CNE gene interaction

To each cluster we assigned the two closest protein-coding genes or lncRNAs (one for each side of the cluster) if they were in cis orientation to the cluster (table 6.4), which will be called associated gene. Except in A. rosae the majority of clusters had no gene in cis direction assigned (table 6.4). Following this protein-coding genes were the next biggest type assigned to cluster (table 6.4). The number of dierentlncRNAs identied next to clusters varied between 36 (N. vitripennis) and 548 (A. mellifera). In some cases one lncRNAwas assigned to two clusters when no other gene was found between the two clusters. The number of uniquelncRNAs was lower. In N. vitripennis we found 27 lncRNAs, 56 in O. abietinus, 292 in A. rosae, and 435 in A. mellifera. In both A.

mellifera and O. abietinus the number oflncRNAs found upstream and downstream of a cluster was comparable (38 in both directions in O. abietinus and 288 to 260 in A.

mellifera), whereas in N. vitripennis alllncRNAswere found upstream. In A. rosae, the majority oflncRNAs was found downstream of CNE clusters (table 6.5).

Table 6.4.: Total amount of CNE clusters per species as well as the count of clusters with at least onelncRNA in cis as the closest gene, number of clusters with protein-coding genes (gene) in cis. N/A shows the number of clusters where no gene was found next to it or were the closest gene was in trans.

Species Cluster Gene lncRNA N/A

Athalia rosae 1,599 1,464 322 1,142 Orussus abietinus 2,088 2,040 76 2,060 Apis mellifera 1,948 1,248 548 2,100 Nasonia vitripennis 1,625 325 36 2,555

As ^lncRNAs are not described as interaction partners with ^CNEs, we looked at the protein-coding gene/lncRNA ratios (table 6.3). In A. mellifera lncRNAs do not occur more often next to a cluster than would be expected. In two species they occur less often than expected, 1.3 times lesser in A. rosae and 12 times lesser in O. abietinus.

In N. vitripennis the were found twice as often as expected neighbouring a cluster in cis.

Table 6.3.: Ratios of lncRNA/protein-coding genes in each species. First number is calcu-lated from all lncRNAs and protein-coding genes present in the OGS, second is calculated from the lncRNAs and protein-coding genes that were found in cis next to aCNEcluster.

Species Ratio whole annotation Ratio CNE cluster neighbours

Athalia rosae 0.30 0.22

Orussus abietinus 0.46 0.037

Apis mellifera 0.44 0.43

Nasonia vitripennis 0.045 0.11

Between 325 (N. vitripennis) and 2,040 (O. abietinus) dierent protein-coding genes were identied as neighbouring a cluster in cis direction. Except for N. vitripennis, over 1,000 genes were identied as neighbouring a cluster in cis: 1,248 (A. mellifera), 1,464 (A. rosae), 2,088 (O. abietinus).

Table 6.5.: CNE clusters with an lncRNA in cis direction next to it. Total includes every occurrence of anlncRNA in the right direction next to aCNEcluster, upstream is the total count of those found upstream, downstream the total count found downstream of a cluster, and unique lncRNAs is the count of dierent lncRNAs identied.

Species total upstream downstream unique lncRNAs

Athalia rosae 322 22 300 292

Orussus abietinus 76 38 38 56

Apis mellifera 548 288 260 435

Nasonia vitripennis 36 36 - 27

For each ^CNE cluster we set a maximum distance of 500 kb in which a gene had to be located. This distance was chosen as other studies showed that genes of interest tend to be located inside this region. The distance between the cluster and the closest gene varied between 1 bp (found in all four species) and 483,349 bp (O. abietinus).

As stated above we found between 36 and 548 cases of lncRNAs next to a CNE cluster in cis direction in one species (table 6.5). The highest number of 548 was found in A. mellifera with 288lncRNAs found upstream and 260 found downstream of a cluster.

Reducing this number to uniquelncRNAs 435 genes remain.

In N. vitripennis all lncRNAs identied as the associated gene of a cluster were found upstream, whereas in all three other species lncRNAs were found both upstream and downstream of CNE clusters. In A. mellifera and O. abietinus the number between upstream and downstream were similar, whereas in A. rosae the majority of lncRNAs

was found downstream (300 genes downstream, 22 upstream) (table 6.5).

We selected the six largest scaolds to get a look at theCNEdistribution. The predicted

CNEswere not equally distributed along the scaolds of each species. This distribution is visualised in gures 6.3, 6.4, 6.5, and 6.6. For each species three graphs are shown, to show the results of the pairwise comparisons. Looking at gure 6.3 a) we see the distribution of theCNEsidentied in the comparison of A. mellifera and A. rosae. Each of the six subplots shows the distribution on one scaold. On the x-axis we see the genomic location of aCNE in Mb and on the y-axis the accumulative number of CNEs. In A. mellifera we see on some of those scaolds only small gaps, meaning locations on

the scaold where noCNEs were identied, and long stretches with no gaps in theCNE

distribution, whereas on others we nd a lot of single ^CNEs (gure 6.3). This uneven distribution is especially noticeable in A. rosae (gure 6.4). A. rosae has less CNEs

identied on the six largest scaold compared to the other three species (up to 60 in A.

rosae compared to up to 600 in the other species). In all four species the distribution of CNEs between all three pairwise comparisons is similar (gures 6.3, 6.4, 6.5, 6.6).

Scaold 1 of O. abietinus is an example where the majority of CNEs were identied in the middle of the scaold. This leads to a high increase of the total number over a small amount of basepairs (gure 6.6). Note that the scale of the y-axis is not unied.

(a) Athalia rosae (b) Nasonia vitripennis

(c) Orussus abietinus

Figure 6.3.: Distribution ofCNEprediction in Apis mellifera dierentiated by species. Only the results for the six longest scaolds are shown. The number of CNEs is the accumulative total amount found on this scaold. x-axis shows the genomic location on the scaold, y-axis the number ofCNEs. The results are for pairwise comparisons between species.

(a) Apis mellifera (b) Nasonia vitripennis

(c) Orussus abietinus

Figure 6.4.: Distribution of CNEprediction in Athalia rosae dierentiated by species. Only the results for the six longest scaolds are shown. The number ofCNEs is the accumulative total amount found on this scaold.

(a) Apis mellifera (b) Athalia rosae

(c) Orussus abietinus

Figure 6.5.: Distribution ofCNEprediction in Nasonia vitripennis dierentiated by species.

Only the results for the six longest scaolds are shown.The number ofCNEs is the accumulative total amount found on this scaold.

(a) Apis mellifera (b) Athalia rosae

(c) Nasonia vitripennis

Figure 6.6.: Distribution of CNE prediction in Orussus abietinus dierentiated by species.

Only the results for the six longest scaolds are shown.The number ofCNEsis the accumulative total amount found on this scaold.