Euromillions lottery winners Chris and Colin Weir have boosted the Scottish National party’s election warchest with another £1m donation, as the party prepares to challenge for dozens of Labour seats.

The Weirs, who won £161m in a then record Euromillions jackpot in July 2011, made the latest £1m gift in early September, just two weeks before the Scottish independence referendum which the SNP’s outgoing leader, Alex Salmond, was convinced his party would win.

The SNP has had a further major gift from its other long-standing multi-millionaire backer, the Stagecoach founder Sir Brian Souter, further swelling the party’s campaign funds. He donated £400,000 two days before the referendum, the electoral commission has reported.

Their latest donation brings the Weirs’ total gifts to the SNP to £3m, in addition to £3.5m they gave to the independence campaign Yes Scotland in the lead-up to the referendum. Souter has now given the SNP nearly £2.6m since March 2007. The scale of these gifts will put the Labour party under intense pressure to match that spending in Scotland during the general election campaign, to protect dozens of seats in central Scotland expected to be under threat from the SNP. A Guardian analysis has identified up to 20 seats at risk, including Dundee West, Edinburgh East, Falkirk, Inverclyde and Ochil and South Perthshire.

Jim Murphy, the favourite to win the Scottish Labour party’s leadership contest, has said he would try to raise £1m from within Scotland and establish a funding stream independent of the party’s UK headquarters.

While opposition parties stepped up their accusations that the SNP are too reliant on multi-millionaire benefactors, the SNP’s income has also been boosted by a record influx of new members, which will further increase its spending power at the May general election.

The party is expected to confirm that about 64,000 new members have joined since it lost the independence referendum, taking its total membership to roughly 90,000, confirming its new status as the UK’s third largest political party.

SNP officials say membership subscriptions and extra monthly direct debit payments play a major part in funding the party, but are sums of money which are individually too small to be recorded by the electoral commission.

“As membership of the SNP surges to over 84,000, the party has never been in better shape. We are very grateful for every donation - large and small - from our supporters which all help us to campaign and work for Scotland,” an SNP spokesman said.