Assessing genetic diversity in the cassava (Manihot esculenta Crantz) germplasm collection in Brazil using PCR-based markers

Knowledge of the origin, organization and nature of the cassava (Manihot esculenta Crantz) germplasm collection in Brazil is incomplete due to lack of critical information on several aspects of the collection. This study verifies the utility of SSR-primed PCR markers for germplasm assessment and then utilizes these markers as well as RAPD's to characterize the Brazilian collection. We specifically address the following questions: 1)what is the relationship of morphologically closely related species to cultivated cassava? 2) What is the genetic diversity of cultivars within and between different habitats in Brazil? 3) Do agronomic traits and molecular markers reveal the same relationship among cassava accessions? 4) How complete is the Brazilian cassava collection and how well is it represented in the Word Core Collection of cassava, maintained by CIAT? Results of the interspecific studies of cassava and its wild relatives confirms the close relationship of cassava, Manihot esculenta ssp. esculenta to Manihot esculenta ssp. flabellifolia as well identifying several other closely related wild species. Next, PCR-based markers indicate a strong grouping of varieties related to the region of cultivation in Brazil. Moreover, important regions such as Cerrados and Amazon are relatively poorly represented in germplasm collections. Interestingly, the relationships of accessions based on agronomic traits are not fully congruent with relationships revealed with RAPD markers. Finally, the genetic diversity of the Brazilian cassava collection is not fully represented in the Core of the Word Core Collection of CIAT.     

Photomorphogenic mutants of tomato

Summary Two cloned cassava genes (pCAS6, pHNL) and a barley gene (pBLT63) which is highly conserved in higher plants, were evaluated as potential multilocus probes for RFLP analysis of phylogenetic relationships of species within the genus Manihot. For M. rubricaulis, M. chlorosticta and M. esculenta subsp. flabellifolia dendrograms based on 13 probe-restriction enzyme combinations show very little variation between accessions of the same species. A close relationship is demonstrated between the Mexican species M. chlorosticta and the M. esculenta subsp. flabellifolia accessions, which does not support the classification of flabellifolia as a true South American wild species.     

Introgression of genes for dry matter content from wild cassava species

Cassava cultivars often have poor resistance to biotic stresses and lack good quality traits. Wild species of cultivated crops have frequently been used as an important source of genetic diversity. Cassava breeders are becoming increasingly interested in incorporating genes of wild relatives. In 2000 the International Center for Tropical Agriculture (CIAT) initiated a programme to introgress genes for several root yield and quality traits from wild cassava relatives into its germplasm collection. The objectives of this study were to evaluate one resulting inter-specific cross with high variability for dry matter content (DMC) and to assess the effect of such a cross on other yield related traits. Crossing of the elite cultivar MTAI 8 to the wild relative Manihot tristis increased the percentage DMC above the normal average of about 35%, with percentage DMC ranging from 34.39 to 42.73. The crosses, however, were accompanied by some detrimental effects, most noticeable the reduction in harvest index (HI). It is apparent that when selecting for DMC, caution should be taken and HI and fresh root yield should be monitored. Regression analysis singled root weight, percentage DMC and fresh root yield out as the most important contributors to dry root yield. Principal component analysis indicated that root weight, roots per plants and DMC contributed most to storage root yield.     

A cassava clonal evaluation trial based on a new cassava breeding scheme

Cassava (Manihot esculenta) breeding at the earlier stages so far has been mainly based on a mass phenotypic recurrent selection, as little data is taken. This has resulted in a long breeding cycle and lack of organised information on the breeding values of parental lines used in the breeding programmes. The International Institute of Tropical Agriculture (CIAT) recently initiated a scheme to try and correct this, by introduction of replication and blocking in the clonal evaluation trial (CET) and collection of data on all genotypes at early stages of breeding. The aim of the study was to evaluate a CET using the new breeding scheme. Replicating and blocking of clones improved the accuracy of the data obtained. High broad-sense heritability values, comparable to those at advanced selection stages, were obtained, as environmental effects were minimised. The highest was for harvest index (HI) (0.80), followed by cassava frogskin disease (CFSD) (0.79), dry matter content (DMC) (0.75) fresh and dry root yields (DRY) (0.73), root weight (RtWt) (0.61), number of commercial roots (0.60) and roots per plant (RtPlt) (0.43). DMC, HI and RtWt were identified as important variables in selection and determination of economic yield at early stages of selection.     

Stability of cassava plants at the DNA level after retrieval from 10 years of in vitro storage

Summary Cassava (Manihot esculenta Crantz) germplasm collections are conventionally maintained by continuous vegetative propagation in the field. Tissue culture techniques provide a more convenient way to conserve germplasm. The cassava in vitro gene bank held in trust at CIAT comprises nearly 6000 accessions. A study was carried out to determine whether any DNA rearrangements resulting from in vitro storage under slow growth could be detected by molecular analysis in retrieved plants. RFLPs with homologous probes, RAPDs with twenty primers and DNA fingerprinting with M13 probe were tested to detect variation at DNA level in cassava plants after ten-years in vitro storage. The molecular marker data obtained in this study supports the stability of the cassava germplasm under the in vitro storage conditions described in this work.     

Introgression of antioxidant activity into cassava (Manihot esculenta C.): an effective technique for extending fresh storage roots shelf life

Physiological postharvest deterioration (PPD) of cassava is the main constraint affecting its nutritional and economical values. PPD is induced by wounds when detaching storage roots from mother plant during harvesting. It is accelerated by the reactive oxygen species (ROS) such as oxygen ion (O₂) and peroxide (O₂)^?2. The carotenoid content and its antioxidant property can help in extending shelf life of cassava storage roots. The primary mode of action of carotenoids as antioxidant is to quench singlet oxygen. Cassava breeding was reported to successfully introgress and improve carotenoid content into cassava. The two types of phytoene synthase (PSY) enzymes (PSY1 and PSY2) are key regulators of carotenoids accumulation in cassava. Carotenoids formation and accumulation in cassava storage roots are induced by a single nucleotide polymorphism in PSY2 which causes a non‐conservative amino acid exchange. This single nucleotide polymorphism in PSY gene is co‐segregated with β‐carotene in cassava storage roots, a phenomenon that could help to unravel the mechanism of introgression of carotenoids into cassava. This article investigates breeding feasibility for improving quality of cassava landraces in developing countries.     

Transfer of high kernel weight and high protein from wild tetraploid wheat (Triticum turgidum dicoccoides) to bread wheat (T. aestivum) using homologous and homoeologous recombination

Summary Wheat pentaploids were produced by hybridizing a high kernel weight (1000 grain wt=56 g), high protein (25.4%) line of wild tetraploid wheat (Triticum turgidum dicoccoides) as male parent, with the three hexaploids (T. aestivum) — normal Chinese Spring and its two homoeologous pairing mutants, ph _1b and ph ₂. The pentaploids were crossed as female parents to the two commercial hexaploid cultivars Warigal and Barkaee and 42-chromosome stable plants selected from the F₁ of the pentaploid x hexaploid crosses.Mean protein content of certain F₃ lines from all six pentaploid x hexaploid crosses was significantly higher than Chinese Spring and the respective commercial hexaploid parent (p<0.005) indicating high protein had been transferred from the tetraploid to the hexaploid level.Kernel weight amongst certain F₃ lines of the three pentaploids x Barkaee was significantly (p<0.0005) higher than either Chinese Spring or Barkaee, indicating the transfer also of high kernel weight from the tetraploid to the hexaploid level. However kernel weight was not significantly increased over Warigal in any F₃ lines of its crosses with the three pentaploids.High levels of homoeologous chromosome pairing in the ph-mutant pentaploids, plus evidence for significant modification of the composition of high-molecular weight (HMW) glutenin subunits of grain protein in certain F₃ derivatives of the ph-mutant pentaploid x hexaploid, crosses indicates that the ph-mutant-derived lines may possess novel (intergenome) genetic recombination, at least for high protein, and possibly kernel weight.     

An SSR-based molecular genetic map of cassava

Summary Microsatellites or simple sequence repeats (SSR) are the markers of choice for molecular genetic mapping and marker-assisted selection in many crop species. A microsatellite-based linkage map of cassava was drawn using SSR markers and a F₂ population consisting of 268 individuals. The F₂ population was derived from selfing the genotype K150, an early yielding genotype from an F₁ progeny from a cross between two non-inbred elite cassava varieties, TMS 30572 and CM 2177-2 from IITA and CIAT respectively. A set of 472 SSR markers, previously developed from cassava genomic and cDNA libraries, were screened for polymorphism in K150 and its parents TMS 30572 and CM 2177-2. One hundred and twenty two polymorphic SSR markers were identified and utilized for linkage analysis. The map has 100 markers spanning 1236.7 cM, distributed on 22 linkage groups with an average marker distance of 17.92 cM. Marker density across the genome was uniform. This is the first SSR based linkage map of cassava and represents an important step towards quantitative trait loci mapping and genetic analysis of complex traits in M. esculenta species in national research program and other institutes with minimal laboratory facilities. SSR markers reduce the time and cost of mapping quantitative trait loci (QTL) controlling traits of agronomic interest, and are of potential use for marker-assisted selection (MAS).     

Breeding cassava for resistance to cassava mosaic disease

Summary Cassava mosaic disease (CMD) is one of the most serious and widespread diseases throughout cassava growing areas in Africa, causing yield reductions of up to 90%. Early research on breeding of cassava (Manihot esculenta Crantz) for resistance to CMD in Africa is reviewed. Changes in population size and in activity of the white-fly vector to CMD (Bemisia tabaci Genn.) in relation to changes in environmental conditions such as amount and distribution of rainfall, light intensity and temperature are discussed in relation to screening for resistance to CMD. Over the past eight years, significant progress has been made at the International Institute of Tropical Agriculture (IITA). Resistance to CMD has been successfully incorporated into high yielding cultivars of acceptable quality. The CMD resistant material has been evaluated and many promising clones have been selected in various countries in tropical Africa and India. The resistance has been effective in those countries.     

Analysis of QTL for yield-related traits in cassava using an F<Subscript>1</Subscript> population from non-inbred parents

A cassava F₁ population raised from the cross SC6 × Mianbao was used to construct a genetic linkage map. The map incorporated 200 polymorphic amplified fragment length polymorphism, sequence-related amplified polymorphism, simple sequence repeat (SSR), and expressed sequence tag (EST)–SSR markers which fit a 1:1 segregation ratio. It comprised 20 linkage groups (LGs) and spanned a genetic distance of 1645.1 cM with an average marker interval of 8.2 cM. Fifty-seven repeatedly detected QTLs (rd-QTLs) for three phenotypic traits (fresh root yield, root dry matter content, and root starch content) were identified in the F₁ population in four trials of year 2003, 2004, 2005, and 2008 by inclusive composite interval mapping. Among the 57 rd-QTLs, 25 rd-QTLs were linked to SSR/EST–SSR markers, which will help to facilitate marker-assisted selective breeding in cassava, and 15 marker intervals on ten LGs showed pleiotropic effects.     

Morphological variation of starch granules in S<Subscript>1</Subscript> cassava progenies

The need to develop new cassava (Manihot esculenta Crantz) varieties with different starch characteristics has greatly motivated new research. The aim of this study was to evaluate morphological alterations in starch granules as well as genetic gains in segregating S₁ cassava populations. Starch from accessions (S₀): BGM0222, BGM1378 and BGM1662 and from their self-fertilized progenies (S₁): F0222, F1378 and F1662, with 78, 80 and 76 individuals, respectively, were evaluated for the following traits: area (ArStGr, µm²), perimeter (PrStGr, µm), length (LeStGr, µm) and number of starch granules (NuStGr). Moreover, some agronomic traits of the S₁ progenies were evaluated at harvest such as: plant height (PlaHei), shoot yield (ShoYi), root yield (RoYi), starch yield (StYi), and root dry matter content (DMC). With the selection of transgressive individuals in S₁ progenies, it is possible to obtain selection gains for values higher and lower than the average of the S₀ genitors, whereas selection gains can reach up to 16.58, 8.39, 7.87 and 34.18% for ArStGr, LeStGr, PrStGr, and NuStGr, respectively. Average size of the starch granule varied from 9.8 to 12.37 µm among individuals in an S₁ progeny. Positive and high correlations between the individuals were found for some morphological characteristics of the cassava starch granule but not strongly correlated with agronomic traits. Therefore, it is possible to select S₁ cassava individuals for agronomic characteristics without losing other important starch traits. Cassava-segregating populations allow the obtainment of individuals with different morphological characteristics as for starch granules, enabling obtainment of genetic gains with the selection.     

Identification of QTL controlling adventitious root formation during flooding conditions in teosinte (Zea mays ssp. huehuetenangensis) seedlings

Adventitious root formation (ARF) at the soil surface is one of the most important adaptations to soil flooding or waterlogging. Quantitative trait loci (QTL) controlling ARF under flooding condition were identified in a 94 F₂ individual population by crossing maize (Zea mays L., B64) × teosinte (Z. mays ssp. huehuetenangensis). A base-map was constructed using 66 SSR and 42 AFLP markers, covering 1,378 cM throughout all ten maize chromosomes. The ARF capacity for seedlings was determined by evaluating the degree of root formation at the soil surface following flooding for 2 weeks. ARF showed continuous variation in the F₂ population. Interval mapping and composite interval mapping analyses revealed that the QTL for ARF was located on chromosome 8 (bin 8.05). Utilising a selective genotyping strategy with an additional 186 F₂ population derived from the same cross combination and 32 AFLP primer combinations, regions on chromosomes 4 (bin 4.07) and 8 (bin 8.03) were found to be associated with ARF. Z. mays ssp. huehuetenangensis contributed all of the QTL detected in this study. Results of the study suggest a potential for transferring waterlogging tolerance to maize from Z. mays ssp. huehuetenangensis.     

Seed Storage Proteins of Triticum turgidum ssp. dicoccoides and Their Effect on the Technological Quality in Durum Wheat

F₄ progenies of a cross between durum wheat cultivar ‘Creso’ and an accession of Triticum turgidum ssp. dicoccoides with high protein content were analysed by two different electrophoretic procedures (A-PAGE and SDS-PAGE). Variation of storage proteins at the Glu-A1, Glu-B1, Glu-B3 and Gli-B1 loci was studied. Electrophoretic analyses have shown that parents contain different alleles at each of the four loci considered and that protein components of T. dicoccoides are uncommon among cultivated wheats. Recombination between the Glu-B3 and Gli-B1 loci was observed. To determine the effects of the allelic variants on gluten properties, F₅ grains from every F₄ line were analysed for protein content and SDS-sedimentation value. Gluten quality was strongly associated with the allelic type of proteins coded by the Glu-B3 locus and, to a lesser extent, to those coded by Glu-A1. Mean sedimentation value of progenies possessing the ‘Creso’Glu-B3 allele was significantly greater than that showing the T. dicoccoides allele. High molecular weight glutenin sub-units coded by the T. dicoccoides Glu-A1 locus were also associated with larger sedimentation values than null form of ‘Creso’. Results of SDS-test, obtained for recombinants between Glu-B3 and Gli-B1, confirmd that specific omega- and gamma-gliadins are only genetic markers of quality, whereas variation for LMW glutenin subunits coded at Glu-B3 is responsible for differences in gluten properties.     

Correlated resistance of cassava to mosaic and bacterial blight diseases

Summary The two most serious diseases of cassava (Manihot esculenta Crantz) are cassava mosaic disease (CMD) and cassava bacterial blight (CBB) (Xanthomonas manihotis Starr). Clone 58308, derived from the third backcross of the interspecific cross of cassava (M. esculenta) x ceara rubber (M. glaziovii), showed a high level of resistance to both diseases. Crosses of 58308 with several other clones which varied from susceptible to moderately susceptible to both diseases gave progenies with a significant genotypic correlation between resistance to both diseases (r=0.90), apparently due to linkage. The heritabilities of resistance to the diseases were estimated at 50–70% for CMD and 25–65% for CBB. Resistance to both diseases is assumed to be polygenic. The correlated response to selection for CMD and for CBB was estimated.     

木薯主要农艺性状的遗传变异、相关性和主成分分析

对广西14个主要木薯品种的9个农艺性状进行了遗传变异分析、相关性分析和主成分分析。结果表明,块根长,块根鲜重、块根数的变异系数较大,分别为13.95%、11.33%、10.22%,块根干物质率和茎径的变异系数最小,分别为7.00%和6.57%;在相关性上,产量与块根数呈极显著的正相关,块根干物质率与块根淀粉含量呈极显著的正相关;主成分分析结果表明,前4个主成分对变异的贡献率达85.69%。在性状选择上,首先对变异大的性状进行选择是非常重要的。在品种选择时,应注意选择产量和淀粉含量均高的品种。     

Origin,diversity and utilization of the Cuban germplasm of common bean (Phaseolus vulgaris L.)

Summary Wheat root characters which influence vital plant processes have scarcely been explored for their genetic control. This study was conducted to i) examine the diversity of root traits and associated shoot traits in spring wheat (Triticum aestivum L.) cultivars; ii) study the nature of genetic control of selected traits; and iii) examine associations among root and shoot traits. Three experiments were conducted in the greenhouse with plants grown in the vermiculite medium in clear plastic tubes. In the first experiment, 42 spring wheat cultivars were grown for three weeks and measurements were taken on root length, leaf length, root number, leaf number, root dry weight, and top dry weight. In the second study, 15F₁'s originating from a partial diallel mating of six cultivars along with the parents were evaluated for 4 weeks. The data on root length, leaf length, and root number were subjected to diallel analysis according to Griffing's method 4, fixed model. A third experiment consisted of studying 2 F₂ populations with 141 plants per population. In the first study the 42 cultivars exhibited a wide range of variation for all six traits. Diallel analysis revealed significant effects of both general combining ability (GCA) and specific combining ability (SCA) for root length, leaf length, and root number. Parents with high GCA estimates were identified. Significant positive correlation coefficients were found among root and shoot traits. Analysis of F₂'s for root length indicated quantitative nature of inheritance of root length.     

Effects of Shading on Chlorophyll Content,Chlorophyll Fluorescence and Photosynthesis of Subterranean Clover

A field‐experiment (2004/2005 and 2005/2006 seasons) was conducted in the coastal plain of south‐eastern Sicily (37°03′N, 15°18′E, 15 m a.s.l.), on a Calcixerollic Xerochrepts soil, aimed at quantifying the effect of shading on chlorophyll (Chl) content, Chl fluorescence, photosynthesis and growth of subterranean clover. Four levels of photosynthetically active radiation reduction (from 0 % to 90 %) were tested on Trifolium brachycalycinum cv. ‘Clare’ and Trifolium subterraneum ecotype ‘Ragalna’. In both species shading progressively increased F_v/F_m, internal CO₂ concentration, diffusive leaf resistance and specific leaf area (up to 8 %, 34 %, 18 % and 68 %, respectively), and decreased Chl content, T_max, photosynthetic rate and plant dry weight (up to 9 %, 24 %, 79 % and 39 %, respectively). As plants aged, characteristic bell‐shaped trends were evident for photosynthetic parameters, with F_v/F_m increasing up until the onset of flowering, and thereafter declining. This implies that F_v/F_m may be a useful indicator of earliness in subterranean clover genotypes. The aboveground dry biomass response to shading was both genotype‐ and season‐dependent, but was predictable from the measurement of relative leaf Chl content. Moreover, our results suggest that an improvement in the interaction between host‐rhizobium may represent a major potential breeding target for enhancing subterranean clover tolerance to shading.     

The effect of cassava mosaic disease on the genetic diversity of cassava in Uganda

Summary Cassava (Manihot esculenta) is a tropical crop that is grown in Africa, Latin America and Southeast Asia. Cassava was introduced from Latin America into West and East Africa at two independent events. In Uganda a serious threat to cassava's survival is the cassava mosaic disease (CMD). Uganda has had two notable CMD epidemics since the introduction of cassava in the 1850s causing severe losses. SSR markers were used to study the effect of CMD on the genetic diversity in five agroecologies in Uganda with high and low incidence of CMD. Surprisingly, high gene diversity was detected. Most of the diversity was found within populations, while the diversity was very small among agroecological zones and the high and low CMD incidence areas. The high genetic diversity suggests a mechanism by which diversity is maintained by the active involvement of the Ugandan farmer in continuously testing and adopting new genotypes that will serve their diverse needs. However, in spite of the high genetic diversity we found a loss of rare alleles in areas with high CMD incidence. To study the effect of the introgression history on the gene pool the genetic differentiation between East and West Africa was also studied. Genetic similarities were found between the varieties in Uganda and Tanzania in East Africa and Ghana in West Africa. Thus, there is no evidence for a differentiation of the cassava gene pool into a western and an eastern genetic lineage. However, a possible difference in the genetic constitution of the introduced cassava into East and West Africa may have been diminished by germplasm movement.     

Changes in extended shelf life of cassava roots during storage in ambient conditions

Cassava roots have a short shelf life due to a process known as post-harvest physiological deterioration (PPD). Within 2–3 d undesirable vascular streaking in the root develops. Tolerance to PPD was recently reported in different cassava genotypes, opening up new opportunities to analyze biochemical changes in stored roots and in the functional properties of their starches. Roots from PPD-susceptible (HMC-1) and tolerant (AM 206-5) clones were harvested and stored for up to 14 d in ambient tropical conditions. AM 206-5 is also characterized by amylose-free starch. Roots and starch were analyzed each day. PPD levels differed significantly between the two clones (35% and 8% at day 14) and showed a relation to scopoletin synthesis, which reached maximum levels around day 3 or 4 of storage. Roots lost weight consistently during storage (≈10% in two weeks). Starch loss per day of root storage was estimated at about 1%. This could be the result of consistent increases in total sugars and respiration of root tissue. Important changes in starch properties were observed. Gel clarity decreased gradually during storage, with more pronounced changes occurring in starches from HMC-1. Swelling power decreased only in the case of AM 206-5. Gel viscosity increased in both genotypes. Improved tolerance to PPD could significantly reduce the economic impact of the short shelf life of ordinary cassava root processing. It remains to be seen, however, whether changes in stored roots positively or negatively affect the quality of the final product.     

Identification of resistant sources and DNA markers linked to genomic region conferring dry root rot resistance in chickpea (Cicer arietinum L.)

A set of 520 chickpea germplasm lines was screened under laboratory conditions using blotter paper technique for reaction to dry root rot caused by Rhizoctonia bataticola (Taub.) Butler. The lines PG06102, BG2094 and IC552137 were identified as resistant for dry root rot. Phenotyping the mapping population consisting of 129 F_2:3 progeny derived from the cross L550 × PG06102 during 2013 winter indicated monogenic inheritance of dry root rot resistance. Fifty‐two of 381 simple sequence repeat (SSR) primers polymorphic between the two parents were used to genotype F₂ resistant and susceptible bulks prepared on the basis of reaction of F_2:3 progeny. Four markers differentiated the resistant and susceptible bulks. All the four polymorphic markers were then assayed on the entire F₂ population. Linkage analysis using 129 F₂ plants revealed that two markers ICCM0299 and ICCM0120b were co‐segregating with resistance to dry root rot. These two markers appeared to have additive effects on resistance and could be potentially utilized in dry root resistance breeding programme.