FAQ

LORE1 is de-repressed during tissue culture. However, new copies occur only in the following generations. The activity of LORE1 was pinpointed to the germline, with highest activity in the male gametophyte. So far no somatic insertions were observed (Fukai, Umehara et al. 2010).

You will receive the R3 generation seeds (3^rd generation of tissue culture regenerated plants). Analysis of insertion sites was done by a high-throughput method in the R2 generation.

R3 is a segregating population. We observe Mendelian segregation of insertions - 1:2:1. In case of recessive mutations the phenotype, if any, should be visible for 25% of the individuals.

The genotyping primers for LORE1 lines are generated using Primer3 with a set of predefined parameters. The PCR product must span the 1000^th position, and the PCR product size should ideally fall between the range of 500-700 base pairs. As follows are the settings we have used for primer design:

Predesigned primers are available for most insertions. We were able to identify at least 95% of the test insertions using abovementioned primers. If you are using predesigned primers, always use the forward and P2 primers for insertion detection. The ±1000bp flanking sequence is reverse complemented when the LORE1 insertion is in the reverse orientation.

The PCR program used for LORE1 insertion line genotyping is known as "touchdown".

To interpret your PCR result, do take note of the following:

• homozygous wild type plants will have gene segments amplified by the forward + reverse primers,
• homozygous LORE1 insertional mutant plants will have gene segments amplified by the forward + P2 primers—the 5kb LORE1 insertion will cause the forward + reverse primers to fail to amplify,
• heterozygous plants will have gene segments amplified by the forward and reverse primers, and by the forward + P2 primers.

The PCR product sizes indicated in the LORE1 download data refers to the expected sizes of amplified gene fragments by the forward + reverse primers ('PCR Product Size in Wild Type') or the forward + P2 primers ('PCR Product Size with Insertion').

Detailed instructions about genotyping by PCR can be found in our publication (Urbanski et al., 2011).

LORE1 has a low frequency of insertions. The R1 generation acquired 3 insertions (two in unknown genes) that do not cause any phenotype. This generation (line G329-3) is used now as a founder line for the whole mutagenized population. The R2 generation has on average 2.9 new LORE1 insertions per plant. The R3 generation comes with approximately 1.9 additional new insertions per plant. Those last insertions will be different among the siblings that are shipped to you.

One case is known when LORE1 was repressed and is not accumulating during the generative propagation (Madsen, Fukai et al. 2005). We did not examine the frequency of new insertion accumulation in the R4 generation.

Non-unique LORE1 insertions occur where multiple plant IDs have been mapped to the same insertional position in the genome. In other words, these plants share identical BLAST headers (in the format of [Chromosome Number]_[Position]_[Orientation]. Usually this is a result of contamination during material collection or genotyping. However, by downloading your LORE1 search results, it is possible to identify the line with the true insertion.

The search results that you have downloaded can be opened in any common spreadsheet program (Table 2a). It contains more information than is displayed on the search results page itself, due to spatial limitations in the latter.

You can see that these lines have LORE1 insertions in the same chromosome, position and orientation. Despite being identical, each plant ID has a unique row coordinate. With that in mind, we look at the hash(#)-separated values in the last two columns, Col Coord Details and Row Coord Details (Table 2b).

We can see that R_47 has the highest count among rows, and therefore we are most confident that the line found at C_12 R_47 has the highest chance of being the line with the true LORE1 insertion at the position chr1_4599382_F. This would be line 30000012 because it has the aforementioned column and row coordinate (Table 2a).

We have not observed any problem with the seed germination, unless a line with a mutation in a known housekeeping gene was examined. We conclude that, due to the low mutational background, the germination and fertility has not deteriorated in the lines.

There might be however, problems with fungal infection. Please examine the seeds carefully before germination and if possible do not germinate all of the seeds at once.

We have recently established a new facility for growing the LORE1 lines. For the following batches of LORE1 lines we are expecting an increase in seed quality and yield.

Yes. LORE1 is a retrotransposon (RNA transposon) that amplifies in genome by a copy-and-paste mechanism. All the insertions will be present in the future generations unless removed by back-crossing.

Yes. Although LORE1 is active in the gametophyte, far less insertions are generated in the female gametophyte. It is than possible to use Gifu as a male partner for crossing with your line to remove accumulated insertions, and have a low chance to generating new ones.

chr0 corresponds to a pseudomolecule made out of contigs that were not assembled into chromosome 1 to 6 pseudomolecules of the Lotus genome release 2.5. This genome release is available on Kazusa institute servers. The sequence of the chr0 as well as .gff files with the gene models for those contigs can be downloaded from the Kazusa FTP server (check pseudomolecules).

chr7 is used for simplicity and describes Lotus chloroplast DNA in legacy versions. In newer versions (≥3.0), chloroplastic DNA are found in the Ljchloro chromosome.

No, so far there is no MTA.

Yes. We are planning to sequence more lines and share the results when they are available.

The routine way is to use the Southern blotting. Sequence-specific amplification polymorphism (SSAP) is however, simpler and faster technique that will additionally allow cloning and sequencing different insertions.

See citation guide.