Assessment of genetic diversity amongst Ugandan sesame (<Emphasis Type="Italic">Sesamum indicum</Emphasis> L.) landraces based on agromorphological traits and genetic markers

Sesame (Sesamum indicum L.) is one of the most important ancient oilseed crops grown throughout the tropical and subtropical regions of the world. In Uganda, most of the cultivated sesame varieties are local landraces which are frequently traded between farmers. Although these traditional landraces are an important source of genetic diversity, knowledge of their genetic diversity is still limited.Agromorphological traits and a set of published and newly developed microsatellite markers were analyzed on a collection of 121 accessions of Ugandan sesame landraces. CpSSR analysis revealed four haplotypes, whereby haplotype B was present in 96% of the individuals. The analysis of nSSR markers from 6 non-coding regions revealed a mean PIC value of 0.56 whereas the PIC value of eight selected EST-derived SSRs was 0.26. Accession-wise, the expected heterozygosity (He) varied from 0 to 0.396. AMOVA revealed that the majority of the variance occurred among the individuals accounting for 75% of the total variation, only 6% was attributed to differences among the districts, pointing towards a high gene flow (Nm = 4.476). These results are supported by the PCoA analysis as well as the NJ tree both of which revealed no clustering of the accessions according to their geographic origin. Also the statistical analysis of 10 agromorphological traits indicated no clear pattern related to the geographic origin. Such a poor grouping, indicative of considerable gene flow across geographic domains, could be explained either by a high outcrossing rate, and/or through extensive seed trading.     

Genetic diversity of physic nut (<Emphasis Type="Italic">Jatropha curcas</Emphasis> L.) revealed by SSR markers

Ten microsatellite markers were used to investigate genetic diversity and genetic structure among 32 accessions of Jatropha curcas. Low levels of average genetic diversity were observed (H _E = 0.160). A dendrogram produced by the Unweighted Pair Group Method with Arithmetic Mean (UPGMA) based on Nei’s genetic distances revealed 3 groups among 32 accessions. The genetic differentiation (F _ST ) among two groups was significant (P < 0.01). The model-based Bayesian clustering method indicated that a population structure (ΔK) was separated into two groups. The analysis of molecular variance (AMOVA) showed higher variability (63.753%) among groups than within groups (36.247%). These findings could assist in defining the best method of genetic conservation and studies in breeding programs for genetic improvement of J. curcas.     

Resistance screening of <Emphasis Type="Italic">Coffea</Emphasis> spp. accessions for <Emphasis Type="Italic">Pratylenchus coffeae</Emphasis> and <Emphasis Type="Italic">Radopholus arabocoffeae</Emphasis> in Vietnam

Coffee varieties with resistance for the plant-parasitic nematodes Pratylenchus coffeae and Radopholus arabocoffeae are limited in Vietnam. A selection of imported varieties and high yield varieties of Arabica coffee in Vietnam were evaluated for resistance to both plant-parasitic nematode species in Northern Vietnam. The same experiments were carried out with hybrid arabica coffee, three selected clones of Coffea canephora and one clone of Coffea excelsa in the Western Highland of Vietnam. The screened coffee accessions from Ethiopia (KH1, KH13, KH20, KH21, KH29, and KH31) were susceptible and good host for P. coffeae. Also accessions 90P4 (Portugal) and Oro azteca (Mexico) had a reproduction factor Rf > 1. Pluma Hidalgo (Mexico), 90/6 (Vietnam), 90P3 (Portugal), 90P2 (Vietnam), Variedad (Mexico), 90T (Portugal), and Garnica (Mexico) were poor hosts (Rf < 1) but not tolerant to P. coffeae, expressed by a reduction of root weight compared to untreated control plants. Most of the coffee accessions tested in Northern Vietnam were intolerant to R. arabocoffeae, except 90T which showed no reduction of root weight, even at high initial nematode densities (4,000/pot). Good hosts for R. arabocoffeae were Variedad, KH1, KH21, KH29, KH20, KH31, and KH13 with Rf > 1. Pluma Hidalgo, 90/6, 90P3, 90P2, 90T, Oro azteca, and Garnica were poor hosts (Rf < 1). In the Western Highland experiment, all arabica coffee accessions were susceptible for P. coffeae with Rf ranging from 1.41 to 1.59. Tolerance to P. coffeae was found in C. liberica var. Dewevrei, Hong34 and Nhuantren. Coffea excelsa, Hong34, Nhuantren, and H1C19 were tolerant to R. arabocoffeae at the highest inoculation density (4,000 nematodes/pot). The most susceptible accessions were Nhuantren and K55. Resistance (Rf < 1) to R. arabocoffeae was found in C. liberica var. Dewevrei and Hong34. This article reports on the first screening for resistance and tolerance to P. coffeae and R. arabocoffeae in coffee accessions in Vietnam and shows promising results for enhanced coffee-breeding.     

<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated genetic transformation of pigeon pea [<Emphasis Type="Italic">Cajanus cajan</Emphasis> (L.) Millsp.] for resistance to legume pod borer <Emphasis Type="Italic">Helicoverpa armigera</Emphasis>

Agrobacterium-mediated genetic transformation was performed using embryonic axes explants of pigeon pea. Both legume pod borer resistant gene (cry1Ac) and plant selectable marker neomycine phosphor transferase (nptII) genes under the constitutive expression of the cauliflower mosaic virus 35S promoter (CaMV35S) assembled in pPZP211 binary vector were used for the experiments. An optimum average of 44.61% successfully hardened dot blot Southern hybridization positive plants were obtained on co-cultivation media supplemented with 200 μM acetosyringone without L-cysteine. The increased transformation efficiency from a baseline of 11.53% without acetosyringone to 44.61% with acetosyringone was further declined with the addition of different concentrations of L-cysteine to co-cultivation media. Transgenic shoots were selected on 50 and 75 mg L⁻¹ kanamycin. Rooting efficiency was 100% on half-strength Murashige and Skoog medium with 20 g L⁻¹ sucrose and 0.5 mg L⁻¹ indole butyric acid in the absence of kanamycin. Furthermore, 100% seed setting was found among all the transgenic events. The plants obtained were subjected to multi- and nochoice tests to determine the behavioral responses and mortality through Helicoverpa armigera bioassays on the leaf and relate their relationship with the expression of cry1Ac protein which was found to be less in leaf as compared to the floral buds, anther, pod, and seed.     

Successful induction of trigenomic hexaploid <Emphasis Type="Italic">Brassica</Emphasis> from a triploid hybrid of <Emphasis Type="Italic">B.</Emphasis><Emphasis Type="Italic">napus</Emphasis> L. and <Emphasis Type="Italic">B. nigra</Emphasis> (L.) Koch

A triploid hybrid with an ABC genome constitution, produced from an interspecific cross between Brassica napus (AACC genome) and B. nigra (BB genome), was used as source material for chromosome doubling. Two approaches were undertaken for the production of hexaploids: firstly, by self-pollination and open-pollination of the triploid hybrid; and secondly, by application of colchicine to axillary meristems of triploid plants. Sixteen seeds were harvested from triploid plants and two seedlings were confirmed to be hexaploids with 54 chromosomes. Pollen viability increased from 13% in triploids to a maximum of 49% in hexaploids. Petal length increased from 1.3 cm (triploid) to 1.9 cm and 1.8 cm in the two hexaploids and longest stamen length increased from 0.9 cm (triploid) to 1.1 cm in the hexaploids. Pollen grains were longer in hexaploids (43.7 and 46.3 μm) compared to the triploid (25.4 μm). A few aneuploid offsprings were also observed, with chromosome number ranging from 34 to 48. This study shows that trigenomic hexaploids can be produced in Brassica through interspecific hybridisation of B. napus and B. nigra followed by colchicine treatment.     

Genetic differentiation and diversity upon genotype and phenotype in cowpea (<Emphasis Type="Italic">Vigna unguiculata</Emphasis> L. Walp.)

The evolution of species is complex and subtle which always associates with the genetic variation and environment adaption during active/passive spread or migration. In crops, this process is usually driven and influenced by human activities such as domestication, cultivation and immigration. One method to discover this process is to analyze the genetic diversity of those crops in different regions. This research first assessed the similarity and differentiation between genetic diversity of genotype and phenotype in 768 world-wild cowpea germplasm which were collected by USDA and US breeding programs. Totally 1048 genotyping by sequencing (GBS) derived single nucleotide polymorphisms (SNPs) and 17 agronomic traits were used to analyze the genetic diversity, distance, cluster and phylogeny. The group differentiation was analyzed based on both the genotype distances from 1048 SNP markers and the phenotypic (Mahalanobis) distance D2 from 11 traits. A consistent result of diversity in genotype (polymorphism information content, PIC) and phenotype (Shannon and Simpson index) indicated that the East Africa and South Asia sub-continents were the original and secondary regions of cowpea domestication. Both dendrograms built by genetic distance present relationship among different regions, and the Mantel coefficient showed medium correlation level (r = 0.58) between genotype and phenotype. The information of both genotypic and phenotypic differentiations may help us to understand evolution and migration of cowpea more comprehensively and also will inform breeders how to use cowpea germplasm in breeding programs.     

Inheritance of time to flowering in cowpea (<Emphasis Type="Italic">Vigna unguiculata</Emphasis> (L.) Walp.)

Time to flowering and maturity is an important adaptive feature in annual crops, including cowpeas (Vigna unguiculata (L.) Walp.). In West and Central Africa, photoperiod is the most important environmental variable affecting time to flowering in cowpea. The inheritance of time from sowing to flowering (f) in cowpeas was studied by crossing a photoperiod-sensitive genotype Kanannnado to a photoperiod-insensitive variety IT97D-941-1. Sufficient seed of F₁, F₂, F₃ and backcross populations were generated. The parental, F₁, F₂, F₃ and the backcross populations were screened for f under long natural days (mean daylength 13.4 h per day) in the field and the parents, F₁, F₂ and backcross populations under short day (10 h per day) conditions. The result of the screening showed that photoperiod in the field was long enough to delay flowering of photoperiod-sensitive genotypes. Photoperiod-sensitivity was found to be partially dominant to insensitivity. Frequency distribution of the trait in the various populations indicated quantitative inheritance. Additive (d) and additive × dominance (j) interactions were the most important gene actions conditioning time to flowering. A narrow sense heritability of 86% was estimated for this trait. This will result in 26 days gain in time to flowering with 5% selection intensity from the F₂ to F₃ generation. At least seven major gene pairs, with an average delay of 6 days each, were estimated to control time to flowering in this cross.     

Sequence characterized amplified region markers tightly linked to the dwarf mosaic resistance gene <Emphasis Type="Italic">mdm1 (t)</Emphasis> in maize (<Emphasis Type="Italic">Zea mays</Emphasis> L.)

Maize dwarf mosaic is one of the devastating and wide spread viral diseases in the world. The present investigation was carried out to develop DNA markers closely linked to the resistance gene mdm1 (t). Linkage between the markers and phenotypes was confirmed by analyzing an F₂ population obtained from a cross between a resistant parent ‘Huangzaosi’ and a susceptible parent ‘Mo17(478)’. Four AFLP markers were found in the maize dwarf mosaic resistant plants. By using (BSA) bulked segregant analysis, two of the four AFLP markers were transformed into Sequence-characterized amplified regions markers (SCARs), nominated Rsun-1 and Rsun-2. The two amplified fragment length polymorphism (AFLP) markers, RHC-1and RHC-2, from the amplification products of primer combination E-AGC/M-CAA and E-AGC/M-GAA, showed linkage with the mdm1 (t) gene in a genetic distance 1.6 and 2.0 cM, respectively. The results indicate that the new SCAR markers will be valuable to distinguish resistant plants from susceptible plants in plantlets growing in seedbeds. The markers developed in this study are suitable for marker-assisted selection for maize dwarf mosaic resistance.     

Phenotypic diversity in cold-tolerant peanut (<Emphasis Type="Italic">Arachis hypogaea</Emphasis> L.) germplasm

Tolerance to low temperature is an important prerequisite for optimal performance of peanut (Arachis hypogaea L.) in a number of temperate peanut-growing environments. One hundred fifty-eight peanut accessions belonging to five botanical types, known to be tolerant to low temperature (12°C) at germination, were evaluated for phenotypic diversity for 15 morphological traits in the 2001 rainy season and for 15 agronomic and two seed quality traits in the 2001 rainy and 2001/2002 post-rainy seasons. Analysis of data, using the residual maximum-likelihood approach indicated that variance components due to genotypes were significant for all traits in the rainy and for all but two traits in the post-rainy season. Clustering based on scores of nine principle components delineated four clusters. The cold-tolerant genotypes and the standard control cultivars in the four clusters differed in mean, variance, and range both during rainy and post-rainy seasons for a range of agronomic traits, indicating the diversity among the clusters. The cold-tolerant accessions were superior to control cultivars for several agronomic traits compared with their respective controls in both the rainy and post-rainy seasons, so their use in breeding should result in genetically diverse cold-tolerant high-yielding peanut cultivars.     

Inheritance mode of male sterility in bunching onion (<Emphasis Type="Italic">Allium fistulosum</Emphasis> L.) accessions

Cytoplasmic male sterility (CMS) is an indispensable trait for F₁ hybrid seed production in bunching onion (Allium fistulosum L.). Expansion of the cytoplasmic diversity of F₁ hybrid cultivars by introduction of various CMS resources has great potential to eliminate vulnerability to cytoplasm type-specific diseases. This study aimed to evaluate appearance frequency of male sterile plants in several bunching onion accessions and to identify CMS resources. In eight (‘Nogiwa Aigara’, ‘Bansei Hanegi’, ‘Amarume’, ‘Kimnung’, ‘Zhangqiu’, ‘INT/CHN/1990/GOTOU’, ‘Natsunegi’ and ‘Guangzhou’) of 135 accessions collected from Japan, China, Mongolia, Korea and Taiwan, male sterile plants appeared with varied frequencies from 1.7% (‘Nogiwa Aigara’ and ‘Bansei Hanegi’) to 24.5% (‘Zhangqiu’). The inheritance mode of Zhangqiu- and Guangzhou-derived male sterility was confirmed to be CMS by sib-crossings and interbreed crossings. Microscopic examination of microsporogenesis in the CMS plants revealed that microspore protoplasm rapidly degenerated without mitotic division after the release of microspores from tetrads. The CMS germplasm described here would be useful for the development of “A” lines to be used in F₁ hybrid seed production of bunching onion. Male fertility in ‘Nogiwa Aigara’, ‘Bansei Hanegi’, ‘Kimnung’, ‘INT/CHN/1990/GOTOU’ and ‘Natsunegi’ was verified to be controlled by a single fertility restoration locus.     

Genetic effects of introgression genomic components from Sea Island cotton (<Emphasis Type="Italic">Gossypium barbadense</Emphasis> L.) on fiber related traits in upland cotton (<Emphasis Type="Italic">G</Emphasis>. <Emphasis Type="Italic">hirsutum</Emphasis> L.)

The germplasm with exotic genomic components especially from Sea Island cotton (Gossypium barbadense L. Gb) is the dominant genetic resources to enhance fiber quality of upland cotton (G. hirsutum L., Gh). Due to low efficiency of phenotypic evaluation and selection on fiber quality, genetic dissection of favorable alleles using molecular markers is essential. Genetic dissection on putative Gb introgressions related to fiber traits were conducted by SSR markers with mapping populations derived from a cross between Luyuan343 (LY343), a superior fiber quality introgression line (IL) with genomic components from Gb, and an elite Upland cotton cv. Lumianyan#22 (LMY22). Among 82 polymorphic loci screened out from 4050 SSRs, 42 were identified as putative introgression alleles. A total of 29 fiber-related QTLs (23 for fiber quality and six for lint percentage) were detected and most of which clustered on the putative Gb introgression chromosomal segments of Chr.2, Chr.16, Chr.23 and Chr.25. As expected, a majority of favorable alleles of fiber quality QTLs (12/17, not considering the QTLs for fiber fineness) came from the IL parent and most of which (11/12) were conferred by the introgression genomic components while three of the six (3/6) favorable alleles for lint percentage came from the Gh parent. Validation of these QTLs using an F₈ breeding population from the same cross made previously indicated that 13 out of 29 QTLs showed considerable stability. The results suggest that fiber quality improvement using the introgression components could be facilitated by marker-assisted selection in cotton breeding program.     

Molecular markers linked to <Emphasis Type="Italic">Bn</Emphasis>;<Emphasis Type="Italic">rf</Emphasis>: a recessive epistatic inhibitor gene of recessive genic male sterility in <Emphasis Type="Italic">Brassica napus </Emphasis>L.

7–7365AB is a recessive genic male sterile (RGMS) two-type line, which can be applied in a three-line system with the interim-maintainer, 7–7365C. Fertility of this system is controlled by two duplicate dominant epistatic genes (Bn;Ms3 and Bn;Ms4) and one recessive epistatic inhibitor gene (Bn;rf). Therefore an individual with the genotype of Bn;ms3ms3ms4ms4Rf_ exhibits male sterility, whereas, plant with Bn;ms3ms3ms4ms4rfrf shows fertility because homozygosity at the Bn;rf locus (Bn;rfrf) can inhibit the expression of two recessive male sterile genes in homozygous Bn;ms3ms3ms4ms4 plant. A cross of 7–7365A (Bn;ms3ms3ms4ms4RfRf) and 7–7365C (Bn;ms3ms3ms4ms4rfrf) can generate a complete male sterile population served as a mother line with restorer in alternative strips for the multiplication of hybrid seeds. In the present study, molecular mapping of the Bn;Rf gene was performed in a BC₁ population from the cross between 7–7365A and 7–7365C. Bulked segregant analysis (BSA) and amplified fragment length polymorphism (AFLP) technique was used to identify molecular markers linked to the gene of interest. From a survey of 768 primer combinations, seven AFLP markers were identified. The closest marker, XM5, was co-segregated with the Bn;Rf locus and successfully converted into a sequence characterized amplified region (SCAR) marker, designated as XSC5. Two flanking markers, XM3 and XM2, were 0.6 cM and 2.6 cM away from the target gene, respectively. XM1 was subsequently mapped on linkage group N7 using a doubled-haploid (DH) mapping population derived from the cross Tapidor × Ningyou7, available at IMSORB, UK. To further confirm the location of the Bn;Rf gene, additional simple sequence repeat (SSR) markers in linkage group N7 from the reference maps were screened in the BC₁ population. Two SSR markers, CB10594 and BRMS018, showed polymorphisms in our mapping population. The molecular markers found in the present study will facilitate the selection of interim-maintainer.     

Fine mapping of a <Emphasis Type="Italic">Xantha</Emphasis> mutation in rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.)

The recessive mutation of the XANTHA gene (XNT) transforms seedlings and plants into a yellow color, visually distinguishable from normal (green) rice. Thus, it has been introduced into male sterile lines as a distinct marker for rapidly testing and efficiently increasing varietal purity in seed and paddy production of hybrid rice. To identify closely linked markers and eventually isolate the XNT gene, two mapping populations were developed by crossing the xantha mutant line Huangyu B (indica) with two wild type japonica varieties; a total of 1,720 mutant type F₂ individuals were analyzed for fine mapping using polymorphic InDel markers and high dense microsatellite markers. The XNT gene was mapped on chromosome 11, within in a fragment of ~100 kb, where 13 genes are annotated. The NP_001067671.1 gene within the delimited region is likely to be a candidate XNT gene, since it encodes ATP-dependent chloroplast protease ATP-binding subunit clp A. However, no sequence differences were observed between the mutant and its parent. Bioinformatics analysis demonstrated that four chlorophyll deficient mutations that were previously mapped on the same chromosome are located outside the XNT region, indicating XNT is a new gene. The results provide useful DNA markers not only for marker assisted selection of the xantha trait but also its eventual cloning.     

Genetic analysis of resistance to flower bud thrips (<Emphasis Type="Italic">Megalurothrips sjostedti</Emphasis>) in cowpea (<Emphasis Type="Italic">Vigna unguiculata</Emphasis> [L.] Walp.)

Cowpea is an important legume in sub-Saharan Africa where its protein rich grains are consumed. Insect pests constitute a major constraint to cowpea production. Flower bud thrips (FTh) is the first major pest of cowpea at the reproductive stage and if not controlled with insecticides is capable of reducing grain yield significantly. Information on the inheritance of resistance to FTh is required to facilitate breeding of resistant cultivars. The genetics of resistance was studied in crosses of four cowpea lines. Maternal effect was implicated while frequency distributions of the F₂ and backcross generations suggest quantitative inheritance. Additive, dominance and epistatic gene effects made large contributions and since improved inbred lines are the desired product, selection should not be too severe in the early generations to allow for desirable gene recombination. This study suggested that some of the genes involved in the control of resistance to FTh are different in TVu1509 and Sanzi. Broad sense heritability ranged from 56% to 73%. Choice of maternal parent in a cross will be critical to the success of resistance breeding.     

DNA profiling and genetic diversity of Korean soybean (<Emphasis Type="Italic">Glycine max</Emphasis> (L.) Merrill) landraces by SSR markers

Approximately 7,000 accessions of Korean soybean (Glycine max (L.) Merrill) landraces, largely composed of three collections, the Korea Atomic Energy Research Institute’s soybean (KAS), the Korean Crop Experiment Station’s soybean (KLS) and the Korean Agricultural Development and Technology Center’s soybean (KADTC) collections, have been conserved at the Rural Development Administration (RDA) genebank in Korea. The accessions within collections were classified based on their traditional uses such as sauce soybean (SA), sprouted soybean (SP), soybean for cooking with rice (SCR), and OTHERS. A total of 2,758 accessions of Korean soybean landraces were used to profile and to evaluate genetic structure using six SSR loci. A total of 110 alleles were revealed by at the six SSR loci. The number of alleles per SSR locus ranged from 9 to 39 in Satt187 and Satt_074, respectively. The number of alleles ranged from 87 in the KADTC collection to 96 in the KLS collection, and from 63 in the SCR group to 95 in the SP group. Nei’s average genetic diversity ranged from 0.68 to 0.70 across three collections, and 0.64 to 0.69 across the usage groups. The average between-group differentiation (G _st) was 0.9 among collections, and 4.1 among the usage groups. The similar average diversity among three collections implies that the genetic background of the three collections was quite similar or that there were a large number of duplicate accessions in three collections. The selection from the four groups classified based upon usage may be a useful way to select accessions for developing a Korean soybean landrace core collection at the RDA genebank. DNA profile information of accessions will provide indications of redundancies or omissions and aid in managing the soybean collection held at the RDA genebank. The information on diversity analysis could help to enlarge the genetic diversity of materials in breeding programs and could be used to develop a core collection.     

<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of faba bean <Emphasis Type="Italic">(Vicia faba</Emphasis> L.) using embryo axes

A system for the production of transgenic faba bean by Agrobacterium-mediated transformation was developed. This system is based upon direct shoot organogenesis after transformation of meristematic cells derived from embryo axes. Explants were co-cultivated with A. tumefaciens strain EHA105/pGlsfa, which harbored a binary vector containing a gene encoding a sulphur rich sunflower albumin (SFA8) linked to the bar gene. Strain EHA 101/pAN109 carrying the binary plasmid containing the coding sequence of a mutant aspartate kinase gene (lysC) from E. coli in combination with neomycinphosphotransferase II gene (nptII) was used as well. The coding sequences of SFA8 and LysC genes were fused to seed specific promoters, either Vicia faba legumin B4 promoter (LeB4) or phaseolin promoter, respectively. Seven phosphinothricin (PPT) resistant clones from Mythos and Albatross cultivars were recovered. Integration, inheritance and expression of the transgenes were confirmed by Southern blot, PCR, enzyme activity assay and Western blot.     

Marker-assisted backcross selection in an interspecific <Emphasis Type="Italic">Cucumis</Emphasis> population broadens the genetic base of cucumber (<Emphasis Type="Italic">Cucumis sativus</Emphasis> L.)

Cucumber (Cucumis sativus L.) is a major cucurbit vegetable species whose genetic base has been drastically reduced during its domestication. The crop’s narrow genetic base (3–12% DNA polymorphism) has resulted from the use of limited genetic material and intense selection during plant improvement. Recently, however, interspecific hybridization has been successful in Cucumis via mating of C. hystrix Chakr. and C. sativus, which resulted in the amphidiploid C. hytivus. We report herein a marker-assisted strategy for increasing genetic diversity in cucumber through introgression backcrossing employing C. hytivus. The comparatively late-flowering but high-yielding, indeterminate, monoecious line WI 7012A (P₁; donor parent) derived from a C. hytivus × C. sativus-derived line (long-fruited Chinese C. sativus cv. Beijingjietou) was initially crossed to the determinate, gynoecious C. sativus line WI 7023A (P₂; recurrent parent 1), and then advanced backcross generation progeny (BC₂) were crossed with the gynoecious indeterminate line WI 9-6A (P₃; recurrent parent 2). More specifically, a single F₁ individual (P₁ × P₂) was backcrossed to P₂, and then BC progeny were crossed to P₂ and P₃, where marker-assisted selection (MAS) for genetic diversity (8 mapped and 16 unmapped markers; designated Sel) or no selection (designated NSel) was applied to produce BC₃P₂ (Sel) and BC₃P₃ (Sel), and BC₂P₂ (NSel) and BC₂P₂S₁ (NSel) progeny. Relative vegetative growth, number of lateral branches (LB), days to flowering (DF), yield (fruit number), and fruit quality [as measured by length:diameter (L:D) and endocarp:total diameter (E:T) ratios] were assessed in parents and cross-progeny. DF varied from ~20 (BC₃P₂Sel) to ~25 days (BC₂P₃Sel) among the populations examined, where progeny derived from P₂ possessed the shortest DF. Differences in cumulative yield among the populations over six harvests were detected, varying from ~8 fruits per plant in BC₃P₂ (Sel) to ~39 fruits per plant in BC₂P₃ (Sel). Although the vigorous vegetative growth of line P₁ was observed in its backcross progeny, highly heterozygous and polymorphic backcross progeny derived from P₃ were comparatively more vigorous and bore many high-quality fruit. Response to selection was detected for LB, DF, L:D, and E:T, but the effectiveness of MAS depended upon the parental lines used. Data indicate that the genetic diversity of commercial cucumber can be increased by introgression of the C. hystrix genome through backcrossing.     

<Emphasis Type="Italic">Agrobacterium</Emphasis> mediated transformation of indica rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.) for insect resistance

The rice leaffolder (RLF), Cnaphalocrocis medinalis is an important pest of rice that causes severe damage in many areas of the world. The plants were transformed with fully modified (plant codon optimized) synthetic Cry1C coding sequences as well as with the hpt and gus genes, coding for hygromycin phosphotransferase and β-glucuronidase, respectively. Cry1C sequences placed under the control of doubled 35S promoter plus the AMV leader sequence, and hpt and gus genes driven by cauliflower mosaic virus 35S promoter, were used in this study. Embryogenic calli after cocultivation with Agrobacterium were selected on the medium containing hygromycin B. A total of 67 hygromycin-resistant plants were regenerated. PCR and Southern blot analyses of primary transformants revealed the stable integration of Cry1C coding sequences into the rice genome with predominant single copy integration. R₁ progeny plants disclosed a monogenic pattern (3:1) of transgene segregation as confirmed by molecular analyses. These transgenic lines were highly resistant to rice leaffolder (RLF), Cnaphalocrocis medinalis as revealed by insect bioassay.     

Screening global <Emphasis Type="Italic">Medicago</Emphasis> germplasm for weevil (<Emphasis Type="Italic">Hypera postica</Emphasis> Gyll.) tolerance and estimation of genetic variability using molecular markers

The genus Medicago encompasses many important forage species for both temperate and tropical regions. M. sativa L., commonly known as lucerne, is one of the most important forage species grown worldwide, but its production suffers seriously from weevil (Hypera postica Gyll.) infestation. The aim of this work was to identify species/accessions with tolerance to weevil and their molecular analysis using simple sequence repeat (SSR) markers. After screening 197 global germplasm encompassing 50 Medicago species for weevil tolerance, 22 lines representing 13 species were identified where leaf damage was ≤15% (P ≤ 0.05). In total, 37 accessions of the 22 lines, five Indian lucerne cultivars with leaf damage ≥75% and 10 accessions of the 13 Medicago species with low to high infestation (>25%) were molecularly assessed using 11 SSR markers (5 newly developed) to delineate closest to lucerne lines for breeding. In total, 33 bands were scored. The SAHN clustering using UPGM algorithm resulted into two main clusters supported with high boot strap values and with genetic similarity ranging from 0.33 to 0.96. Two accessions of M. tenoreana were observed closest to Indian lucerne cultivars. The rich variability revealed can be used as potential resource for transferring genes across species. Although the inter-specific hybridization is difficult preposition in genus Medicago largely due to post fertilization barrier, the identified species/accessions can be utilized on priority in breeding programs especially employing biotechnological tools like culturing of fertilized pods, ovule-embryo culture and electroporation.     

Genetic variation in mutants of black gram (<Emphasis Type="Italic">Vigna mungo</Emphasis> (L.) Hepper) evaluated by RAPD markers

Genetic variation among four mutants with parent of black gram (high seed protein, tall, bushy, and dwarf mutants) was investigated. The mutants were generated with physical gamma rays and chemical ethyl methane sulphonate. These mutants had some advantages like high protein content, lodging resistance, and high seed yield compared with parent cultivars (cvs.). The objective of the present investigation is to find genetic variation in mutants of black gram with their parents. The genetic variation was evaluated with 20 random primers, generated total 202 fragments scored with 58 polymorphic alleles, and the average was 10.1 alleles per locus and a range of 1–9 alleles. The average polymorphic rates were 38.37 among the mutants and parents through the 20 primers. Primers OPA-14 and OPI-04 revealed 35% of DNA polymorphism in this investigation. The genetic distance (GD) among the genotypes was 0.19 suggesting a significant degree of genetic diversity. The five genotypes were used to construct a dendrogram based on the similarity matrix, revealing a genetic distance varying from 0.600 to 0.725. This variation was due to the mutation induced by gamma rays and ethyl methane sulphonate. Further research is needed to analyze the sequence and expression of these mutated genes and to develop and improve black gram via genetic engineering to evaluate their use.