers

In North African oases Medicago sativa ssp. sativa is likely to have been cultivated for long time without contacts with ssp. falcata . In order (i) to verify the genetic distinctiveness of the alfalfa germplasm of the oasis and (ii) to examine their internal variation, 17 farm landraces were collected in Siwa oasis (Egypt) and studied with four Egyptian and three Italian cultivars (120 plants/population) by means of bio-agronomic characters in 2 years cultivation and SSR markers in 'bulk' samples. Siwa germplasm was similar to the cultivars for dry matter yield but differed for plant form, earliness and reaction to fall conditions; SSR analysis based on 31 polymorphic DNA fragments distinguished Siwa landraces from the two groups of varieties. The variation within Siwa group was of continuous type based on bio-agronomic traits; average genetic similarity was high (0.8959) and UPGMA dendrogram suggests poor isolation among the landraces, although all, except two, were uniquely fingerprinted. The use of putative 'pure' subspecies of high agronomic value in the alfalfa variety construction process is discussed.     

Genetic diversity of feral alfalfa (<Emphasis Type="Italic">Medicago</Emphasis> <Emphasis Type="Italic">sativa</Emphasis> L.) populations occurring in Manitoba,Canada and comparison with alfalfa cultivars: an analysis using SSR markers and phenotypic traits

Feral populations of cultivated crops may act as reservoirs for novel traits and aid in trait movement across the landscape. Knowledge on the genetic diversity of feral populations may provide new insights into their origin and evolution and may help in the design of efficient novel trait confinement protocols. In this study, the genetic diversity of 12 feral alfalfa (Medicago sativa) populations originating from southern Manitoba (Canada) and 10 alfalfa cultivars and a M. falcata germplasm were investigated using eight SSR markers (i.e., microsatellites) and 14 phenotypic traits. We found that the genetic diversity observed in feral populations was similar to the diversity detected among the 10 cultivars. Analysis of molecular variance revealed that there was great genetic variation within (99.8%) rather than between different feral populations. Cluster analysis (unweighted pair-group method using arithmetic average) revealed no differentiation between feral populations and cultivars for neutral loci. High levels of population differentiation for phenotypic traits (and not for neutral markers) suggest the occurrence of heterogeneous selection for adaptive traits. The phenotypic traits we studied did not distinctly separate feral populations from cultivars but there was evidence of natural selection in feral populations for traits including winter survivability, rhizome production, and prostrate growth habit. Our results suggest that feral alfalfa populations need to be considered in the risk assessment of alfalfa containing novel genetically modified (GM) traits. Further, feral alfalfa populations may be regarded as a source of new germplasm for plant improvement.     

Identification and validation of an over‐dominant QTL controlling soybean seed weight using populations derived from Glycine max × Glycine soja

Heterosis, or hybrid vigour, has been used to improve seed yield in several important crops for decades and it has potential applications in soybean. The discovery of over‐dominant quantitative trait loci (QTL) underlying yield‐related traits, such as seed weight, will facilitate hybrid soybean breeding via marker‐assisted selection. In this study, F₂ and F_2 : 3 populations derived from the crosses of ‘Jidou 12’ (Glycine max) × ‘ZYD2738’ (Glycine soja) and ‘Jidou 9’ (G. max) × ‘ZYD2738’ were used to identify over‐dominant QTL associated with seed weight. A total of seven QTL were identified. Among them, qSWT_13_1, mapped on chromosome 13 and linked with Satt114, showed an over‐dominant effect in two populations for two successive generations. This over‐dominant effect was further examined by six subpopulations derived from ‘Jidou12’ × ‘ZYD2738’. The seed weight for heterozygous individuals was 1.1‐ to 1.6‐fold higher than that of homozygous individuals among the six validation populations examined in different locations and years. Therefore, qSWT_13_1 may be a useful locus to improve the yield of hybrid soybean and to understand the molecular mechanism of heterosis in soybean.     

Molecular marker analysis of genetic variation characterizing a grasspea (Lathyrus sativus) collection from central Italy

Levels of genetic similarity characterizing 20 grasspea (Lathyrus sativus L.) populations collected in central Italy (17 populations in the Marche region and three populations in the Abruzzo region) were analysed with amplified fragment length polymorphism (AFLP) molecular markers. Two main clusters were found: one included large‐seeded populations from farms that were not market‐oriented (named Household populations) and the second, small‐seeded populations, cultivated in market‐oriented farms (named Commercial populations). Relationships among populations collected in different regions were found, although one population of the Abruzzo region was placed between the two main clusters, suggesting a possible further genetic differentiation within this grasspea germplasm collection. Principal component analysis based on AFLP marker frequency was effective in identifying polymorphic markers showing high discriminating ability between clusters H and C. In particular, seven markers showing high positive and three markers with low negative PC1 scores showed an almost cluster‐specific distribution. These results will be useful for enhancing Italian grasspea germplasm use in plant‐breeding programmes and for extending grasspea cultivation within the sustainable agricultural systems of central Italy.     

Estimating genetic relationships among historical sources of alfalfa germplasm and selected cultivars with sequence related amplified polymorphisms

Fifteen alfalfa populations consisting of six public cultivars and nine historically recognized sources of alfalfa germplasm in North American cultivars were examined using sequence related amplified polymorphisms (SRAPs). Three bulk DNA samples from each population were evaluated with fourteen different SRAP primer pairs. This resulted in 249 different amplicons, of which over 90% were polymorphic. A dendrogram from the analysis suggests that the public cultivars are quite diverse from all the historical sources of germplasm. The highest mean genetic similarity among the nine original sources of Medicago germplasm was 0.85 between PI 536535 (Peruvian) and 536536 (Indian), while the lowest (0.47) was between PI 560333 (M. falcata) and 536539 (African). The highest mean genetic similarity among the nine original sources of Medicago germplasm and the public alfalfa cultivars was 0.78 between PI 536532 (Ladak) and Vernal, while the lowest (0.59) was between PI 536539 (African) and Oneida. Relationships based on SRAP analysis appear to generally concur with expected relationships based on fall dormancy. This report demonstrates that SRAPs are a promising marker system for detecting polymorphisms in alfalfa.     

Development of molecular markers for crown rot resistance in wheat: mapping of QTLs for seedling resistance in a '2-49' × 'Janz' population

Crown rot, caused by Fusarium pseudograminearum, is an important disease of wheat in Australia and elsewhere. In order to identify molecular markers associated with partial seedling resistance to this disease, bulked segregant analysis and quantitative trait loci (QTL) mapping approaches were undertaken using a population of 145 doubled haploid lines constructed from ‘2‐49’ (partially resistant) × ‘Janz’ (susceptible) parents. Phenotypic data indicated that the trait is quantitatively inherited. The largest QTLs were located on chromosomes 1D and 1A, and explained 21% and 9% of the phenotypic variance, respectively. Using the best markers associated with five QTLs identified by composite interval mapping, the combined effect of the QTLs explained 40.6% of the phenotypic variance. All resistance alleles were inherited from ‘2‐49’ with the exception of a QTL on 2B, which was inherited from ‘Janz’. A minor QTL on 4B was loosely linked (19.8 cM) to the Rht1 locus in repulsion. None of the QTLs identified in this study were located in the same region as resistance QTLs identified in other populations segregating for Fusarium head blight, caused by Fusarium graminearum.     

Difference in Allelopathic Potential as Influenced by Root Periderm Colour of Sweet Potato (Ipomoea batatas)

Laboratory bioassay and high‐performance liquid chromatography (HPLC) analysis were conducted to determine the allelopathic potentials of aqueous or methanol extracts from three different coloured sweet potato [Ipomoea batatas L. (Lam)] cultivars by plant part. The aqueous extracts applied on filter paper significantly inhibited root growth of alfalfa (Medicago sativa L.). Aqueous leaf leachates at 40 g dry tissue l^?1 (g l^?1) from white sweet potato cultivar ‘Sinyulmi’ showed the highest inhibition against alfalfa, followed by yellow ‘Sinhwangmi’ and purple ‘Jami’. Alfalfa root growth was significantly inhibited by methanol extracts of the same plants as the concentration increased. Aqueous and methanol extracts from leaves showed the most inhibitory effect on alfalfa root growth followed by stems and roots. By means of HPLC analysis, leaf samples of sweet potato had the highest amount of phenolic compounds followed by stems and roots. Total content of these compounds was highest for leaf extracts (37.7 mg 100 g^?1), detected in EtOAc fraction, especially trans‐cinnamic acid (20.9 mg 100 g^?1). These results suggest that sweet potato plants are allelopathic and that their activities differ depending on plant part as well as root periderm colour.     

基于cpSSR标记的甘薯品种亲缘关系及遗传多样性分析

在甘薯遗传多样性研究中,目前主要采用细胞核基因组的遗传信息。而放任授粉(集团杂交)是甘薯育种的重要手段,但基于叶绿体微卫星(chloroplast simple sequence repeat,cpSSR)标记技术的甘薯的遗传多样性分析和指纹图谱构建的研究报道较少。本研究基于cpSSR标记技术,对16份甘薯品种进行分子鉴别,并建立相对应的指纹图谱。利用在19对cpSSR引物中筛选出的11对特异性引物进行cpSSR扩增,通过聚丙烯酰胺凝胶电泳检测,结果显示,共扩增出43个条带,其中多态性条带33条,平均每对引物扩增出3个多态性条带,多态性百分率为76.74%。16份甘薯品种的遗传距离变异范围为0.0912-0.5067,平均遗传距离是0.2301。聚类分析表明,cpSSR标记能大致反映出不同品种之间的亲缘关系。该指纹图谱的建立,可为中国甘薯品种数据库的完善、近缘甘薯品种的鉴定、育种亲本的选择和品种保护提供参考依据。     

Responses to Selection for Root Dimension in a South European Variety of Perennial Ryegrass)*

The objective of the study was to determine the effect of selection for large root dimension on root and shoot characteristics of an Italian variety of Lolium perenne L. (cv. ‘Vejo’). Two cycles of recurrent restricted phenotypic selection (RRPS) were carried out in 1983 and 1984 at Perugia (Italy). In each cycle, 20 out of 1,500 plants were selected and intercrossed under isolation. In 1985, 120 plants from‘Vejo selected’ and an equal number of plants from‘Vejo’ initial seed-lot were grown hydroponically for 92 days; at the end of the experiment, the plants were measured for their number of leaves and stems, leaf length, length of the longest root, root volume and shoot and root dry weight. Sufficient genetic variability existed among plants of cv. ‘Vejo’ to allow highly significant responses to selection for root dimension (+25 % in root length, +39 % in root volume, + 67 % in root dry weight and +31 % in root: shoot dry weight ratio). Furthermore, two cycles of RRPS for root dimension did not show negative responses of the above-ground parts of the plant.     

Development of cleaved amplified polymorphic sequence (CAPS) markers linked to a green rice leafhopper resistance gene, Grh3(t)

The chromosomal location of the resistance gene for green rice leafhopper (GRLH), an injurious insect for rice, has been determined and RFLP markers closely linked to this gene have been identified. The susceptible japonica rice variety ‘Nipponbare’ was crossed with a resistant japonica rice line ‘Aichi42’, in which green rice leaf hopper resistance had been introduced from an indica variety ‘Rantaj‐emas2’, and the 100 F₂ plants obtained were used for linkage analysis. The green rice leafhopper resistance gene, Grh3(t), was mapped between RFLP markers C288B and C133A on chromosome 6 and co‐segregated with C81. Of the RFLP markers tightly linked to Grh3(t), C81 was converted to a SCAR marker and C133A to a cleaved amplified polymorphic sequence marker that could distinguish the heterozygous genotype to establish an effective marker‐aided selection system for the GRLH resistance gene.     

Variable resistance of bread wheat (Triticum aestivum) lines carrying 2NS/2AS translocation to wheat blast

Wheat blast disease, caused by Magnaporthe oryzae (anamorph Pyricularia oryzae), produces severe damage to wheat production in South America. It was observed that many resistant cultivars contain the 2NS/2AS translocation from Triticum ventricosum. In this study, we evaluate the presence of the 2NS/2AS translocation in 57 advanced breeding lines and one variety ‘Caninde 1’ from Paraguayan wheat germplasm, using VENTRIUP‐LN2 primers. The germplasm ‘Caninde 1 and 22’ of the breeding lines, found positive for the presence of 2NS/2AS translocation, were inoculated with a single aggressive Magnaporthe pathotype P14‐039, to assess their response to wheat blast infection under controlled conditions. Based on the disease infection score, ten of the breeding lines, ‘Caninde 1’ and ‘Milan’ (positive control), were classified as resistant. Three of the remaining breeding lines were classified as moderately resistant, five as moderately susceptible and other four as susceptible. Our results show that the expression of 2NS/2AS‐based blast resistance is more dependent on genetic background of the inserted germplasm than previously envisioned.     

Identification of a molecular marker linked to the closed capsule mutant trait in sesame using AFLP

The identification of an amplified fragment length polymorphism (AFLP) marker linked to an agronomically useful trait in sesame is reported. A bulked segregant analysis (BSA) approach was adopted on segregating progenies of a cross between the closed capsule mutant line ‘cc3’, and the Turkish variety ‘Muganli‐57′. A total of 72 primer combinations were screened for linkage to the trait, but only one closely linked amplified fragment length polymorphism (AFLP) marker was identified. The linkage was confirmed by analysing the AFLP profile from single plants. The marker has the potential to accelerate breeding programmes aimed at modifying unwanted side‐effects of the closed capsule mutation by marker‐assisted selection.     

Evidence of a major genetic factor conditioning freezing sensitivity in winter wheat

The inheritance of freezing tolerance in F₂‐derived F₄ populations from all possible crosses of winter wheat cultivars ‘Kestrel’, ‘Eltan’, ‘Tiber’, ‘Froid’ and germplasm line Oregon Feed Wheat #5 (ORFW) was investigated. When frozen to a temperature equal to the LT₅₀ of the least freezing tolerant parent (ORFW), survival frequency distributions were skewed to greater survival in six of the 10 crosses, however, very few of the progeny from the four crosses to ORFW survived. The inheritance of this freezing sensitivity was investigated with freezing of F_2:4 populations from the crosses of ORFW to ‘Eltan’ or ‘Tiber’ to the LT₅₀ of the hardier parent. Very few of the F_2:4 populations survived as well as ‘Eltan’ or ‘Tiber’, indicating a small number of strongly dominant genetic factors in ORFW that conditioned freezing sensitivity. Molecular analysis indicated these factors were not spring‐type vernalization alleles.     

Inbreeding Depression and Other Genetic Effects in Populations of Maize Containing Highland Tropical Germplasm

A variety cross diallel of five populations of maize (Zea mays L.) was evaluated in a cool, temperate environment. Two of the parental populations were Corn Belt Dent synthetics (AS3 and BS22), one was developed from selected lines from a CIMMYT population developed for highland regions of the tropics and four Corn Belt Dent inbred lines (NZS3), and the other two had the pedigrees ‘Criollo de Toluca’× AS3² (NZS1) and ‘San Gerónimo’× BS222 (NZS2). ‘Criollo de Toluca’ is from the highlands of Mexico and ‘San Gerönimo’ is from the highlands of Peru. The objective was to study the influence of introgressing highland tropical germplasm into elite Corn Belt Dent synthetics on genetic effects, especially inbreeding depression, and to develop recommendations for the utilization of the introgressed populations. Inbreeding depression explained the greatest proportion of the variation among entries for grain yield, days to silking and plant height, but not for grain moisture at harvest and root lodging. For grain yield, NZS1, NZS2, and NZS3 had high levels of inbreeding depression and the difference between NZS1 and AS3 was statistically significant. High levels of root lodging were associated with NZS1 and high grain moisture at harvest with NZS2. These results indicate that a recurrent selection method, such as S₁ selection, which effectively reduces inbreeding depression, should be used in early cycles of selection with introgressed populations containing highland tropical germplasm.     

Quantitative trait loci for grain-quality traits across a rice F2 population and backcross inbred lines

Improving grain-quality is an important goal in rice breeding programs. One vital step is to find major quantitative trait loci (QTLs) for quality related traits and then investigate the relationships among them. We crossed ‘N22’, an indica variety with good appearance but low grain weight, to a japonica variety, ‘Nanjing35’, with superior grain yield but poor appearance. This enabled us to construct an F₂ population and a set of backcross inbred lines (BILs) for QTL-mapping for the traits related grain appearance. In all, 37 QTLs were identified for grain length (GL), grain width (GW), grain thickness (GT), thousand-grain weight (TGW), and the percentage of grains with chalkiness (PGWC). Of these, 17 QTLs detected from 184 plants in the F₂ population explained 4.97–27.26 % of the phenotypic variance, another 20 QTLs were identified using BILs from 2009 to 2010. Quantitative trait loci for major effects were detected in different populations and across years. A new QTL hot spot (marker interval RM504–RM520) was found on Chromosome 3, which harbored QTLs for GL, GW, GT, and TGW. Among our five examined traits, grain shape was significantly correlated with TGW and PGWC. The PGWC values of two heavier grains BILs, L93, and L145 are much lower than Nanjiing35, the analysis of genotype showed that this greater weight may due to the locus for GL occurring within RM504–RM520 on Chromosome 3. Therefore, those two lines will allow us to develop a long-grain high-yield rice variety with less chalkiness.     

Correlated effects of exotic pollen‐fertility restorer genes on agronomic and quality traits of hybrid rye

In CMS (cytoplasmic male sterility)‐based hybrid rye (Secale cereale L.) breeding, effective pollen‐fertility restoration is an essential prerequisite for achieving maximum grain yield on the one hand and for minimizing ergot (Claviceps purpurea) infestation on the other. Restorer genes for the CMS‐inducing ‘Pampa’ cytoplasm derived from landraces collected in Iran and Argentina are used by breeders for achieving this goal. Here, restorer genes from four germplasm sources (‘Altevogt 14160’, ‘IRAN III’, ‘Trenelense’ and ‘Pico Gentario’) were analysed by producing three‐way cross hybrids between an elite CMS single cross and pollinators with and without a given restorer gene. Materials were evaluated on large drilled plots for restorer index (RI), grain yield, plant height and other traits in six environments. In experiment 1, a restorer gene from ‘Altevogt 14160’ was used. Seven pairs of marker‐selected carrier and non‐carrier backcross lines served as pollinators. In experiment 2, the pollinators were 17 backcross line pairs from the other three germplasm sources. These lines were grouped as high (RI > 67%) and low restorers (RI < 30%), respectively, using testcrosses with a highly diagnostic CMS tester. Hybrids carrying an exotic restorer gene suffered from a significant grain yield reduction by 4.4% and 9.4% and were 9.3 and 4.8 cm taller in experiments 1 and 2, respectively. Thousand‐kernel weight was reduced, whereas quality traits were only slightly affected. For all traits, significant genetic variance existed among the testcrosses to the presence vs. absence of a given exotic restorer gene. This offers a chance for the breeder to reduce or ultimately overcome the presently observed performance reductions brought about by exotic restorer genes.