Genetic mapping of seven knobs from the inbreds B73, Ki3, and CML277
In maize, knobs are named systematically. The letter “K” is followed by the chromosome number and arm designation. For example, K1S is the name for the knob on the short arm of chromosome 1. Multiple knobs have been identified on some chromosome arms and have been given additional numbers, for example, K4L1 and K4L2, where the highest appended number identifies the knob closest to the telomere. Not indicated is the fact that knobs vary in size (often qualitatively designated as small, medium, and large) and repeat type (primarily 180-bp repeat, TR-1 repeat, or a mixture of both). Even if both parents contain a knob at the same site, when they are different in size or repeat content, the knob can be mapped. The publically available maize germplasm includes 25 different recombinant inbred line (RIL) populations known as the NAM diversity panel (McMullen et al. 2009). Each of these RIL populations involves the B73 reference inbred and a different inbred for which basic cytological descriptions are known (Albert et al. 2010). We chose two RIL sets that provided high value in terms of polymorphic knobs: the B73 × Ki3 population and the B73 × CML277 population which together allow the mapping of nine knobs (Fig. 1; Table 1)
Both the CML277 and Ki3 populations were used to map K1S, K4L2, K5L, K6L, K8L, and K9S. In addition, K3L was mapped in the Ki3 population and K4L1 in the CML277 population (Table 1). A total of 128 (Ki3) and 126 (CML277) individuals were scored for the presence or absence of the segregating knobs. We were not able to map the knobs on 1S or 6L due to low LOD scores and/or non-concordance of the map locations between the two populations. We were able to genetically resolve (but not reliably map) two different 6L knobs in CML277, both of which are proximal to the very small B73 6L knob (Fig. 1). Ki3 has one 6L knob proximal to the B73 knob, and this knob may or may not be the same as one of the two knobs in CML277. We note that there also appears to be TR-1-rich knob on chromosome 6S; however, this region does not condense into a visible knob at the pachytene substage of meiosis like other TR-1-rich areas and thus has not been considered a knob (Kato 1976).
Knobs lie in gene-dense areas
We mapped four knobs from the B73 parent: K4L2 (primarily TR-1), K5L (primarily 180 bp), K8L (primarily 180 bp), and K9S (primarily 180 bp). B73 has a large knob on chromosome 7L, but this knob is also found in Ki3 and CML277 so it could not be mapped. All four knobs map to regions that are gene-dense and normally experience high levels of crossing over (Fig. 2). In the intervals containing K4L2, K5L, and K9S, the B73 genome assembly shows large numbers of the expected repeats (Fig. 2). K8L is less clear as it maps to an interval on the genome assembly that includes two separate TR-1 arrays and one 180-bp array. The distal boundary of the interval lies within several scattered 180-bp arrays. These results make it difficult to identify the location of K8L with certainty, particularly when taken in the context of the fact that other inbreds contain mixed knobs at the same location (both knobs 180 and TR-1; Table 2). Based on these data, we have annotated the B73 RefGen_v2 assembly locations on the MaizeGDB Genome Browser (Sen et al. 2009) to indicate the locations of K4L2, K5L, and K9S, for which we have high confidence.
Knobs affect local recombination
For three knobs, K2L, K3L, and K5L, there was sufficient knob polymorphism in the NAM population (Table 2) to carry out statistical comparisons of the effects of knobs on recombination. These diverse lines, however, cannot be compared directly because each line has a different set of polymorphic markers (SNPs) relative to B73. We therefore chose to calculate centiMorgans (cM) per megabase (Mb) values over three intervals for each knob: an interval directly proximal to the knob (on the centromere side), an interval that includes the knob, and an interval directly distal to the knob (on the telomere side; Table 3).
The data reveal a clear suppression of recombination around K2L and K3L when paired with knobless chromosomes (Table 3; Supplementary Table 1). For both of these knobs, recombination was reduced up to twofold on a cM/Mb scale. This difference is particularly notable considering that the values represent averages among intervals of different sizes depending on the closest polymorphic SNPs. The reductions are also evident in graphs that compare recombination to physical distances (Fig. 3). For both chromosomes, knobs are associated with a local reduction in the slope of the recombination curves. On chromosome 5L, we compared recombination in lines heterozygous for a large and small knob to lines that were homozygous for the small knob. These data suggest that knob size polymorphism may slightly reduce recombination, although the difference was not significant (Table 3). Interestingly, in both the large/small and small/small 5L knob pairings, graphical displays show a flattening of the recombination curves over the knob site (Fig. 3).
We also assayed the NAM lines for changes in linkage disequilibrium (LD) around knob repeat arrays. It seemed possible that at local scales, i.e., within kilobase distances from knob repeats, recombination may be severely reduced. If this were the case, we would find that knob-linked alleles would be highly correlated with each other at a population scale (that is, they would show high levels of LD). LD can be accurately interpreted from published SNP profiles of the diverse inbreds used in the NAM metapopulation (McMullen et al. 2009). As shown in Fig. 4, we calculated and plotted LD for regions flanking each of the three knobs that were mapped to the B73 reference genome assembly (K4L2, K5L, and K9S; Fig. 4). These plots did not reveal any notable changes in LD around knobs relative to the rest of the genome.