Meiotic crossover reduction by virus-induced gene silencing enables the efficient generation of chromosome substitution lines and reverse breeding in Arabidopsis thaliana.

Plant breeding applications exploiting meiotic mutant phenotypes (like the increase or decrease of crossover (CO) recombination) have been proposed over the last years. As recessive meiotic mutations in breeding lines may affect fertility or have other pleiotropic effects, transient silencing techniques may be preferred. Reverse breeding is a breeding technique that would benefit from the transient downregulation of CO formation. The technique is essentially the opposite of plant hybridization: a method to extract parental lines from a hybrid. The method can also be used to efficiently generate chromosome substitution lines (CSLs). For successful reverse breeding, the two homologous chromosome sets of a heterozygous plant must be divided over two haploid complements, which can be achieved by the suppression of meiotic CO recombination and the subsequent production of doubled haploid plants. Here we show the feasibility of transiently reducing CO formation using virus-induced gene silencing (VIGS) by targeting the meiotic gene MSH5 in a wild-type heterozygote of Arabidopsis thaliana. The application of VIGS (rather than using lengthy stable transformation) generates transgene-free offspring with the desired genetic composition: we obtained parental lines from a wild-type heterozygous F1 in two generations. In addition, we obtained 20 (of the 32 possible) CSLs in one experiment. Our results demonstrate that meiosis can be modulated at will in A. thaliana to generate CSLs and parental lines rapidly for hybrid breeding. Furthermore, we illustrate how the modification of meiosis using VIGS can open routes to develop efficient plant breeding strategies

Study of variation in microRNA regions and early head development in Arctic charr (Salvelinus alpinus)

ABSTRACT Arctic charr (Salvelinus alpinus) is a northern freshwater fish that inhabits the sea, lakes and rivers. In Iceland those lakes have been all formed after the last glaciation (10000 years ago). In Lake Thingvallavatn, Iceland, Arctic charr shows as many as four sympatric morphs. Two of them are limnetic (Planktivorous and Piscivorous) and the rest are benthonic (Small benthic and Large benthic). One remarkable feature is the high variability these morphs present regarding to their morphology, behaviour and life history. One part of this research will be focused on the study of genetic variation in different microRNAs genomic region of Planktivorous (PL), Small Benthic (SB) and Large Benthic (LB) population. These regions will also be compared between Salmo salar and Salvelinus alpinus. Another aim is to study the developmental basis of morphological differences observed in the three morphs previously mentioned plus one more, an Aquaculture (AC) stock. The main goal is to describe eye size, position and shape changes along variation in the strucutures of the splacnocranium in early stages of these morphs. After that different morphs and stages will be compared, ossification of these structures will be also explained comparing morphs and stages. Samples used in the study of genetic variation where collected in different years from Lake Thingvallavatn, in Iceland. DNA isolation, amplification by PCR procedure and sequencing were done in order to identify and describe variations in the miRNA regions. Once these variations were found, I tested for genotype and allele frequency differences between morphs. Using morphometrics, I tested morph/stage differences in changes of the structures. In conclusion significant allele and genotype frequency between morphs were found comparing LB with SB and PL in the microRNA genomic region of 19c,20b-3p,18b and 18b*. Embryos used for morphometrics had the bigger stage frame for AC. Comparison was possible in 4 stages of the four morphs finding significant differences regarding to morphs and stages. Also significant changes occur differantially on the stage depending on the morph.  Bleikja (Salvelinus alpinus) lifir á norðlægum slóðum, í sjó og ferskvatni. Á Íslandi eru nokkrir stofnar bundnir við ferskvatn, læki og vötn, sem mynduðust í kjölfar síðustu ísaldar (fyrir um 10000 árum). Í Þingvallavatni eru mestur fjölbreytileiki í bleikjustofninum, þar finnast fjögur samsvæða afbrigði. Tvö þeirra eru sviflæg, sílableikja og murta, en tvö botnlæg, dvergbleikja og kuðungableikja. Sérstakt er hversu ólík afbrigðin eru í útliti, háttum og lífssögu. Fyrri hluti rannsóknarinnar lýtur að greiningu á miRNA svæðum í þremur bleikjuafbrigðum, með það að markmiði að kanna hvort þau séu til staðar í bleikju og hvort að erfðabreytileiki á þessum svæðum fylgi afbrigðum. Við skoðum hversu svipuð svæðin eru milli bleikju og lax Salmo salar. Seinni hluti rannsóknarinnar snýr að greiningu á formi og staðsetningu augna í bleikjufóstrum. Við skoðum murtu, dverg og kuðungableiku, auk eldisbleikju frá Hólum, og könnum staðsetningu, afstöðu og stærð augna og beina á fyrstu vikum og mánuðum þroskunar. Sýnin fyrir erfðarannsóknina voru öll tekin úr Þingvallavatni, en á ólíkum árum. Ákveðin svæði voru mögnuð upp með PCR og raðgreind með Sanger aðferð, með það að markmiði að finna og skilgreina erfðabreytileika í þeim. Sérstaklega var prófað hvort að breytileiki í ákveðnum stöðum genanna væri misalgengur í bleikuafbrigðunum þremur. Niðurstöðurnar sýna að breytileiki á einu miRNA svæðinu er marktækt algengari í kuðungableikju, en í dvergbleikju og murtu. Þetta svæði inniheldur genin 19c,20b,18b and 18b*. Sumar breytingar í formi sýna mjög sterka fylgni við þroskastig og stærð fóstursins, en aðrar breytast lítið meðan á þroskun stendur. Samanburður á afbrigðum leiðir í ljós að sumar formbreytingar eru bundnar við ákveðin afbrigði, sem sýnir að hluti af þeim svipfarsmun sem greinir að afbrigðin kann að eiga sér rætur í snemmþroskun bleikjunar

Genotyping Data Hyperrecombinant offspring VIGS

Offspring generated from the crosses of F1 lerxcol plants in which RECQ4 and/or FIGL1 were pressumably knocked-down. Expected lines were expected to be hyperrecombinant but they display the same recombination events as compared to a wild-type population. The genoyping markers used can be seen in the first two rows while the first two columns display the total number of lines used. The markers in blue (B) corresopnd to homozygous Ler alleles while the green ones (H) correspond to the presence of a Col-Ler alleles

Genotyping data MiMe offspring VIGS

Genotyping data regarding the offpsring generated from crosses of F1LerxCol hybrids to the transgenic line male sterile Ler. The genotyping markers used for genotyping are listed in the first rows. The first two columns contain the different lines genotyped. The blue color (B) identify a marker corresponding to a homozygous Ler allele while the green marker (H) corresponds to heterozygous Col-Ler

Knock-down of gene expression throughout meiosis and pollen formation by virus-induced gene silencing in Arabidopsis thaliana

Through the inactivation of genes that act during meiosis it is possible to direct the genetic make-up of plants in subsequent generations and optimize breeding schemes. Offspring may show higher recombination of parental alleles resulting from elevated crossover (CO) incidence, or by omission of meiotic divisions, offspring may become polyploid. However, stable mutations in genes essential for recombination, or for either one of the two meiotic divisions, can have pleiotropic effects on plant morphology and line stability, for instance by causing lower fertility. Therefore, it is often favorable to temporarily change gene expression during meiosis rather than relying on stable null mutants. It was previously shown that virus-induced gene silencing (VIGS) can be used to transiently reduce CO frequencies. We asked if VIGS could also be used to modify other processes throughout meiosis and during pollen formation in Arabidopsis thaliana. Here, we show that VIGS-mediated knock-down of FIGL1, RECQ4A/B, OSD1 and QRT2 can induce (i) an increase in chiasma numbers, (ii) unreduced gametes and (iii) pollen tetrads. We further show that VIGS can target both sexes and different genetic backgrounds and can simultaneously silence different gene copies. The successful knock-down of these genes in A. thaliana suggests that VIGS can be exploited to manipulate any process during or shortly after meiosis. Hence, the transient induction of changes in inheritance patterns can be used as a powerful tool for applied research and biotechnological applications

Meiotic crossover reduction by virus-induced gene silencing enables the efficient generation of chromosome substitution lines and reverse breeding in Arabidopsis thaliana

Plant breeding applications exploiting meiotic mutant phenotypes (like the increase or decrease of crossover (CO) recombination) have been proposed over the last years. As recessive meiotic mutations in breeding lines may affect fertility or have other pleiotropic effects, transient silencing techniques may be preferred. Reverse breeding is a breeding technique that would benefit from the transient downregulation of CO formation. The technique is essentially the opposite of plant hybridization: a method to extract parental lines from a hybrid. The method can also be used to efficiently generate chromosome substitution lines (CSLs). For successful reverse breeding, the two homologous chromosome sets of a heterozygous plant must be divided over two haploid complements, which can be achieved by the suppression of meiotic CO recombination and the subsequent production of doubled haploid plants. Here we show the feasibility of transiently reducing CO formation using virus-induced gene silencing (VIGS) by targeting the meiotic gene MSH5 in a wild-type heterozygote of Arabidopsis thaliana. The application of VIGS (rather than using lengthy stable transformation) generates transgene-free offspring with the desired genetic composition: we obtained parental lines from a wild-type heterozygous F1 in two generations. In addition, we obtained 20 (of the 32 possible) CSLs in one experiment. Our results demonstrate that meiosis can be modulated at will in A. thaliana to generate CSLs and parental lines rapidly for hybrid breeding. Furthermore, we illustrate how the modification of meiosis using VIGS can open routes to develop efficient plant breeding strategies.</p