Well, It is rather hard to tell orientation of somebody without outright asking them. But, I like to think it has something a bit to do with their personalities. As that is sort of the reason we have six main characters in this show, to get different viewpoints and avenues for character development.
Now the easiest to tell, by far, is Rarity. The show has made no light work of showing her interest in stallions albeit in a way to make a jab at her manipulative flirting. Plus, considering her rather high view of her reputation, she really does seem the Straight Stereotype to balk & get huffy at the implication that she might be gay. Does that mean she can't be gay, of course not, some of the best shipping fics I've come across have involved the ensuing hilarity of Rarity!
As for Pinkie, its rather a bit harder to tell. I do get the feeling that many in the fandom see her irrepressible energy & her self-given title of a Party Pony and assume she's a loose mare. While I understand that, I've seen her capable of calming down on the odd occasion to fully assess a situation & consider the feelings of others. Not...always...I do have to admit...but regardless she strikes me as the sort to care too much for others to hurt their feelings by ditching them after a one-night stand. Though, as she holds as many parties as she clearly has she has undoubtedly had to deal with those types with ill intentions or the occasional drunk mare and I like to think she's had the practice to flirt with the best of them. Madame de Pie does strike me as someone that'd be open with their sexuality, were this not a show for kids I fully expect her to be that one friend who just won't stop with all the crass sexual jokes. Though I wouldn't be surprised if Pinkie was still a virgin. Would however be surprised if a certain other previously-mentioned mare was...
The next two girls are significantly harder to place. Applejack has always seemed to care more for working on her farm then even spending time with other ponies. She gets a ticket for the biggest shindig around and what does she do? Get profits for her farm. Rarity goes far out of her way to spend time with her in one of the more recent episodes & AJ cares more for work, yet again. I'd put Applejack more as an Asexual. Sure, if you took the effort and straight-up asked for a date with her she'd probably accept without bothering twice to think of what your gender was; just reckon ah'm gon' have me a hard time fittin' ya inta mah shed-ule on account of the eighteen things ah gotta do at this farm before noon!
And now for the big one! I do agree with that sentiment, that everyone wants a piece of Rai-Rai. Confidence is attractive, doubt many can argue that point, but I don't think Rainbow Dash cares that way. She loses her temper on a hairstring. Sure, I can agree she's protective and kind to Fluttershy and with her personality that IS saying alot! But this is the ever-fluffin' FLUTTERSHY we're talkin' about! Her personality trope grates on me and I still want to protect & hug her! Way I see it, Dash has extraordinarily high expectations. Both in herself, as we've seen her collapse under pressure a few times in the show despite her bluster, and in others with as often as we've seen her lose her temper. Dragonshy, one of my favorite episodes, sort of shut the door in my mind on Flutterdash. RD spent the entire episode grumbling & scowling at ol' Fluttershy instead of helping her with her fear. In my opinion, RD doesn't look like she'd accept anyone who would hold her back. But! In the newer episode of Stranger than Fanfiction, we saw RD meet somepony with a common interest, a stallion that could meet on her ends & make her happy! To me, that darn near seemed like the writers attempt to say "No, no guys! She straight, honestly! Stop with all the shipping!" . You can't TELL me that wasn't a date!...well, until he proved just as mouthy as she was to add hilarity and drama.
...still yet to see if this will challenge my Raridash...Rairibow?...Rainity?...
So that leaves us with what, Twilight and Fluttershy? Aye, sort of lump them a bit together, as they are both the clearest introverts of the group. Maybe it's just my own thoughts, but i'm inclined to think that introverts are more likely to give consideration to being gay. As much as we've seen Twiliey avidly research anything regardless of its importance (she has a book that tells you how to RUN for fluff's sake!) no doubt she has some bookcase of personal books she keeps well-hidden from anypony who wanders in and especially any young dragons. I like the notion of some comic strip jokes you see about DeviantArt that portray Twilight as an avid maker & reader of lurid fanfiction. Twilight knows about sex, and has thought about it in depth. Anypony who spends that much time reading & thinking has had to at some point! She is Bi- at the LEAST!
As for Fluttershy, well...have to agree. She spends most of her time holed up in her house and is afraid to meet most anypony. But still, if you took the time to ask her, i'm sure she wouldn't have the heart to turn you down because she's not attracted to your gender. What she needs is somepony just outgoing enough to make plans and pull her into an adventure, but introverted just enough to stop & listen to her.
That one episode about the Smokey Mountains or whatever. Where it's middle of a battlefield & Twilight is sitting there depressed at her failure and Fluttershy comes up, rests a hoof on her shoulder & gives her a calm, gentle solution. ....darn near broke my heart, there.
Twi-shy All The Way!!!