Backcross Generations Research Articles

Development and validation of multiplex-PCR assay for simultaneous detection of rare alleles of crtRB1 and lcyE governing higher accumulation of provitamin A in maize

Vitamin A deficiency is a widely prevalent health disorder among millions of people worldwide. Introgression of crtRB1 and lcyE favourable alleles that enhance concentration of provitamin A in maize endosperm have been employed in maize biofortification programmes. To make marker-assisted selection (MAS) more effective, we have developed rapid and convenient multiplex-polymerase chain reaction (PCR) assay to simultaneously discover the allelic combinations among the segregants. Validation of the multiplex assay was done in two backcross-derived populations developed using elite inbreds viz., HKI193-1 and HKI193-2 carrying unfavourable alleles of crtRB1 (296 bp) and lcyE (300 bp) and HarvestPlus inbreds viz., HP704-22 and HP704-23 possessing favourable alleles of crtRB1 (543 bp) and lcyE (650 bp). We also standardized the uniplex-PCR assays for both the genes that gave robust and reproducible results in sub-tropical populations. Gel profiles of BC1F1, BC2F1 and BC2F2 revealed that these assays identified the backcross progenies homo-or hetero-zygous for the favourable- or unfavourable-alleles. Multiplex-PCR assay also precisely confirmed the results of individual uniplex assays in different backcross generations. Cost and time analyses showed that multiplex-PCR assay has potential to save 41% of cost, and 50% of time compared to two uniplex assays in a MAS programme. It has also saved 50% of the manpower. The multiplex assay possesses significant advantage over uniplex assays and enhances the efficiency of selection. This is the first report of development and validation of multiplex-PCR assay of crtRB1 and lcyE for utilization in maize biofortification programme.

Transfer of Downy Mildew Resistance from Wild Basil (Ocimum americanum) to Sweet Basil (O. basilicum).

Sweet basil (Ocimum basilicum) is susceptible to downy mildew caused by the oomycete foliar pathogen Peronospora belbahrii. No resistant varieties of sweet basil are commercially available. Here, we report on the transfer of resistance gene Pb1 from the highly resistant tetraploid wild basil O. americanum var. americanum (PI 500945, 2n = 4x = 48) to the tetraploid susceptible O. basilicum 'Sweet basil' (2n = 4x = 48). F1 progeny plants derived from the interspecific hybridization PI 500945 × Sweet basil were resistant, indicating that the gene controlling resistance (Pb1) is dominant, but sterile due to the genetic distance between the parents. Despite their sterility, F1 plants were pollinated with the susceptible parent and 115 first backcross generation to the susceptible parent (BCs1) embryos were rescued in vitro. The emerging BCs1 plants segregated, upon inoculation, 5:1 resistant/susceptible, suggesting that resistance in F1 was controlled by a pair of dominant genes (Pb1A and Pb1A'). Thirty-one partially fertile BCs1 plants were self-pollinated to obtain BCs1-F2 or were backcrossed to Sweet basil to obtain the second backcross generation to the susceptible parent (BCs2). In total, 1 BCs1-F2 and 22 BCs2 progenies were obtained. The BCs1-F2 progeny segregated 35:1 resistant/susceptible, as expected from a tetraploid parent with two dominant resistant genes. The 22 BCs2 progenies segregated 1:1 resistant/susceptible (for a BCs1 parent that carried one dominant gene for resistance) or 5:1 (for a BCs1 parent that carried two dominant genes for resistance) at a ratio of 4:1. The data suggest that a pair of dominant genes (Pb1A and Pb1A') residing on a two homeologous chromosomes is responsible for resistance of PI 500945 against P. belbahrii.

Marker assisted breeding for introgression of opaque-2 allele into elite maize inbred line BML-6

Improvement of QPM (Quality Protein Maize) along with high content of lysine and tryptophan had foremost importance in maize breeding programme. The efficient and easiest way of developing QPM hybrids was of backcross breeding in marker aided selection. Hence the present investigation was aimed with an endeavour to convert elite maize inbred line BML-6 into QPM line. CML-181 was identified to be a donor variety as it revealed high-quality polymorphism with BML-6 for opaque-2 gene specific marker umc1066. Non-QPM inbred line BML-6 was crossed with QPM donor CML-181 and produced F1 followed by BC1F1 and BC2F1 population was developed. Foreground selection was carried out with umc1066 in F1, and selected plants were used for BC1F1 and BC2F1 populations. Two hundred plants were screened in both BC1F1 and BC2F1 population with umc1066 for foreground selection. The selected plants were screened for foreground selection with amino acid modifiers. Foreground selected plants for both opaque-2 and amino acid modifiers were screened for background selection for BML-6 genome. Recurrent parent genome (RPG) was determined for BC2F1 population plants. Three plants have shown with RPG 90–93% in two generation back cross population. Three selected BC2F2 populations were screened for foreground and back ground selection followed by agronomical and biochemical evaluation. The QPM converted version of BML-6 contains 0.97% of tryptophan and 4.02% of lysine concentration in a protein. Agronomical and biochemical screened BC2F2 plants were selfed for BC2F3. QPM version of BML-6 line can be used for the development QPM version of maize single cross hybrids.

Genetic variability in CMS backcross generations and their maintainers in eggplant (Solanum melongena L.)

Twelve genotypes of eggplant in BC3, BC4 and BC5 generations of CMS-lines derived from Solanum ae-thiopicum × Solanum melongena cross along with their maintainers were evaluated for eighteen quantitative charac-ters at Punjab Agricultural University Ludhiana. Analysis of variance depicted significant variation (P ? 0.05) for all the characters in all generations. High PCV and GCV values were observed for fruit length, fruit girth, fruit weight, number of fruits plant-1 and yield plant-1 in all generations indicating high variability in the germplasm. High heritability coupled with high genetic advance was found for peduncle length, fruit length, fruit girth, fruit weight, number of fruits plant-1 and yield plant-1 in all generations indicating the predominance of additive gene action for these traits. Thus, selections can be made from present germplasm for the development of improved CMS inbred lines with varying fruit traits.

Rapid and targeted introgression of fgr gene through marker-assisted backcrossing in rice (Oryza sativa L.).

While it is crucial for developing countries like Malaysia to achieve self-sufficiency in rice (Oryza sativa L.), it is equally critical to be able to produce high-quality rice, specifically fragrant rice, which demands are often met through importation. The present study was aimed at developing high-yielding fragrant rice, in a timely and cost-effective manner. A marker-assisted backcross (MABC) approach was optimised to introgress the fragrance gene (fgr) into two high-yielding Malaysian varieties, MR84 and MR219, within two years utilising less than 50 molecular markers. Coupled with phenotypic screening, one single foreground marker (fgr-SNP) and 48 background markers were selected and utilised, revealing recovery of at least 90% of recurrent parent genome (RPG) in merely two backcross generations. Collectively, the yield potential of the developed BC2F2 lines (BLs) was higher (P > 0.05) than the donor parent, MRQ74, and similar (P < 0.05) to both the recurrent parents, MR84 and MR219. In addition, some of the developed BLs showed good grain quality, such as having long grain. We believe that this is the first report comprising the validation and utilisation of the single functional marker system (fgr-SNP) in introgressing the fgr gene into different rice varieties.

Introgression of heat shock protein (Hsp70 and sHsp) genes into the Malaysian elite chilli variety Kulai (Capsicum annuum L.) through the application of marker-assisted backcrossing (MAB).

Backcrossing together with simple sequence repeat marker strategy was adopted to improve popular Malaysian chilli Kulai (Capsicum annuum L.) for heat tolerance. The use of molecular markers in backcross breeding and selection contributes significantly to overcoming the main drawbacks such as increase linkage drag and time consumption, in the ancient manual breeding approach (conventional), and speeds up the genome recovery of the recurrent parent. The strategy was adopted to introgress heat shock protein gene(s) from AVPP0702 (C. annuum L.), which are heat-tolerant, into the genetic profile of Kulai, a popular high-yielding chilli but which is heat sensitive. The parents were grown on seed trays, and parental screening was carried out with 252 simple sequence repeat markers. The selected parents were crossed and backcrossed to generate F1 hybrids and backcross generations. Sixty-eight markers appeared to be polymorphic and were used to assess the backcross generation; BC1F1, BC2F1 and BC3F1. The average recipient allele of the selected four BC1F1 plants was 80.75% which were used to produce the BC2F1 generation. BC1-P7 was the best BC1F1 plant because it had the highest recovery at 83.40% and was positive to Hsp-linked markers (Hsp70-u2 and AGi42). After three successive generations of backcrossing, the average genome recovery of the recurrent parent in the selected plants in BC3F1 was 95.37%. Hsp gene expression analysis was carried out on BC1F1, BC2F1 and BC3F1 selected lines. The Hsp genes were found to be up-regulated when exposed to heat treatment. The pattern of Hsp expression in the backcross generations was similar to that of the donor parent. This confirms the successful introgression of a stress-responsive gene (Hsp) into a Kulai chilli pepper variety. Furthermore, the yield performance viz. plant height, number of fruits, fruit length and weight and total yield of the improved plant were similar with the recurrent parent except that the plant height was significantly lower than the Kulai (recurrent) parent.

Marker-Assisted Development and Evaluation of Near-Isogenic Lines for Broad-Spectrum Powdery Mildew Resistance Gene Pm2b Introgressed into Different Genetic Backgrounds of Wheat.

At present, most of released wheat cultivars or breeding lines in China are susceptible to powdery mildew (Pm) (caused by Blumeria graminis f. sp. tritici, Bgt), so there is an urgent need to rapidly transfer effective and broad-spectrum Pm resistance genes into elite cultivars/lines. Near-isogenic lines (NILs) with short target gene region are very important in molecular breeding and map-based cloning and can be developed by combining marker-assisted selection and conventional phenotypic identification. However, no Pm gene NILs were reported by using this method in the previous studies. A new broad-spectrum dominant resistance gene Pm2b, derived from the Chinese wheat breeding line KM2939, conferred high resistance to Pm at both the seedling and adult stages. In this study, with the aid of forward and background selection (FS and BS) using molecular markers, the Pm2b gene was introgressed into three elite susceptible commercial cultivars Shimai 15, Shixin 828, and Kenong 199 through the back-crossing procedure. With the appropriate backcrossing generations, selected population sizes and marker number for BS, the homozygous resistant BC3F2:3 NILs of Pm2b gene in the three genetic backgrounds with the highest recipient genome composition of about 99%, confirmed by simple sequence repeat markers and 660K single nucleotide polymorphic array, were developed and evaluated for the powdery mildew resistance and agronomic traits. The different resistance and similar or improved agronomic performance between Pm2b NILs and their corresponding recurrent parents indicated their potential value in the marker-assisted breeding of the Pm2b gene. Moreover, the development of four flanked diagnostic markers (CFD81, BWM25, BWM20, and BWM21) of the Pm2 gene can effectively assist the forward selection and accelerate the transfer and use of this resistance gene.

Pairing analysis and in situ Hybridisation reveal autopolyploid-like behaviour in Solanum commersonii\xa0\xd7\xa0S. tuberosum (potato) interspecific hybrids

Wild potato relatives are rich sources of desirable traits for introgressive hybridisation into cultivated potato. One of them, Solanum commersonii (2n = 2x = 24, 1EBN, endosperm balance number), is an important species belonging to the potato tertiary genepool. It can be used in potato breeding through bridge crosses and 2n gamete production. Triploid F1 hybrids between S. commersonii (through spontaneous 2n egg formation) and diploid 2EBN Solanum tuberosum Group Phureja were crossed with S. tuberosum Group Tuberosum, resulting in successive backcross 1, 2 and 3 progenies. The main aim of this study was to determine if there are any barriers to homoeologous pairing and recombination in the allotriploid (S. commersonii × potato) hybrids and their backcrosses, and so to predict if S. commersonii chromosomes can be transmitted to the next generation and introgressed into their recipient potato chromosomes. Microscopic observations of spread pollen mother cells suggested no preferential pairing in the triploid hybrids, while chromosome transmission and segregation in further meiotic stages were fairly balanced. Fluorescent in situ hybridisation with BAC probes (BAC FISH) was used to obtain markers to trace the meiotic behaviour of specific chromosome pairs. Moreover, genomic in situ hybridisation (GISH) demonstrated no obvious differences in fluorescence signals between the homoeologues suggesting that repetitive sequences did not diverge much between the parental species. As a consequence, we were not able to trace the course of the S. commersonii chromosomes in the successive introgressive hybridisation backcross generations. Our results strongly point at a high genomic similarity between the homoeologous chromosomes promising high suitability of S. commersonii in introgressive hybridisation breeding of potato.

Molecular-agronomic characterization and genetic study reveals usefulness of refined Ogura cytoplasm based CMS lines in hybrid breeding of cauliflower (Brassica oleracea var. botrytis L.)

Cytoploasmic male sterility (CMS) is the most widely used system for hybrid seed production in vegetable brassicas. Information of morphological and molecular divergence in combination with combining ability is instrumental in selecting suitable parent in hybrid development. Twenty five CMS lines developed after more than 9 generations of backcrossing were analysed for different agronomic and floral traits. Besides, they were also evaluated for combining ability to reveal their breeding potential. Two CMS lines, Ogu402-6A and Ogu76-4A were distantly placed from rest of the CMS genotypes based on morphological characterization. Molecular analysis through SSR primers also revealed the genetic distance of the CMS lines Ogu402-6A from rest of the CMS pool. Three genotypes (Ogu402-6A, Ogu76-4A and Ogu119-2A) with early maturity were identified for their use in development of short duration hybrids. Similarly, 3 CMS lines Ogu118-2A, OguHL-3A and Ogu126-1A were identified with yield potential of more than 60t/ha. Based on 13 polymorphic SSR markers 25 CMS lines were grouped into 4 major clusters. Molecular diversity in combination with agronomic characterization will be very useful in diverse parental lines. Introgression of Ogura cytoplasm into cauliflower nuclear background caused variety of flower deformities. However, these deformities were genotype specific. General combining ability (GCA) of the CMS lines revealed their potential for use in hybrid breeding. Few genotypes were identified based on combining ability for their use in development of short duration and high yielding F1 hybrids.

Development of a cytoplasmic male‐sterile line NJCMS4A for hybrid soybean production

AbstractCytoplasmic male‐sterile (CMS) lines are being used to produce hybrid seeds. Thus far, four CMS sources in soybean [Glycine max (L.) Merr.] have been reported in China. However, they are not sufficient or efficient in meeting the requirements of commercial soybean hybrid seed production. In this study, 33 varieties were tested for CMS using 45 crosses among 37 landraces and 17 annual wild soybean accessions (Glycine soja Sieb. et Zucc.). The cross of N23661 × N23658 showed partial to complete male sterility in backcross generations, while the corresponding reciprocal cross showed normal male fertility. Thus, the cytoplasm of N23661 is male‐sterile, the continuously backcrossed line is a male‐sterile line (designated NJCMS4A), and N23658 is its maintainer (designated NJCM4B). The male fertility of NJCMS4A was restored by another accession, Nansheng9403. Accordingly, NJCMS4A along with its maintainer and restorer composes a complete set of three lines for producing hybrid soybean. Using mitochondrial markers and sequence analyses, NJCMS4A is a CMS line with its cytoplasm not identical to the four previously reported CMS sources in soybean.

&lt;b&gt;Inheritance of resistance to &lt;i&gt;Meloidogyne incognita&lt;/i&gt; race 3 in cotton accession TX 25

Cotton producers worldwide suffer with the losses caused by the presence of phytonematodes. The aim of the present study was to investigate the inheritance of resistance to Meloidogyne incognita race 3 in Gossypium hirsutum variety punctatum accession TX 25. Accessions of Gossypium sp. were obtained from the germplasm bank of Embrapa Cotton. Two experiments were performed in two consecutive years. In the first experiment, a susceptible parental line, FiberMax 966, a resistant parental line, TX 25, and their F 1 , F 2 and backcross generations were tested. In the second experiment, parental lines FiberMax 966 and TX 25, their F 2 generation, and genotypes M315 (resistant), LA887 and DeltaOpal (moderately resistant) were tested. In both experiments, plants were inoculated with 2000 eggs and J2 of M. incognita race 3. The gall index, egg mass index and reproduction factor were evaluated 120 days following inoculation. In the first experiment, plants from the F 1 and backcross generations were susceptible. Plants from the F 2 generation presented a 3:1 resistant-to-susceptible ratio in the two experiments, indicating oligogenic resistance.

Molecular genetic analysis of the melanoma regulatory locus in Xiphophorus interspecies hybrids.

Development of spontaneous melanoma in Xiphophorus interspecies backcross hybrid progeny, (X. hellerii × [X. maculatus Jp 163 A × X. hellerii]) is due to Mendelian segregation of a oncogene (xmrk) and a molecularly uncharacterized locus, called R(Diff), on LG5. R(Diff) is thought to suppresses the activity of xmrk in healthy X. maculatus Jp 163 A parental species that rarely develop melanoma. To better understand the molecular genetics of R(Diff), we utilized RNA-Seq to study allele-specific gene expression of spontaneous melanoma tumors and corresponding normal skin samples derived from 15 first generation backcross (BC1 ) hybrids and 13 fifth generation (BC5 ) hybrids. Allele-specific expression was determined for all genes and assigned to parental allele inheritance for each backcross hybrid individual. Results showed that genes residing in a 5.81 Mbp region on LG5 were exclusively expressed from the X. hellerii alleles in tumor-bearing BC1 hybrids. This observation indicates this region is consistently homozygous for X. hellerii alleles in tumor bearing animals, and therefore defines this region to be the R(Diff) locus. The R(Diff) locus harbors 164 gene models and includes the previously characterized R(Diff) candidate, cdkn2x. Twenty-one genes in the R(Diff) region show differential expression in the tumor samples compared to normal skin tissue. These results further characterize the R(Diff) locus and suggest tumor suppression may require a multigenic region rather than a single gene variant. Differences in gene expression between tumor and normal skin tissue in this region may indicate interactions among several genes are required for backcross hybrid melanoma development.

Molecular stacking of wide compatibility gene, S5 n and elongated uppermost internode (eui) gene into IR 58025B, an elite maintainer line of rice

One of the most widely used and stable wild-abortive cytoplasmic male sterile (WA-CMS) line is IR 58025A. We had earlier transferred the gene (eui1) for elongated uppermost internode into the elite WA-CMS line named IR 58025eB with the help of markers. To overcome incomplete panicle exsertion in the WA-CMS line and better exploitation of indica/japonica heterosis, we have stacked a major gene for wide-compatibility (S5 n ) into IR 58025eB through marker-assisted breeding. SSR markers (RM5970) tightly linked to eui1 gene and (S5-InDel) functional maker of S5 n gene were used for foreground selection, while ninety-one microsatellite markers polymorphic between IR 58025eB and donor Dular (S5 n ) were utilized for background selection at each backcross generation and backcrossing was continued till BC3 generation. Phenotypic observation previous to background selection at each generation provided supplementary information for identifying the novel recombinants and the fast-tracked recurrent parent genome recovery which ranged from 94.50 to 96.70% in selected BC3F4 plants. At BC3F5 generation, seven stable, backcross-derived lines possessing S5 n and eui genes, high yield, and long-slender grain type have been identified; their maintainer ability was confirmed through test crosses. These improved lines are being converted to CMS lines by marker-assisted backcrossing.

QTL mapping for haploid male fertility by a segregation distortion method and fine mapping of a key QTL qhmf4 in maize.

Four QTL related to haploid male fertility were detected by a segregation distortion method and the key QTL qhmf4 was fine mapped to an interval of ~800kb. Doubled haploid (DH) technology enables rapid development of homozygous lines in maize breeding programs. However, haploid genome doubling is a bottleneck for the commercialization of DH technology and is limited by haploid male fertility (HMF). This is the first study reporting the quantitative trait locus (QTL) analysis of HMF in maize. Four QTL, qhmf1, qhmf2, qhmf3, and qhmf4, controlling HMF have been identified by segregation distortion (SD) loci detection in the selected haploid population derived from 'Yu87-1/Zheng58'. Three loci, qhmf1, qhmf2, and qhmf4, were also detected in the selected haploid population derived from '4F1/Zheng58'. The QTL qhmf4 showed the strongest SD in both haploid populations. Based on the sequence information of 'Yu87-1' and 'Zheng58', thirteen markers being polymorphic between the two lines were developed to saturate the qhmf4 region. A total of 8168 H1BC2 (haploid backcross generation) plants produced from 'Yu87-1' and 'Zheng58' were screened for recombinants. All the 48 recombinants were backcrossed to 'Zheng58' to develop H1BC3 progeny. The heterozygous H1BC3 individuals were crossed with CAU5 to induce haploids. In each H1BC3 progeny, haploids were genotyped and evaluated for anther emergence score (AES). Significant (or no significant) difference (P < 0.05) between haploids with or without 'Yu87-1' donor segment indicated presence or absence of qhmf4 in the donor segment. The analysis of the 48 recombinants narrowed the qhmf4 locus down to an ~800kb interval flanked by markers IND166 and IND1668.

Identification of a novel cytoplasmic male sterile line M2BS induced by partial-length HcPDIL5-2a transformation in rice (Oryza sativa L.)

A cytoplasmic male sterile line (designated as M2BS) was obtained from an indica rice maintainer M2B induced by partial-length HcPDIL5-2a (Hibiscus cannabinus protein disulfide isomerase-like) transformation. The anther of M2BS was short, slender, hygrophanous, and fissured. I2-KI staining method showed that there was typical and spherical abortion in pollen grains. M2BS was found abortive at middle and late stage of monocyte by the modified carbol fuchsin stained observation and paraffin section observation. The tapetum was observed pre-degenerated in M2BS. Hereditary analysis indicated that the male sterility of M2BS was a maternally inherited inability after six backcross generations with M2B and the combinations of M2BS hybridized with other two male fertile materials. The M2BS could be affirmed a cytoplasmic male sterile (CMS) type. Moreover, it was a transgenic plant confirmed by PCR, Southern blot and RT-PCR detection. M2BS could be distinguished from M2B and its CMS line M2A by RFLP analysis. The overall mitochondrial genome sequencing results showed, that in M2BS, the main differences of mitochondrial gene sequence were located in nad4, nad5, nad7, orf194 and intergenic region, relatively to those of M2A. The obtained results indicate that M2BS is a novel cytoplasmic male sterile line.

Development of opaque-2 introgression line in maize using marker assisted backcross breeding

Two elite normal maize inbreds that produced heterotic experimental hybrid (OML 17-3×OML 42-9) with 45% yield advantage over the standard check Vivek QPM 9, were used for conversion to QPM status using opaque-2 donors (CML 176 and CML 186) using marker assisted back cross breeding. Each of the normal maize parental inbreds was used as female parent in separate conversion programme using cross combination OML 17-3×CML 176 and OML 42-9×CML 186. Foreground selection (using opaque-2 specific marker phi 057) combined with phenotypic selection for recipient parent at early back cross generations (BC1 to BC3) was carried out for rapid recovery of recurrent parent genotype. The BC3F1 plants showing heterozygous status (O2o2) were selfed and advanced to BC3F2 to enable for identification of homozygous recessive opaque-2 segregants at seedling stage. The most promising BC3F3 introgression lines from either cross combinations were finally selected based on higher tryptophan and lysine content. The newly developed introgression lines (OQL 176-17-3 and OQL 186-42-9) were at par with the QPM donors (CML 176 and CML 186) and nearly double the lysine and tryptophan content as compared to respective normal inbreds (OML 17-3 and OML 42-9). These may serve as valuable breeding materials for development of QPM hybrids.

Studying the Genetics of Resistance to CyHV-3 Disease Using Introgression from Feral to Cultured Common Carp Strains

Sustainability and further development of aquaculture production are constantly challenged by outbreaks of fish diseases, which are difficult to prevent or control. Developing fish strains that are genetically resistant to a disease is a cost-effective and a sustainable solution to address this challenge. To do so, heritable genetic variation in disease resistance should be identified and combined together with other desirable production traits. Aquaculture of common carp has suffered substantial losses from the infectious disease caused by the cyprinid herpes virus type 3 (CyHV-3) virus and the global spread of outbreaks indicates that many cultured strains are susceptible. In this research, CyHV-3 resistance from the feral strain “Amur Sassan” was successfully introgressed into two susceptible cultured strains up to the first backcross (BC1) generation. Variation in resistance of families from F1 and BC1 generations was significantly greater compared to that among families of any of the susceptible parental lines, a good starting point for a family selection program. Considerable additive genetic variation was found for CyHV-3 resistance. This phenotype was transferable between generations with contributions to resistance from both the resistant feral and the susceptible cultured strains. Reduced scale coverage (mirror phenotype) is desirable and common in cultured strains, but so far, cultured mirror carp strains were found to be susceptible. Here, using BC1 families ranging from susceptible to resistant, no differences in resistance levels between fully scaled and mirror full-sib groups were found, indicating that CyHV-3 resistance was successfully combined with the desirable mirror phenotype. In addition, the CyHV-3 viral load in tissues throughout the infection of susceptible and resistant fish was followed. Although resistant fish get infected, viral loads in tissues of these fish are significantly lesser than in those of susceptible fish, allowing them to survive the disease. Taken together, in this study we have laid the foundation for breeding CyHV-3-resistant strains and started to address the mechanisms underlying the phenotypic differences in resistance to this disease.

Hybridisation between cherry tomato (small fry) and Petomech for shortened fruit maturation, size and earliness

Tomato (Lycopersicon esculentum var. cerasiforme) is among the most important vegetable crops grown world wide providing good nutritional requirements and boost incomes, especially in sub-Saharan Africa. The objective of this study was to determin the heritability of shortened fruit maturation (SFM) period in a hybrid from a cross between cherry (Small fry) tomato and Petomech. A field experiment was carried out at the Crops Research Institute (CRI), Kwadaso. A cross was made between Petomech and Small fry. Fruit maturation period (FMP) (days from anthesis to the breaker stage of fruit Colour) was 42 days for Small fry and 52 days for Petomech. Parental, F1, F2, and backcross generations differed in FMP, yield, estimates of broad- and narrow-sense SFM heritabilities of 61.4 and 38.8%, respectively, on a single-plant basis. A test for midparent heterosis also showed significance (P&lt;0.05). Genetic control of SFM was quantitative in nature, and highly dominant. The F1 and F2 generations had FMP of 47.2 and 49.5 days, respectively. F1, F2, and backcross generations all differed in number of fruits per plant, giving broad- and narrow-sense heritabilities of 69.73 and 55.67%, respectively. With a mean per fruit weight of 150.1 g Petomech recorded the highest fruit weight, while Small Fry recorded the lightest fruit weight of 81.4 g. Estimated broad-and narrow sense heritability for fruit weight was 71.76 and 41.68%, respectively. There was a significant difference between Small Fry and Petomech, as well as the BC1 and BC2 in terms of fruit size. The highest heterotic effect was found in number of fruits per plant (27.6%) over mid-parent. The highest yield was recorded by Petomech (18.9 t ha-1), while Small Fry recorded the lowest value of (14.3 t ha-1).Keywords: Genetics, heterosis, heritability, Lycopersicon esculentum

High-level intrathymic thyrotrophin receptor expression in thyroiditis-prone mice protects against the spontaneous generation of pathogenic thyrotrophin receptor autoantibodies.

The thyrotrophin receptor (TSHR) A-subunit is the autoantigen targeted by pathogenic autoantibodies that cause Graves' hyperthyroidism, a common autoimmune disease in humans. Previously, we reported that pathogenic TSHR antibodies develop spontaneously in thyroiditis-susceptible non-obese diabetic (NOD).H2h4 mice bearing a human TSHR A-subunit transgene, which is expressed at low levels in both the thyroid and thymus (Lo-expressor transgene). The present study tested recent evidence that high intrathymic TSHR expression protects against the development of pathogenic TSHR antibodies in humans. By successive back-crossing, we transferred to the NOD.H2h4 background a human TSHR A-subunit transgene expressed at high levels in the thyroid and thymus (Hi-expressor transgene). In the sixth back-cross generation (>98% NOD.H2h4 genome), only transgenic offspring produced spontaneously immunoglobulin (Ig)G class non-pathogenic human TSHR A-subunit antibodies. In contrast, both transgenic and non-transgenic offspring developed antibodies to thyroglobulin and thyroid peroxidase. However, non-pathogenic human TSHR antibody levels in Hi-expressor offspring were lower than in Lo-expressor transgenic mice. Moreover, pathogenic TSHR antibodies, detected by inhibition of TSH binding to the TSHR, only developed in back-cross offspring bearing the Lo-expressor, but not the Hi-expressor, transgene. High versus low expression human TSHR A-subunit in the NOD.H2h4 thymus was not explained by the transgene locations, namely chromosome 2 (127-147 Mb; Hi-expressor) and chromosome 1 (22.9-39.3 Mb; low expressor). Nevertheless, using thyroiditis-prone NOD.H2h4 mice and two transgenic lines, our data support the association from human studies that low intrathymic TSHR expression is associated with susceptibility to developing pathogenic TSHR antibodies, while high intrathymic TSHR expression is protective.

Obtaining advanced generations from Solanum peruvianum PI 126944 in the genetic background of S. lycopersicum by immature seed culture

The accession Solanum peruvianum PI 126944 has been reported as resistant to different biotic and abiotic stresses. In previous works of the group, advanced generations were derived from two interspecific hybrids, including up to pseudo-F6 generations and backcrosses to the cultivated species of some of the pseudo-Fn generations. Moreover, resistance to Tomato yellow leaf curl virus (TYLCV) and Tomato spotted wilt virus (TSWV) was confirmed in some of the plant materials obtained. Here we describe the development of some advanced backcross generations between the cultivated tomato and S. peruvianum. Three backcross assays were carried out. In the first assay, 16 plants belonging to pseudo-F4, pseudo-F5 and pseudo-F6 generations were backcrossed as male parents to cultivated tomato. Immature seeds from the 408 fruits obtained were platted and 77 plants were regenerated. A total of 21 descendants obtained in the first assay were backcrossed as male parents in the second assay, which allowed the obtaining of 599 fruits and regeneration of 365 plants. The 19 male parents used in the third assay were obtained either in the first or in the second one. Seventeen of them were obtained in the first assay and two in the second. A total of 339 plants were regenerated from 519 fruits. A high number of the plants obtained in these assays has been acclimated. Some plants set fruits with viable seeds.