#!/usr/bin/perl
use strict;
use warnings;

my (@genes, %tested);
while (<>) {
    s/^>//;
    
    my ($col1, $col2) = split;
    push @genes, $col1;
    $tested{$col2}++; 
}

{
    local $\ = "\n";
    for (@genes) {
        print if not $tested{$_};  
    }
}

##</code><code>##

C:\Old_Data\perlp>type o33.txt
>chr9:133738100-133738472_0 chr20:62159728-62161126_840
>chr9:133738100-133738472_60    chr2:215589720-215676478_59220
>chr9:133738100-133738472_120   chr2:215589720-215676478_59160
>chr9:133738100-133738472_180   chr15:99500240-99507809_0
>chr9:133738100-133738472_240   chr2:215589720-215676478_59100
>chr9:133738100-133738472_253   chr1:162745876-162746210_215
>chr9:133747466-133747650_0 chr5:108523084-108532592_960
>chr9:133747466-133747650_60    chr20:62159728-62161126_900
>chr9:133747466-133747650_65