Genetic diversity in groundnut (<Emphasis Type="Italic">Arachis hypogaea</Emphasis> L.) genotypes and detection of marker trait associations for plant habit and seed size using genomic and genic SSRs

Groundnut (Arachis hypogaea L.) an important oilseed crop in India is known to have narrow genetic base. Therefore, the assessment of genetic diversity and detection of marker-trait association are important objectives for the genetic improvement of groundnut. The present study involved the development of 192 SSR markers from Arachis genomic survey sequences. From these, seven polymorphic SSRs along with 15 other genomic SSRs, 19 genic SSRs, and three STS markers were used to detect genetic diversity among 44 groundnut genotypes. These polymorphic SSR markers amplified 155 bands (76 genomic and 79 genic), of these 128 bands (67 genomic and 61 genic) were polymorphic. The genomic SSR exhibited 88.1% and genic SSRs displayed 77.2% allelic polymorphism. The polymorphic information content (PIC) of the markers ranged from 0.04 to 0.95. The pair-wise genetic similarity ranged from 24.2 to 90.7% for genomic SSR and 32.9 to 97.9% for genic SSR markers. Cluster analysis based on the pooled data from both genomic and genic SSRs revealed a dendrogram which could distinguish all the genotypes. Further, the AMOVA analysis detected 16.7% genetic variation due to differences in seed size and 13.0% due to plant habit. Based on locus-by-locus AMOVA and Kruskal-Wallis ANOVA and further confirmation by discriminant analysis and general linear model, six markers were found to be associated with plant habit and four markers with seed size.     

Developing and validating microsatellite markers in elephant grass (<Emphasis Type="Italic">Pennisetum purpureum</Emphasis> S.)

Elephant grass [Pennisetum purpureum S.; syn. Cenchrus purpureus (Schumach.) Morrone] is an important global forage crop and is recognized for high yields of herbage with good nutritive value. It also has high biomass potential to be utilized as a biofuel feedstock. Whereas several previous genetic studies adapted simple sequence repeat (SSR) markers from pearl millet [Pennisetum glaucum (L.) R.Br.] for investigations in elephant grass, the present study developed SSR markers from 3536 DNA sequences derived from 16 elephant grass entries. A total of 3866 SSRs were identified including 1028 monomeric, 2019 dimeric, 735 trimeric, 49 tetrameric, 20 pentameric and 15 hexameric repeat motifs. Three hundred and seven sequences contained more than one repeated motif, and 154 SSRs were present in compound formation. Susequenctly,  four elephant grass and two pearl millet genotypes were chosen to validate 727 SSR markers. Of these, 628 markers produced visually detectable amplification products, including 73 (11.6%) polymorphic ones across all six genotypes. Polymorphism between the four elephant grass genotypes was revealed by 316 (50.6%) markers with diversity index values ranging from 0.75 to 0.38. Dimeric SSRs had the highest polymorphic rate (48.7%). These validated SSR markers had 58.6% (368 of 628) transferability rate to pearl millet. The availability of these polymorphic SSR markers will support advanced genetic studies in P. purpureum and its relatives.     

Development of linkage map and mapping of QTLs for oil content and yield attributes in linseed (<Emphasis Type="Italic">Linum usitatissimum</Emphasis> L.)

Linseed (Linum usitatissimum L.) is an important oilseed as well as stem fiber crop and rich source of omega-3 fatty acid. The present study aims to develop linkage map based on Indian genotypes and utilize it for mapping QTLs for important agronomic traits. Two diverse parental genotypes (KL-213 and RKY-14) of linseed showed wide range of variability for oil content and yield attributes. These parental genotypes also showed reasonable level of SSR polymorphism (~ 9.0%). The mapping population showed normal distribution of phenotypic traits. One hundred forty-six SSR markers were mapped on 15 linkage groups with marker density ranging from 3 to 18 markers per linkage group at average distance of 14.2 cM. A total of 11 QTLs were identified for six quantitative traits. Three QTLs for capsules/plant, 2 QTLs each for plant height, seeds/capsule and oil content and 1 QTL each for branches/plant and seed weight/plant were detected. Phenotypic variability explained by these QTLs varied from 1 to 15.23%. This study provides framework linkage map of linseed using Indian genotypes, which needs to be enriched further for future application in marker assisted breeding of linseed.     

Phenotypic and genotypic screening for rust resistance in common bean germplasm in Uganda

Rust caused by Uromyces appendiculatus (Pers., Pers.) Unger is one of the major foliar diseases of common bean (Phaseolus vulgaris) in Uganda. The use of host resistance remains the best option in managing this disease. The objective of this study was to identify sources of broad-spectrum rust resistance in common bean germplasm including landraces, commercial cultivars and introduced genotypes using a combination of phenotypic and genotypic screening with 22 simple sequence repeat (SSR) markers located on chromosome Pv04. A total of 138 genotypes were field screened from 2014 and 2015 using an alpha lattice design. The variance and correlation of disease incidence, area under the disease progression curve (AUDPC) and total grain yield were computed using GenStat. The polymorphism information content of the genotypes was determined, and the association of the markers and the disease resistance traits were analyzed using PowerMarker and TASSEL respectively. Resistance of each genotype was compared to the presence and absence of amplified markers. There were highly significant differences (P < 0.001) among the genotypes for disease incidence, AUDPC and total grain yield and a strong correlation (P < 0.001) between disease incidence and AUDPC in both years. The SSR markers, BARC_PV_SSR04725, bean_ssr_0778 and bean_ssr_2892 were associated (P ≤ 0.05) with rust resistance. Fifteen 15 genotypes which included the landraces Nabufumbo, and Kapchorwa white, and the commercial cultivar NABE 2 were identified as new sources of rust resistance that would be useful in future bean breeding programmes in Uganda.     

Development of a core set of SSR markers for the characterization of <Emphasis Type="Italic">Gossypium</Emphasis> germplasm

Molecular markers such as simple sequence repeats (SSR) are a useful tool for characterizing genetic diversity of Gossypium germplasm. Genetic profiles by DNA fingerprinting of cotton accessions can only be compared among different collections if a common set of molecular markers are used by different laboratories and/or research projects. Herein, we propose and report a core set of 105 SSR markers with wide genome coverage of at least four evenly distributed markers per chromosome for the 26 tetraploid cotton chromosomes. The core marker set represents the efforts of ten research groups involved in marker development, and have been systematically evaluated for DNA polymorphism on the 12 genotypes belonging to six Gossypium species [known collectively as the cotton marker database (CMD) panel]. A total of 35 marker bins in triplex sets were arranged from the 105 markers that were each labeled with one of the three fluorescent dyes (FAM, HEX, and NED). Results from this study indicated that the core marker set was robust in revealing DNA polymorphism either between and within species. Average value of polymorphism information content (PIC) among the CMD panel was 0.65, and that within the cultivated cotton species Gossypium hirsutum was 0.29. Based on the similarity matrix and phylogenetic analysis of the CMD panel, the core marker set appeared to be sufficient in characterizing the diversity within G. hirsutum and other Gossypium species. The portability of this core marker set would facilitate the systematic characterization and the simultaneous comparison among various research efforts involved in genetic diversity analysis and germplasm resource preservation.     

Identification of QTLs for agronomic traits using association mapping in lentil

A diverse panel of 96 genotypes of lentil was used in this study to identify QTL for nine agronomic traits through marker-trait association analysis. This study showed significant genetic variability among the lentil genotypes for nine agronomic traits and had medium to large broad sense heritability estimates (h² =?0.58–0.95). Screening of 534 SSR markers resulted in 266 polymorphic loci that generated 697 alleles ranging from 2 to 16 alleles per locus across the genotypes. The model-based population structure analysis identified two distinct subpopulations among lentil genotypes and each subpopulation did not show any admixture. Marker trait association (MTA) analysis following ML model resulted in the identification of 24 MTAs for nine traits at P?<?0.01. The per cent of phenotypic variation explained by each associated marker with particular agronomic trait ranged from 7.3 to 25.8%. The highest proportion of total phenotypic variation (23.1–25.8%) was explained by the QTLs controlling the primary branches/per plant. In the present study, few EST-SSR markers showed significant association with days to maturity, pods/plant, secondary branches/plant, 100 seed weight, yield/plant and reproductive duration and explained large phenotypic variation (7.3–23.8%). Hence, these markers can be used as functional markers in lentil breeding program for developing improved cultivars.     

Analysis of population structure and genetic diversity reveals gene flow and geographic patterns in cultivated rice (<Emphasis Type="Italic">O. sativa</Emphasis> and <Emphasis Type="Italic">O. glaberrima</Emphasis>) in West Africa

To fully exploit the diversity in African rice germplasm and to broaden the gene pool reliable information on the population genetic diversity and phenotypic characteristics is a prerequisite. In this paper, the population structure and genetic diversity of 42 cultivated African rice (Oryza spp.) accessions originating from West Africa (Benin, Mali and Nigeria, Liberia etc.) were investigated using 20 simple sequence repeats (SSR) and 77 amplified fragment length polymorphisms (AFLP). Additionally, field trials were set up to gain insight into phenotypic characteristics that differentiate the genetic populations among rice accessions. The analysis revealed considerably high polymorphisms for SSR markers (PIC mean?=?0.78) in the germplasm studied. A significant association was found between AFLP markers and geographic origin of rice accessions (R?=?0.72). Germplasm structure showed that Oryza sativa accessions were not totally isolated from Oryza glaberrima accessions. The results allowed identification of five O. glaberrima accessions which grouped together with O. sativa accessions, sharing common alleles of 18 loci out of the 20 SSR markers analyzed. Population structure analysis revealed existence of a gene flow between O. sativa and O. glaberrima rice accessions which can be used to combine several interesting traits in breeding programs. Further studies are needed to clarify the contributions of this gene flow to valuable traits such as abiotic and biotic stresses including disease resistance.     

Production and characterization of intergeneric diploid cybrids derived from symmetric fusion between Microcitrus papuana Swingle and sour orange (Citrus aurantium)

Plants were regenerated from intergeneric somatic hybridization between embryogenic protoplasts of Microcitrus papuana Swingle and leaf-derived protoplasts of sour orange (Citrus aurantium L.) via electrofusion. The regenerated plants were morphologically similar to the leaf parent in growth vigor, leaf and branch structure. FCM analysis showed that they were diploids. Simple-sequence-repeat (SSR) and cleaved-amplified-polymorphic-sequence(CAPS) were employed for hybridity characterization. SSR banding patterns of the regenerated plants were identical to the leaf parent, sour orange, indicating that they possessed nuclear component derived from sour orange. DNA amplification with chloroplast and mitochondrial universal primers, followed by restriction endonuclease digestion, revealed polymorphism between the fusion parents. Therefore, this method was used to determine the cytoplasmic compositions of the regenerated plants. Banding patterns for all the polymorphic primer/enzyme combinations of the regenerated plants were similar to those of the embryogenic parent, M. papuana, suggesting that only the cytoplasmic components derived from the embryogenic parent were present in the regenerated plants. FCM, SSR and CAPS demonstrated that intergeneric diploid cybrids have been successfully obtained by symmetric fusion. Related results concerning nuclear and cytoplasmic composition of previous diploid somatic hybrids and potential mechanism for regeneration of such kind of plants are discussed herein. This revised version was published online in July 2006 with corrections to the Cover Date.     

Genetic diversity and association mapping of agronomic yield traits in eighty six synthetic hexaploid wheat

Association mapping is a method to identify associations between target traits and genetic markers based on linkage disequilibrium (LD) of a quantitative trait locus. Synthetic hexaploid wheat (SHW) is derived from a cross between Triticum durum Desf. and Aegilops tauschii Coss. that enhances genetic diversity and broadens breeding resources. In this study, phenotypic diversity in 110 wheat accessions (86 SHW germplasm specimens and 24 conventional wheat varieties) was evaluated quantitatively for yield characteristics of grains per spike, thousand kernel weight, and spike length. Phenotypic data were collected over two years at two locations, and 1785 alleles were detected (mean 6.59), ranging from 3 to 11 alleles per locus. The average genetic diversity index was 0.749, with a range from 0.239 to 0.923. The polymorphic information content (PIC) ranged from 0.145 to 0.968, with a mean value of 0.695. The genetic diversity index and PIC indicated that genome B > D > A. Accessions were grouped into three subgroups based on STRUCTURE and unweighted pair-group with arithmetic mean clustering. The mean LD decay across the genome was 11.78 cM. Association mapping between traits and simple sequence repeat markers was performed using the generalized linear model approach. Forty-six SSR loci were significantly associated with the measured agronomic traits in two geographic locations. Together, these results broaden our knowledge of how to harness elite genes and genetic diversity in SHW in genomic and marker-assisted selection.     

Identification of simple sequence repeat markers for sweetpotato weevil resistance

The development of sweetpotato [Ipomoea batatas (L.) Lam] germplasm with resistance to sweetpotato weevil (SPW) requires an understanding of the biochemical and genetic mechanisms of resistance to optimize crop resistance. The African sweetpotato landrace, ‘New Kawogo’, was reported to be moderately resistant to two species of SPW, Cylas puncticollis and Cylas brunneus. Resistance has been associated with the presence of hydroxycinnamic acids esters (HCAs), but the underlying genetic basis remains unknown. To determine the genetic basis of this resistance, a bi-parental sweetpotato population from a cross between the moderately resistant, white-fleshed ‘New Kawogo’ and the highly susceptible, orange-fleshed North American variety ‘Beauregard’ was evaluated for SPW resistance and genotyped with simple sequence repeat (SSR) markers to identify weevil resistance loci. SPW resistance was measured on the basis of field storage root SPW damage severity and total HCA ester concentrations. Moderate broad sense heritability (H² = 0.49) was observed for weevil resistance in the population. Mean genotype SPW severity scores ranged from 1.0 to 9.0 and 25 progeny exhibited transgressive segregation for SPW resistance. Mean genotype total HCA ester concentrations were significantly different (P < 0.0001). A weak but significant correlation (r = 0.103, P = 0.015) was observed between total HCA ester concentration and SPW severity. A total of five and seven SSR markers were associated with field SPW severity and total HCA ester concentration, respectively. Markers IBS11, IbE5 and IbJ544b showed significant association with both field and HCA-based resistance, representing potential markers for the development of SPW resistant sweetpotato cultivars.     

Hybrids of <Emphasis Type="Italic">Passiflora</Emphasis>: <Emphasis Type="Italic">P. gardneri</Emphasis> versus <Emphasis Type="Italic">P. gibertii,</Emphasis> confirmation of paternity,morphological and cytogenetic characterization

Two new varieties of interspecific hybrids of Passiflora have been developed from the cross between P. gardneri versus P. gibertii, both registered under the Passiflora Society International. Twelve putative hybrids were analyzed. Hybridization was confirmed using RAPD and SSR markers. Primer UBC11 (5′-CCGGCCTTAC-3′) generated informative bands. Primer SSR Pe75 has amplified species-specific fragments and a heterozygote status was observed with two parent bands 300 and 350 bp. The molecular markers generated have been analyzed for the presence or absence of specific informative bands. Based on the morphological characterization, we have identified two hybrid varieties: P. ‘Gabriela’ and P. ‘Bella’. P. ‘Gabriela’ produced flowers in bluish tones, bluish petals on the adaxial and abaxial faces, light blue sepals on the adaxial and light green on the abaxial faces, corona with the base of filaments in intense lilac color and white apex. P. ‘Bella’ produced flowers in lilac tones, intense lilac petals on the adaxial and abaxial faces, dark lilac sepals with whitish edges on the adaxial and light green on the abaxial faces, corona with the base of filaments in intense lilac color and white apex. The cytogenetic analysis verified that the hybrids have the same chromosomal number as the parents (2n = 18); the formation of bivalents between the homeologous chromosomes (n = 9) was observad, leading to regular meiosis, which allows the sexual reproduction and use of these hybrids in breeding programs.     

Estimation of genetic diversity among 34 genotypes in the genus <Emphasis Type="Italic">Cajanus</Emphasis> with contrasting host response to the pod borer and its allied pests

The genetic divergence among 34 genotypes belonging to 12 species of genus Cajanus were carried out using plant pest interaction and DNA marker analysis. Principal component analysis based on average percentage of pod damage caused by pod borer, plume moth, and blue butterfly in the field conditions, and growth of their larva and pupa on an artificial diet in vitro dispersed these genotypes into four coordinates evincing high genetic divergence as expected. DNA marker analysis using 11 pairs of SSR and nine ISSR primers showed higher polymorphism at the species level, and these primers exhibited variation with regard to average band informativeness, resolving power, and PIC value. No single primer was able to distinguish between all the 34 genotypes of Cajanus but nine species specific amplified fragments were generated by five ISSR primers. The pairwise Jaccard’s similarity coefficient and Nei’s genetic distance values revealed a higher level of inter-specific genetic variation in the genus Cajanus. The clustering of genotypes based on Jaccard’s similarity coefficient vis-a-vis Nei’s genetic distance agreed with the sectional classification of the genus Cajanus. Seven cultivars of C. cajan and the genotypes of their wild progenitor C. cajanifolius remained in one cluster, whereas accessions of C. platycarpus and C. scarabaeoides were out grouped. The rest of the genotypes belonging to nine species of Cajanus formed another cluster. The principal coordinate analysis also supported this clustering pattern. Moreover, these findings have good many implications for future breeding endeavors aimed at the introgression of pod borer resistance alleles.     

Mining of favorable marker alleles for flag leaf inclination in some rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.) accessions by association mapping

Flag leaf angle (FLA) in rice (Oryza sativa L.) is one of the important traits affecting F₁ seed production by mechanization. To elucidate the genetic mechanism of FLA and mine favorable marker alleles for F₁ seed production in rice, we performed a genome-wide association study using phenotypic data over 2 years and genotypic data of 262 pairs of simple sequence repeat (SSR) markers collected from 441 rice accessions. We detected seven SSR marker loci associated with FLA and four loci were novel. The four newly found loci were RM6266 on chromosome 3, RM348 on chromosome 4, RM258 on chromosome 10 and RM7303 on chromosome 11. We found a total of 27 favorable alleles, of which four, i.e., RM348-130 bp, RM7303-90 bp, RM258-180 bp, and RM4835-230 bp, had phenotypic effects larger than 10°. Nine combinations, which increased FLA by 45.7°–94.7° through pyramiding the favorable alleles contained in seven typical accessions, were predicted.     

Interspecific hybridization in <Emphasis Type="Italic">Sarcococca</Emphasis> supported by analysis of ploidy level,genome size and genetic relationships

Knowledge of ploidy level differences, genome size and genetic relationships between species facilitates interspecific hybridization in ornamentals. For Sarcococca (Buxaceae) only limited (cyto)genetic information is available. The aim of this study was to determine the genome size and chromosome number and to unravel the genetic relationships of a breeder’s collection using AFLP marker analysis. Based on these results, interspecific crosses were made and the efficiency and hybrid status was verified. Two groups of diploid plants (2n = 2x = 24) were observed, with either a genome size of 4.11–4.20 or 7.25–9.63 pg/2C. All the tetraploid genotypes (2n = 4x = 48) had genome sizes ranging from 7.91 to 8.18 pg/2C. In crosses between parents with equal ploidy level and genome size a higher crossing efficiency (on average 58% of the hybridizations resulting in fruits) and more true hybrids (on average 96% of the offspring) were obtained compared to crosses between plants with different genome size and ploidy level (on average 23% fruits and 24% hybrids, respectively). In none of the cross combinations, the ploidy level or genome size was found to be a complete hybridization barrier, although unilateral incongruity was found in some cross combinations. Distant genetic relationships did not hamper the hybridization within Sarcococca genotypes. Our findings will contribute to a more efficient breeding program and a faster achievement of hybrids with an added value.     

QTL mapping and identification of corresponding genomic regions for black pod disease resistance to three <Emphasis Type="Italic">Phytophthora</Emphasis> species in <Emphasis Type="Italic">Theobroma cacao</Emphasis> L.

The cacao tree (Theobroma cacao L.) is a species of great importance because cacao beans are the raw material used in the production of chocolate. However, the economic success of cacao is largely limited by important diseases such as black pod, which is responsible for losses of up to 30–40% of the global cacao harvest. The discovery of resistance genes could extensively reduce these losses. Therefore, the aims of this study were to construct an integrated multipoint genetic map, align polymorphisms against the available cacao genome, and identify quantitative trait loci (QTLs) associated with resistance to black pod disease in cacao. The genetic map had a total length of 956.41 cM and included 186 simple sequence repeat (SSR) markers distributed among 10 linkage groups. The physical “in silico” map covered more than 200 Mb of the cacao genome. Based on the mixed model predicted means of Phytophthora evaluation, a total of 6 QTLs were detected for Phytophthora palmivora (1 QTL), Phytophthora citrophthora (1 QTL), and Phytophthora capsici (4 QTLs). Approximately 1.77–3.29% of the phenotypic variation could be explained by the mapped QTLs. Several SSR marker-flanking regions containing mapped QTLs were located in proximity to disease regions. The greatest number of resistance genes was detected in linkage group 6, which provides strong evidence for a QTL. This joint analysis involving multipoint and mixed-model approaches may provide a potentially promising technique for detecting genes resistant to black pod and could be very useful for future studies in cacao breeding.     

High-throughput targeted genotyping using next-generation sequencing applied in <Emphasis Type="Italic">Coffea canephora</Emphasis> breeding

The use of molecular markers to detect polymorphism at DNA level is one of the most significant developments in molecular biology techniques. With the development of new next-generation sequencing technologies, the discovery of SNP became easier and faster, and the costs of data point were reduced. The development and use of SNP markers for coffee have provided new perspectives for the evaluation of genetic diversity and population structure via different statistical approaches. In this study, 72 Coffea canephora genotypes were analyzed to identify the SNP markers and apply them to genetic studies and selection of parents/hybrids in genetic breeding. As many as 117,450 SNP were identified using the RAPiD Genomics platform. After quality analyses, 33,485 SNP were validated for analyses of genetic diversity and population structure. Genotypes were separated based on their varietal groups, and Hybrids were differentiated using the clustering and Bayesian approach. Coffee accessions mistakenly identified in the germplasm and breeding program were detected. The Conilon varietal group presented the lowest genetic dissimilarity values, suggesting the introduction of new accessions in the germplasm bank. The highest genetic distances values were observed among genotypes of the heterotic groups (Conilon and Robusta). The markers were efficient in evaluating the genetic diversity and population structure of C. canephora. Promising crosses were selected within and between the varietal groups. Hybrids with greater genetic distances were selected, which were important for C. canephora breeding programs.