Table 1 Summary of lat mutants isolated in this studya
Mutant alleleMutant isolate numberChange in DNA sequencebChange in protein sequencecMethods used to confirm mutationdLinkage of mutation to LatB sensitivityeSynthetic lethality with ida5?f
lat1-18GCC to GACA1083Dg,hP, B, ASBN.D.
lat1-215CAG/ to CAASplice acceptor, intron 5PN.D.N.D.
lat1-320GAG to TAGE1350-STOPgP, ASN.D.N.D.
lat1-421TCG to dCGS159-FS-22aa-STOPgPN.D.N.D.
lat1-524CAG to TAGQ1289-STOPgP, B, AS7 tetrads; BYes
lat1-635GCG to dCGA45-FS-12aa-STOP or upstream mutationgP, BBiN.D.
lat2-19CAG to TAGQ55-STOPP, B, AS4 tetrads; BYes
lat2-210CAG/ to CGGSplice acceptor, intron 3P, BBYes
lat2-333AAC to ATCN304IPN.D.N.D.
lat3-117CGC AAA to CGT TAAR326R, K327-STOPB, AS4 tetrads; BYes
lat3-219AAG CCC to AGG dddK269R, P279-DELP, BBN.D.
lat3-331CAG to TAGQ400-STOPPN.D.N.D.
nap1-1 (lat4-1)16GGG to AAGG371KP, S, AS4 tetradsYes
nap1-2 (lat4-2)18CAG/ to CACjSplice acceptor, intron 6P, S, AS4 tetradsYes
nap1-3 (lat4-3)59kTGA to CGASTOP-381R-27aa-STOPSN.D.Yes
nap1-4 (lat4-4)108kTGA to TGGSTOP-381W-27aa-STOPSN.D.N.D.
NAP1-5 (LAT4-5)106kGAC to GTTD158VSN.D.N.D.
nap1-6113kGAT to GTTD15VSN.D.N.D.
nap1-7114k/GTGCGTT to GTGCCATSplice donor, intron 2SN.D.N.D.
  • a In addition to the mutants listed, complementation and linkage analysis were used to assign mutants 54, 55, 85, 92, 104, 105, 111, and 115 to lat1; mutants 102, 110, and 117 to lat2; and mutants 45, 56, 62, 68, 101, 107, 109, and 116 to lat3 (see Figure 4B).

  • b Shown is the altered codon(s), splice-acceptor, or splice-donor sequence at the position in the gene indicated in the next column. The new base(s) is indicated by boldface type; /, the site of the splice; d, deletion.

  • c Missense mutations are indicated by the change in predicted amino acid. STOP, normal stop codon or nonsense mutation; FS, frameshift; DEL, deletion. For mutations that result in additional predicted amino acids before a stop codon is encountered, the length of such sequence is indicated. Lengths of predicted wild-type products: LAT1 (Cre10.g464550), 1547 (based on the translation start site manually assigned in this study) or 1476 amino acids (as currently annotated in Phytozome 10.3, see text for detail); LAT2 (Cre10.g438250), 1065 amino acids; LAT3 (Cre02.g076000), 507 amino acids; NAP1 (LAT4; Cre03.g176833), 380 amino acids.

  • d P, pool sequencing by Illumina; B, bulked-segregant sequencing by Illumina; S, Sanger sequencing of individual gene or transcript after amplification by PCR; AS, allele-specific PCR. See Materials and Methods for details and Figure 9B for an illustration of the allele-specific PCR.

  • e Number of tetrads examined for cosegregation of LatB sensitivity and the putative lat mutation (as determined by allele-specific PCR) in a cross of the mutant to wild type; perfect cosegregation was seen in all tetrads examined. Note that bulked-segregant sequencing (B), where performed, also confirms the linkage of the putatively causative mutation to the LatB-sensitive phenotype. N.D., not determined.

  • f Indicated are results of tests for synthetic lethality with ida5-1. See text, Figure 9, and Table 3 for details. N.D., not determined.

  • g See text about the uncertainty in regard to the translation start site for this gene. The amino acid numbers shown assume that the more upstream start site is correct; if the currently annotated, downstream start site is correct, these numbers would change, and the lat1-6 mutation would lie in the 5′-UTR.

  • h The uncertainty from the original pool-sequencing results (see text) was resolved by the bulked-segregant sequencing and allele-specific PCR.

  • i Bulked-segregant reads from 10 mutant segregants had one wild-type and 34 mutant reads at the indicated position, which is embedded in an ∼0.3-Mb region containing three other uniformly read noncoding SNPs. Thus, the one wild-type read is presumed to be an Illumina sequencing error, as it would otherwise require a gene conversion or a double crossover in a very small region.

  • j The mutational alteration could be a point mutation or a deletion of one AC pair from an ACAC…AC run in the intron just upstream of the acceptor. Either possibility would result in a loss of the splice-acceptor sequence.

  • k These mutants were not part of the original set of 14 on which pooled sequencing was performed but were assigned to NAP1 (LAT4) by complementation and linkage analysis and then analyzed by Sanger sequencing.