The very end of Harvest Moon: A Wonderful Life. No, not your character dying, or even your child getting married. The very last line your character speaks in the game. When your wife observes how you've both gotten old, and aren't the glamorous young things you used to be.
Your Character:You're still beautiful.
Previous to that; given what a Sugar Bowl the series usually is, Ellen's death in Harvest Moon 64 doubles as this and the closest thing the series has to a Wham Episode. Elli's reaction just drives the trope home.
Even worse if you played the original title. That cute, friendly young woman you befriended and maybe even married? Dead
Screwing up anyone's love life. For example: making a character leave the game (like Kai or Cliff), turning down a character, choosing a bad option in a love event, or (sometimes) courting a character. It's even worse in the Wonderful Life series, especially in the first game. If you court a character and marry another, they'll go to your house crying and basically give you a "You Suck"speech.
Out of all the "betrayed" scenes in A Wonderful Life, the saddest has got to be Muffy's. Let's just say that it's hard not to feel bad about getting Muffy to four hearts and then proposing to someone else.:
Muffy:I thought I didn't want to see you again. But I had to go and do something stupid. Coming here... I was serious about you.
Animal Parade: What Gill says in the Japanese version of the game if you tell him you don't like him back when he confesses to you.
Gill:I'm sorry. I wasn't trying to push my feelings on you. If there's someone else, you should tell him how you feel... Thanks for today.
In Animal Parade, after marrying the Harvest King and maxing out his affection for you, his maximum-affection line drives home the full effect of your very different lives:
"After you are gone... I shall love what you loved... I shall sing the songs that you sang."
"After you are gone, I will be alone once more. When I think about that, it makes me sad."
In Harvest Moon DS, most of Galen's dialogue post Nina's death is pretty sad, especially when you imagine him going to bed and living alone in their little house every single night.
In Connect to the New World/Story of Seasons, Edda will die on the first day of winter, no matter what you do. You can't even prevent it, unlike Ellen's death. For how much of a Sugar Bowl the games have been lately, it's especially shocking.
In A Wonderful Life, it's unavoidable. No matter what girl you pick to marry, the other two will have sad lives. Muffy remains a single waitress all her life and will occasionally be seen on the beach at night crying about losing her beauty and never getting a chance to have a child. Celia grows into an angry bitter man-hater who seems to give Marlin hell at the farm. And Nami runs out of money and tearfully has to leave town to live with her deadbeat dad, and years later when she returns she spends all her time drinking herself into a stupor.
The revelation that Gotz from Back to Nature and Friends of Mineral Town had lost his wife and child to a storm. You can see why not he sometimes act rough.